Journal of Pathology J Pathol 2014; 234: 152–163 Published online 6 August 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.4341
ORIGINAL PAPER
Morgana acts as a proto-oncogene through inhibition of a ROCK–PTEN pathway Federica Fusella,1 Roberta Ferretti,1† Daniele Recupero,2 Stefania Rocca,1 Augusta Di Savino,1 Giusy Tornillo,1‡ Lorenzo Silengo,1 Emilia Turco,1 Sara Cabodi,1 Paolo Provero,1 Pier Paolo Pandolfi,1,3 Anna Sapino,2 Guido Tarone1 and Mara Brancaccio1* 1
Department of Biotechnology and Health Sciences, University of Torino, Torino, Italy Department of Medical Sciences, University of Torino, Torino, Italy 3 Cancer Genetics Program, Division of Genetics, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 2
*Correspondence to: Mara Brancaccio, Molecular Biotechnology Center, Via Nizza, 52, 10126 Torino, Italy. e-mail: [email protected] † ‡
Current address: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Current address: Cardiff School of Biosciences, The Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, UK.
Abstract Morgana/CHP-1 is a ubiquitously expressed protein able to inhibit ROCK II kinase activity. We have previously demonstrated that morgana haploinsufficiency leads to multiple centrosomes, genomic instability, and higher susceptibility to tumour development. While a large fraction of human cancers has shown morgana down-regulation, a small subset of tumours was shown to express high morgana levels. Here we demonstrate that high morgana expression in different breast cancer subtypes correlates with high tumour grade, mitosis number, and lymph node positivity. Moreover, morgana overexpression induces transformation in NIH-3T3 cells and strongly protects them from various apoptotic stimuli. From a mechanistic point of view, we demonstrate that morgana causes PTEN destabilization, by inhibiting ROCK activity, hence triggering the PI3K/AKT survival pathway. In turn, morgana down-regulation in breast cancer cells that express high morgana levels increases PTEN expression and leads to sensitization of cells to chemotherapy. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: breast cancer; chemoresistance; morgana; PTEN; ROCK
Received 15 October 2013; Revised 11 February 2014; Accepted 13 February 2014
No conflicts of interest were declared.
Introduction Morgana/CHP-1 (coded by the CHORDC1 gene) is a ubiquitous protein [1] with chaperone activity [2,3], and plays a key regulatory role in centrosome duplication and genomic stability through ROCK II inhibition [4,5]. Morgana is highly conserved during phylogenesis [1,6,7] and has an essential role during developmental processes [4]. The absence of morgana in Drosophila neuroblasts causes a strong mitotic phenotype, consisting of a high frequency of polyploid cells, with most diploid cells displaying supernumerary centrosomes. Moreover, morgana haploinsufficiency in mouse embryonic fibroblasts (MEFs) derived from morgana +/− embryos causes a higher frequency of polyploid cells, supernumerary centrosomes, and multipolar spindles [4]. Genomic instability is a typical feature of cancer cells and is known to favour cancer onset and progression [8–10]. In line with these considerations, morgana +/− MEFs are transformed by oncogenic Ras, rather than undergoing senescence; in addition, tumour Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
susceptibility in response to chemical mutagens is significantly higher in morgana +/− mice than in wild-type mice [4]. The role of morgana as a tumour suppressor in humans is supported by the fact that morgana expression is strongly reduced in a large percentage of human breast and lung cancer samples compared with normal tissues [4]. However, our tissue array analysis also indicated that morgana is overexpressed in a small fraction of human breast and lung cancers [4]. To further investigate this intriguing result, we analysed morgana levels in human breast cancer samples, and here we show that morgana is overexpressed in different breast cancer subtypes, in particular in triple-negative breast cancers (TNBCs), and that its overexpression correlates with tumour grade, mitosis number, and lymph node positivity. Furthermore, we found that morgana overexpression is able to transform NIH-3T3 cells and to protect them from different apoptotic stimuli. Accordingly, morgana down-regulation in T47D and MDA-MB-231 breast cancer cells that express high morgana levels confers J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
Morgana proto-oncogenic function via PTEN
sensitivity to chemotherapeutic agents. On a molecular level, inhibition of ROCK by high morgana levels causes down-regulation of phosphatase and tensin homologue (PTEN), leading to enhanced AKT activation and cell survival.
Materials and methods Tissue array preparation and immunohistochemistry (IHC) This study was conducted in compliance with the ethical regulatory requirements for the handling of biological specimens after appropriate informed consent and Institutional Review Board approval. A series of 208 breast cancer samples and 51 TNBC samples was collected at the Breast Unit of the San Giovanni Battista Hospital of Torino, Italy. Tissue array preparation and morgana detection are described in the Supplementary materials and methods. In the tissue array analysed, three cores were obtained for each breast cancer sample in the areas of higher cellularity and one core from normal adjacent breast tissue. The analysis of morgana staining intensity in the normal breast epithelium of different patients indicated a comparable expression level and was used as a reference. High and low levels were defined when the staining intensity was respectively higher or lower compared with the staining intensity in normal tissue. Slides were scored independently by two pathologists.
Analysis of copy number alteration Previously generated somatic copy number alteration data [11] were used to determine the fraction of breast tumours with morgana coding gene (CHORDC1) amplification. The data were obtained from the website associated with the paper [11] and included 773 breast cancer samples, 499 of which were classified into gene expression-based molecular subtypes.
Antibodies and reagents Western blotting was performed using the following primary antibodies: anti-morgana P1/PP0 (1 μg/ml) [4], actin (sc-1615; 1 : 3000), ROCK I (sc-5560; 1 : 1000), ROCK II (sc-5561; 1 : 1000), MLC2 (sc-15370; 1 : 1000), cyclin D1 (sc-753; 1 : 1000), and p70S6K (sc-230; 1 : 1000), which were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); Bad (9292; 1 : 1000), ROCK I (4035; 1 : 1000), caspase 3 (9665; 1 : 1000), phospho-MLC2 (3671; 1 : 1000), PTEN (9559; 1 : 1000), phospho-AKT Ser473 (9271; 1 : 1000), AKT (4691; 1 : 1000), phospho-GSK3β (9336; 1 : 1000), GSK3β (9315, 1 : 1000), phospho-p70S6K (9208, 1 : 1000), phospho-4E-BP1 (2855, 1 : 1000), and 4E-BP1 (9452, 1 : 1000), which were from Cell Signaling (Danvers, MA, USA). Vinculin (SAB4200080, 1 : 5000) and α-tubulin (T5168; 1 : 8000) were from Sigma-Aldrich Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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(St Louis, MO, USA). GAPDH (MAB374, 1 : 3000), phospho-MYPT1 (07–251; 1 : 1000), and GFP were from Millipore (Billerica, MA, USA). Finally, ROCK II (07–443; 1 : 1000) and MYPT1 (07–672; 1 : 1000) were from Upstate Biotechnology (Lake Placid, NY, USA). Fetal bovine serum (FBS), fetal calf serum (FCS), penicillin–streptomycin (PS), and basic fibroblast growth factor (bFGF) were from Invitrogen (Carlsbad, CA, USA). Epidermal growth factor (EGF), heparin, insulin, hydrocortisone, Akt inhibitor GSK690693, docetaxel, epirubicin, and etoposide were from Sigma-Aldrich.
Cell culture NIH-3T3 and human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and propagated and maintained according to protocols supplied by ATCC. The experiments were performed within 6 months after resuscitation. NIH-3T3 cells were maintained in DMEM with 10% FCS and 5% PS. MDA-MB-231 and 293 cells were cultured in DMEM with 10% FBS and 5% PS. MCF-7 and T47D were cultured in RPMI with 10% FCS and 5% PS. NIH-3T3 cells stably overexpressing morgana or GFP were obtained as previously described [2]. MCF-7 cells overexpressing morgana were obtained using a pLVX lentiviral vector coding for mouse morgana. Morgana knockdown in MDA-MB-231 and T47D was performed by infecting cells with pGIPZ lentiviral particles expressing two different morgana shRNAs, together with the TurboGFP (Thermoscientific Open Biosystems, Chesterville, AL, USA). Expression of ROCK I deletion mutants fused with GFP was obtained by calcium phosphate transfection of 293 cells.
Transforming assays To assess the NIH-3T3 cell proliferation rate, 5 × 105 cells were seeded in 35 mm plates and counted at the indicated times. MDA-MB-231 and T47D cell growth curves were generated by plating 50 000 cells and staining cells with crystal violet at the indicated times, as previously described [4]. Soft agar colony formation assays were performed by resuspending cells in complete DMEM containing 0.3% low gelling agarose (Sigma-Aldrich) and seeding cells in triplicate into six-well plates containing a 2 ml layer of solidified 0.6% agar (Sigma-Aldrich). The Akt inhibitor, GSK690693, was added every 24 h with a daily change of medium at the final concentration of 2 nM. After 4 weeks, colonies were stained with nitroblue tetrazolium (Sigma-Aldrich), photographed with Zeiss microscopy, and counted using the ImageJ Software.
Assessment of tumourigenicity in nude mice The use of animals complied with the Guide for the Care and Use of Laboratory Animals published by the J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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US National Institutes of Health and was approved by the Animal Care and Use Committee of the University of Torino. A total of 106 NIH-3T3 or 106 MDA-MB-231 cells were resuspended in 200 μl of PBS and injected subcutaneously into the left flank of female nude athymic mice or SCID mice. Tumours were measured using a caliper.
Cell death treatments NIH-3T3 cells were detached and kept in suspension for 12 h, serum-starved for 24 h, or treated with 125 μM etoposide for 16 h. Apoptosis was assayed by annexin V staining and by TUNEL assay (In Situ Cell Death Detection Kit TMR red; Roche, Mannheim, Germany). Cells treated with etoposide were stained for annexin V. Apoptosis of T47D and MDA-MB-231 cells treated with etoposide was detected by staining cells with anti-cleaved caspase3 antibody (Cell Signaling). Apoptosis of T47D and MDA 231 treated with docetaxel was assayed using a TUNEL assay. Apoptosis of T47D and MDA-MB-231 cells treated with epirubicin was detected by annexin V staining (BD Biosciences, San José, CA, USA).
ROCK kinase assay Kinase assays were performed as previously described [12] and as described in the Supplementary materials and methods. Kinase activity was detected by western blotting using anti-phospho-MYPT1 and anti-MYPT1 antibodies.
Immunoprecipitation and western blot analysis For immunoprecipitation, cells were lysed in NP-40 lysis buffer [20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.5% NP-40, 10 mM NaF, 1 mM PMSF, 1 mM Na3 VO4 , and protease inhibitors]. Five micrograms of the selected antibody was added, along with protein G-coated Sepharose, to the protein extracts and processed as previously described [12]. For western blot analysis, cells were lysed in Tris-buffered saline with 1% Triton X-100, plus phosphatase and protease inhibitors. To detect phosphorylation of MLC2, cells were lysed with SDS buffer [1% SDS, 60 mM Tris–HCl (pH 6.8)]. Total protein extracts (30 μg) were analysed by western blotting and detected by the chemiluminescent reagent LiteAblot (Euroclone, Milano, Italy). Band intensities were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).
Statistical significance The data are presented as means ± SEM. For statistical analyses, significance was tested using a two-tailed Student’s t-test or, when required, one- or two-way ANOVA with Bonferroni’s correction. A minimum value of p < 0.05 was considered to be statistically significant. Statistical analyses were performed using GraphPad Prism 4. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
F Fusella et al
Results Morgana overexpression is associated with aggressive phenotypes in human breast cancers We evaluated morgana expression levels in a tissue array containing 208 human breast cancer specimens. Of the tumours analysed, 63% had levels of morgana lower than the normal breast epithelium, confirming that morgana is frequently down-regulated in human breast cancers [4]. However, 17% of tumour samples had higher morgana expression levels compared with normal tissue (Figure 1A). Interestingly, morgana levels correlated positively with tumour grade (Figure 1B), mitosis number (Figure 1C), mean Ki67 labelling index (Figure 1D), and lymph node involvement (Figure 1E). Although morgana overexpression did not correlate with oestrogen (ER), progesterone (PR) or HER2 status, we noticed that seven triple-negative cancer samples out of the nine included in the tissue array expressed high levels of morgana. Triple-negative breast cancers do not express ER or PR, lack HER2 overexpression, and are associated with a shorter median time-to-relapse and increased mortality [13]. To expand the analysis to a relevant number of tumours, we analysed morgana expression levels in a tissue array containing 51 specimens from human triple-negative breast cancers. The results indicated that 36% of the samples showed strong overexpression of morgana compared with normal breast epithelium (Figure 1F). In accordance, analysis of the data within the Cancer Genome Atlas project [11] highlighted that the morgana gene (CHORDC1) locus is amplified in 19% of basal-like triple-negative breast cancers (Figure 1G).
Morgana overexpression induces transformation in NIH-3T3 cells and protects cells from apoptotic stimuli To investigate the possible role of morgana overexpression in tumourigenesis, we chose the NIH-3T3 cell line, which represents a widely used model system for testing oncogene function. We infected NIH-3T3 cells with a lentivirus coding for morgana fused to a myc epitope (NIH MORGANA) or GFP as a control (NIH GFP) (Figure 2A). NIH MORGANA showed the same growth rate as control cells during the exponential phase, but continued to proliferate once they reached confluence (Figure 2B), reaching a 3.5-fold higher saturation density compared with controls (Figure 2C). Accordingly, NIH MORGANA failed to down-regulate cyclin D1 at confluence (Figure 2D) and formed foci when subjected to a focus-forming assay. In addition, NIH MORGANA formed ten times more colonies than control cells in a soft agar assay (Figure 2E). When injected into nude mice, NIH MORGANA invariably formed tumours within 2 weeks, while mice injected with control cells did not develop tumours, even 5 weeks after injection (Figures 2F and 2G). J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 1. Morgana overexpression is associated with an aggressive phenotype in human breast cancers. (A) Morgana expression levels in breast cancer tissue arrays. Representative pictures of IHC staining of normal tissue and cancer samples that are strong, normal or low for morgana expression are shown. (B) Correlation between morgana expression and tumour grade, (C) mitosis number, (D) proliferative index, and (E) lymph node involvement in human breast cancer samples. (F) Comparison between the percentage of strong, normal, and low morgana expression levels in samples from non-triple-negative breast cancers (non-TNBCs) and TNBCs. Representative pictures of IHC staining of normal tissue and TNBC samples that are strong, normal or low for morgana expression are shown. (G) The table shows the incidence of amplifications involving the CHORDC1 locus in different breast cancer subtypes obtained from the data within the Cancer Genome Atlas project. Graph bars represent standard errors (*p < 0.05; ***p < 0.001).
Furthermore, we subjected NIH MORGANA and control cells to three distinct apoptotic stimuli, namely suspension, serum withdrawal, and DNA damage. In all cases, morgana overexpression conferred resistance to apoptosis, as assessed by cytofluorimetric analysis of annexin V and propidium iodide-stained cells (Figure 3A) and by TUNEL assay (Figure 3B). Accordingly, the apoptotic molecular markers BAD and cleaved caspase 3 were strongly reduced in NIH MORGANA compared with control cells (Figure 3C). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Morgana destabilizes the tumour suppressor PTEN by inhibiting ROCK Given that morgana haploinsufficiency leads to centrosome overduplication and genomic instability [4] and that these features have been demonstrated to critically contribute to cell transformation [8–10], we investigated the possibility that morgana overexpression affects centrosome number. Interestingly, NIH-3T3 cells and primary MEFs overexpressing morgana did J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 2. Morgana overexpression induces NIH-3T3 cell transformation. All analyses were performed on NIH-3T3 cells infected in three independent experiments and overexpressing morgana to similar levels. (A) Western blot showing that morgana levels in NIH-3T3 cells overexpressing morgana (NIH MORGANA) are approximately three times higher than in uninfected NIH-3T3 cells (NIH) or cells infected with a control vector coding for GFP (NIH GFP). (B) Growth curves of NIH, NIH GFP, and NIH MORGANA. (C) Saturation density of NIH, NIH GFP, and NIH MORGANA after 4 days of confluence. (D) Western blot analysis of cyclin D1 on NIH, NIH GFP, and NIH MORGANA protein extracts; vinculin was used as loading control. The graph shows the average intensity of the cyclin D1 bands normalized to vinculin. (E) Soft agar colony formation assay of NIH, NIH GFP, and NIH MORGANA. (F) Representative images of nude mice injected subcutaneously with 1 × 106 NIH, NIH GFP, and NIH MORGANA at day 20 post-injection. The table shows the results of the tumour growth in nude mice. (G) Tumour volume curves in nude mice. The data are the results of three independent experiments. Graph bars represent standard errors (*p < 0.05; **p < 0.01; ***p < 0.001).
not show supernumerary centrosomes (Supplementary Figures 1A and 1B) or signs of mitotic defects, suggesting that genomic instability is not involved in morgana-mediated NIH-3T3 cell transformation. The centrosome is also an essential component of the cellular machinery, which directs spindle orientation and governs asymmetric cell division. However, morgana overexpression did not cause stem cell amplification in mammosphere generation assays [14,15] (Supplementary Figures 1C–1E). In an effort to understand the molecular mechanisms by which morgana plays a role in cancer, we discovered Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
that NIH MORGANA showed a significantly higher level of AKT phosphorylation, together with its downstream targets, GSK3β, p70S6K, and 4E-BP1, compared with control cells (Figure 4A). In addition, PTEN, the phosphatase that antagonizes PI3K, was less abundant in NIH MORGANA compared with control cells (Figure 4A). Given that the PI3K–AKT pathway is well known to play a crucial role in tumourigenesis and chemoresistance [16], we decided to further investigate this aspect. It has been reported that ROCK I can phosphorylate PTEN on Ser 229 and Thr 321 [17,18], causing J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 3. Morgana protects cells from apoptosis. Percentage of apoptotic uninfected NIH-3T3 cells (MOCK) or infected with an empty vector (EMPTY) or morgana (MORGANA) subjected to suspension for 12 h or starvation for 24 h or etoposide treatment for 16 h (black bars), assessed by annexin V and propidium iodide staining (A) or TUNEL assay (B). The basal level of apoptosis in NIH-3T3 cells is also shown for all the experiments (NT, white bars). (C) Western blot analysis of extracts from NIH, NIH EMPTY, and NIH MORGANA subjected to suspension, starvation or etoposide treatment, immunostained for morgana, caspase 3, BAD, and GAPDH as loading control. The data are the results of three independent experiments. Graph bars represent standard errors (**p < 0.01; ***p < 0.001).
increased PTEN stability and activity [19]. We previously demonstrated the ability of morgana to bind and inhibit ROCK II kinase activity [4]. However, morgana also binds ROCK I [4], and co-immunoprecipitation experiments with different ROCK I domains revealed that morgana binds the coiled coil domain of ROCK I (Supplementary Figures 2A and 2B), similarly to other known regulators of ROCK activity, such as Gem, Rad, Shroom 3, and Rho [20]. In line with our previous findings [4], the total ROCK activity in NIH-3T3 cells overexpressing morgana is reduced compared with controls, as assessed by analysing MLC2 phosphorylation in total cell extracts Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
(Figure 4B). Similar results were obtained using MEFs overexpressing morgana, demonstrating that morgana also inhibits ROCK in primary cells (Figure 4C). In vitro kinase assays on ROCK I immunoprecipitated from NIH MORGANA and NIH GFP showed that ROCK I activity was significantly lower in morgana-overexpressing cells than in control cells (Figure 4D), leading us to conclude that morgana is able to inhibit both ROCK I and ROCK II kinase activity. The expression of constitutively active ROCK (amino acids 106–553) induced PTEN stabilization per se in NIH-3T3 cells (EMPTY versus EMPTY ROCK in Figure 5A). Morgana overexpression induced a J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 4. Morgana overexpression inhibits ROCK I activity and destabilizes the tumour suppressor PTEN. (A) Western blot analysis of P-AKT, AKT, PTEN, P-GSK3β, GSK3β, P-p70S6K, p70S6K, P-4E-BP1, 4E-BP1, morgana, and vinculin on protein extracts of NIH-3T3 infected with GFP (GFP) or morgana (MORGANA). The graphs show the average intensity of the P-AKT bands normalized to AKT and PTEN bands normalized to vinculin. (B) Western blot analysis of protein extracts from NIH (MOCK), NIH GFP, and NIH MORGANA immunostained for P-MLC2, MLC2, ROCK II, ROCK I, morgana, and tubulin. The graph shows the average intensity of the P-MLC2 bands on MLC2. (C) Western blot analysis of extracts from primary WT MEFs (MOCK), MEFs infected with morgana (MORGANA) or with GFP (GFP) immunostained for P-MLC2, MLC2, ROCK II, ROCK I, morgana, and tubulin as loading control. The graph shows the average intensity of the P-MLC2 bands to MLC2. (D) ROCK I was immunoprecipitated from NIH GFP and NIH MORGANA, and subjected to an in vitro kinase assay; ROCK I, phospho-MYPT1 (P-MYPT1), and MYPT1 were detected by western blot. The ROCK inhibitor, Y-27632, was added to a ROCK I immunoprecipitate from NIH GFP, as control. Immunoprecipitation with unrelated immunoglobulins (IgG) from NIH GFP total extract was performed as a control. TE, total extract from NIH GFP. The graph shows the average intensity of the P-MYPT1 bands normalized to MYPT1. The data are the results of three independent experiments. Graph bars represent standard errors (*p < 0.05; **p < 0.01).
significant reduction in PTEN levels (EMPTY versus MORGANA). Notably, the expression of constitutively active ROCK in morgana-overexpressing cells restored PTEN levels (EMPTY versus MORGANA ROCK), as well as MLC-2 phosphorylation (Figure 5A) and apoptosis sensitivity (Figure 5B), to the levels of control cells. The results indicate that morgana acts via ROCK to decrease PTEN expression and to protect cells from apoptosis. Furthermore, NIH MORGANA treated with the Akt inhibitor, GSK690693, lose their ability to form colonies in soft agar (Figure 5C), demonstrating that the activation of the Akt pathway is crucial for morgana-dependent transformation. In summary, high morgana levels, resulting in decreased ROCK I activity, cause reduced PTEN stability that in turn, by counteracting PI3K, leads to increased AKT activation. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Morgana confers chemoresistance to breast cancer cells by destabilizing PTEN We analysed morgana expression in normal breast epithelium-derived cell lines (MCF-10) and in some breast carcinoma cells (MCF-7, T47D, and MDA-MB-231). Interestingly, morgana is expressed at low levels in MCF-10 and MCF-7, and at higher levels in the more aggressive T47D and MDA-MB-231 cell lines (Figure 6A). We first investigated if morgana overexpression in the less aggressive MCF-7 breast cancer cells can increase their tumourigenic potential (Figure 6B). Morgana overexpression did not influence MCF-7 growth rate in the exponential phase (not shown), but it strongly enhanced the ability of MCF-7 to form colonies in soft agar (Figure 6C) and to survive to apoptosis (Figure 6D). J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 5. Morgana provides protection from apoptosis via the ROCK–PTEN axis. (A) Western blot analysis of PTEN, P-MLC2, MLC-2, morgana, and vinculin on NIH EMPTY and NIH MORGANA, uninfected or infected with a lentivirus coding for a constitutively active ROCK I (amino acids 106–553, ROCK). The graph shows the average intensity of the PTEN bands normalized to vinculin. (B) Percentage of apoptotic NIH EMPTY, NIH MORGANA, and NIH MORGANA ROCK treated with 125 μM etoposide for 16 h (black bars). The data were normalized by setting the percentage of NIH EMPTY apoptotic cells to 100%. The basal level of apoptosis in NIH EMPTY cells is also shown (NT, white bars). (C) Western blot analysis of P-GSK3β, GSK3β, morgana, and vinculin on untreated NIH MORGANA and NIH EMPTY or treated with 2 nM of the Akt inhibitor, GSK690693. The graph shows the number of colonies grown in a soft agar assay in the presence or absence of 2 nM of the Akt inhibitor, GSK690693. The data are the results of three independent experiments. Graph bars represent standard errors (*p < 0.05; **p < 0.01; ***p < 0.001).
On the other hand, by using shRNAs, morgana expression was reduced in T47D and MDA-MB-231 cell lines (Figure 6E). Notably, while morgana down-regulation did not alter the growth ability of these cells, neither in vitro (Figure 6F) nor in vivo (Figure 6G), it strongly influenced their susceptibility to apoptosis. In fact, silencing of morgana caused increased apoptosis of both T47D and MDA-MB-231 cells in response to different chemotherapeutic agents (Figures 6H–6M), among them docetaxel and epirubicin, two drugs used in breast cancer neoadjuvant chemotherapy. We next evaluated the relevance of morgana-dependent PTEN regulation in T47D and MDA-MB-231 cancer cells, which carry an intact PTEN gene [21]. In accordance with our previous results, in both T47D and MDA-MB-231 cells, morgana down-regulation caused an increase in ROCK activity detected as a higher level of phosphorylated MLC2 in the absence of changes in ROCK I and II expression (Figures 7A and 7B). Moreover, both breast cancer cells, in which morgana was down-regulated by shRNA, showed increased PTEN expression levels Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
and decreased AKT phosphorylation when compared with control cells (Figures 7A and B). Given that T47D harbours a constitutively activating PI3K mutation, we demonstrated that PTEN modulation was still able to impact on T47D chemoresistance (Supplementary Figures 3A and 3B). In accordance with a role of PTEN in morganadependent chemoresistance, PTEN down-regulation in MDA-MB-231 cells expressing shRNA targeting morgana (Figure 7C) restored apoptosis resistance to the level of MDA-MB-231 control cells, both with docetaxel and with epirubicin (Figure 7D). Overall, these results demonstrated that morgana acts via ROCK to decrease PTEN expression levels and confers resistance to chemotherapeutic agents in cancer cells.
Discussion We have previously characterized morgana as a protein involved in the regulation of the centrosome cycle and therefore responsible for genomic stability maintenance. J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 6. Morgana confers chemoresistance to breast cancer cells. (A) Western blot analysis of MCF-10, MCF-7, T47D, and MDA-MB-231 protein extracts immunostained for morgana and vinculin. (B) Western blot analysis of protein extracts from uninfected MCF-7 (MOCK) or infected with a vector coding for morgana (MORGANA) or with an empty vector (EMPTY) immunostained for morgana and vinculin. (C) Soft agar colony formation assay of MCF-7 MOCK, EMPTY, and MORGANA. (D) Percentage of apoptotic MCF-7 EMPTY or MORGANA subjected to starvation for 24 h (black bars). The basal level of apoptosis in MCF-7 cells is also shown (NT, white bar). (E) Western blot analysis on uninfected T47D and MDA-MB-231 protein extracts (MOCK) or infected with an empty vector (EMPTY) or shRNAs against morgana (shMORG1 and shMORG2) immunostained for morgana and vinculin. (F) Growth curves of T47D and MDA-MB-231 MOCK, EMPTY, shMORG1, and shMORG2. (G) Tumour weight graph after 1 month from subcutaneous injection of MDA-MB-231 MOCK, EMPTY, shMORG1, and shMORG2 in SCID mice (n = 4 mice per group). (H) Percentage of apoptotic T47D EMPTY and shMORG1 treated with 500 μM etoposide for 24 h (black bars). (I) Percentage of apoptotic T47D EMPTY and shMORG1 treated with docetaxel 100 μM for 24 h (black bars). (J) Percentage of apoptotic T47D EMPTY and shMORG1 treated with epirubicin 50 μM for 24 h (black bars). The basal level of apoptotic T47D cells is shown (NT, white bars). (K) Percentage of apoptotic MDA-MB-231 EMPTY, shMORG1, and shMORG2 treated with 500 μM etoposide for 24 h (black bars). (L) Percentage of apoptotic MDA-MB-231 EMPTY, shMORG1, and shMORG2 treated with 50 μM docetaxel for 24 h (black bars). (M) Percentage of apoptotic MDA-MB-231 EMPTY, shMORG1, and shMORG2 treated with 50 μM epirubicin for 24 h (black bars). The basal level of apoptotic MDA-MB-231 cells is shown (NT, white bar). The data are the results of three independent experiments. Graph bars represent standard errors (**p < 0.01; ***p < 0.001). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Figure 7. Morgana confers chemoresistance to breast cancer cells by destabilizing PTEN. (A) Western blot analysis of extracts from T47D infected with an empty vector (EMPTY) or shRNA targeting morgana (shMORG) immunostained with phosphorylated and total MLC2, PTEN, phosphorylated and total AKT, ROCK II, ROCK I, morgana, and vinculin. The graphs show the average intensity of the P-AKT bands normalized to AKT and of PTEN normalized to vinculin. (B) Western blot analysis of MDA-MB-231 EMPTY or shMORG protein extracts immunostained with phosphorylated and total MLC2, PTEN, phosphorylated and total AKT, ROCK II, ROCK I, morgana, and vinculin. The graphs show the average intensity of the P-AKT bands normalized to AKT and of PTEN normalized to vinculin. (C) Western blot analysis of extracts from MDA-MB-231 EMPTY or shMORG infected with an empty vector (EMPTY) or shRNA against PTEN (shPTEN) immunostained with PTEN, morgana, and vinculin. (D) Percentage of apoptotic MDA-MB-231 EMPTY EMPTY, shMORG EMPTY, and shMORG shPTEN treated with 50 μM docetaxel for 24 h or treated with 50 μM epirubicin for 24 h. (E) Morgana overexpression, via ROCK I inhibition, leads to PTEN destabilization and consequently to AKT overactivation, inducing cell survival and chemoresistance. The data are the results of three independent experiments. Graph bars represent standard errors (*p < 0.05; **p < 0.01; ***p < 0.001).
Morgana haploinsufficiency leads to multiple centrosomes and a higher tumourigenicity in response to chemical carcinogen exposure in mice [4]. The relevance of morgana in human carcinogenesis is highlighted by the fact that morgana expression is strongly down-regulated in a large proportion of human Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
lung and breast tumours [4]. All of these lines of evidence qualify morgana as a bona fide tumour suppressor. However, a small percentage of human tumours have shown strong and homogeneous expression of morgana. Here we show that high morgana expression levels in human breast cancers correlate with tumour J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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grade, proliferative capacity, and lymph node involvement. Morgana overexpression does not correlate with ER, PR, and HER2 status; however, triple-negative breast cancers (TNBCs) overexpress morgana with a higher incidence. Notably, TNBCs are very aggressive tumours associated with metastases, high recurrence after chemotherapy, and high mortality rates [13,22]. In accordance, the morgana coding gene was found to be amplified in 19% of basal-like triple-negative breast cancers present in the Cancer Genome Atlas [11]. Overall, these data strongly suggest that high morgana levels enhance cancer aggressiveness. Indeed, three-fold overexpression of morgana induced transformation of NIH-3T3 cells, resulting in loss of contact inhibition and acquisition of substrate-independent growth and formation of tumours in nude mice. The oncogenic features of morgana-overexpressing NIH-3T3 cells were not related to alterations in the centrosome cycle or function. In fact, morgana-overexpressing cells showed a normal centrosome number, correct formation of bipolar spindles during mitosis, and no difference in symmetric cell division. However, morgana overexpression clearly provides protection from apoptosis and confers chemoresistance to fibroblasts and breast cancer cells. We have demonstrated that morgana is able to bind and inhibit ROCK I and II kinase activity. A specific role for ROCK I is well established in apoptosis. In fact, the expression of a constitutively active form of ROCK I induces apoptotic membrane blebbing responsible for cell contraction and nuclear disintegration [23,24]. Increasing evidence shows that ROCK I is also involved in the induction of the first apoptotic phase, upstream of caspase 3 activation. In particular, it has been demonstrated that expression of a constitutively active ROCK I mutant is sufficient to induce caspase 3 cleavage and activation in neonatal rat cardiomyocytes [19,25]. Moreover, it has been shown that in haematopoietic and cardiac cells, ROCK I can phosphorylate PTEN on Ser 229 and Thr 321 [17,18], increasing PTEN stabilization and activity [19]. We have demonstrated that morgana can inhibit ROCK I kinase activity, and that high morgana expression results in reduced PTEN levels, both in NIH-3T3 and in breast cancer cell lines. The ability of morgana to regulate PTEN levels depends on ROCK I, since constitutively active ROCK I restores normal PTEN levels and susceptibility to apoptotic stimuli in morgana-overexpressing cells. PTEN is a potent tumour suppressor that acts as a lipid phosphatase of phosphatidylinositol (3,4,5)-triphosphate (PIP3), thus opposing PI3K function. PTEN loss leads to PIP3 accumulation and activation of AKT and mammalian target of rapamycin (mTOR) signalling. Interestingly, PTEN is a haploinsufficient tumour suppressor and subtle down-regulation of PTEN has been demonstrated to strongly impact on cancer susceptibility and progression [26]. Moreover, sustained AKT activation is a potent mechanism for enhancing cell survival and chemotherapy resistance in cancer [16]. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Notably, it has been reported that a high percentage of TNBCs show low PTEN expression [22,27–29], and that PTEN loss predicts early recurrence in TNBCs [30]. It is also known that PTEN expression and function are modulated by different mechanisms, such as genetic mutations, epigenetic silencing, microRNA regulation, competitive endogenous RNA, post-translational modification, and alteration in localization [26]. Here we describe a new mechanism adopted by cancer cells to destabilize PTEN by post-translational modification. Increased morgana expression levels result in ROCK I inhibition, which in turn leads to PTEN destabilization and enhancement of the AKT pro-survival pathway, conferring resistance to chemotherapeutic agents. In accordance with our results, morgana transcript levels have been found to be up-regulated in a microarray analysis of ovarian cancers that recur after chemotherapy, compared with primary tumours [31]. Overall, our findings indicate that both underexpression of morgana [4] and overexpression of morgana sustain tumourigenesis. Very few other proteins have been described to play both tumour suppressive and oncogenic functions, the most famous example being nucleophosmin (NPM) [32,33]. On the one hand, morgana acts as a tumour suppressor, the down-regulation of which results in centrosome amplification and genomic instability through ROCK II activation. On the other hand, morgana overexpression, through ROCK I inhibition, destabilizes PTEN and induces cell survival, thus acting as an oncogene (Figure 7E). Neoadjuvant chemotherapy, based on docetaxel and epirubicin, is widely used in breast cancer to increase tumour resectability, decrease the risk of recurrence, and increase overall survival. However, neoadjuvant treatment is ineffective in half of the patients treated and often results in toxicity. For this reason, identification of markers able to predict tumour resistance to neoadjuvant chemotherapy is of great clinical interest. To this end, high morgana expression levels, conferring resistance to both epirubicin and docetaxel, represent a novel indicator of cancer resistance to neoadjuvant chemotherapy. Finally, the presence of high morgana expression levels, unveiling PTEN down-regulation and overactivation of the PI3K downstream pathways, will provide valuable information for directing targeted therapy.
Acknowledgments We would like to thank Flavio Cristofani and Antonellisa Sgarra for assistance with animal experiments; and Shuh Narumiya, Claudio Brancolini, Ferdinando Di Cunto, and Enzo Calautti for sharing DNA constructs. We also thank Radhika Srinivasan for manuscript revision. This work was supported by an AIRC 2011 (IG 11456) grant to MB and by an AIRC 2011 (IG 11346) grant to SC. AD was supported by a fellowship from AIRC (annual fellowship ‘Marco Fabio Sartori’). J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Author contribution statement FF designed the study and performed the experiments. RF, SR, GT, and AD performed the experiments. DR performed tissue array analysis. DR and AS scored breast cancer slides. PP performed bioinformatics analysis. LS, ET, SC, PPP, AS, and GT planned the experiments. MB supervised the project and planned the experiments. FF and MB wrote the article.
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16. Hafsi S, Pezzino FM, Candido S, et al. Gene alterations in the PI3K/PTEN/AKT pathway as a mechanism of drug-resistance (review). Int J Oncol 2012; 40: 639–644. 17. Li Z, Dong X, Wang Z, et al. Regulation of PTEN by Rho small GTPases. Nature Cell Biol 2005; 7: 399–404. 18. Vemula S, Shi J, Hanneman P, et al. ROCK1 functions as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability. Blood 2010; 115: 1785–1796. 19. Chang J, Xie M, Shah VR, et al. Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis. Proc Natl Acad Sci U S A 2006; 103: 14495–14500. 20. Morgan-Fisher M, Wewer UM, Yoneda A. Regulation of ROCK activity in cancer. J Histochem Cytochem 2013; 61: 185–198. 21. Perez-Tenorio G, Alkhori L, Olsson B, et al. PIK3CA mutations and PTEN loss correlate with similar prognostic factors and are not mutually exclusive in breast cancer. Clin Cancer Res 2007; 13: 3577–3584. 22. Lehmann BD, Pietenpol JA. Identification and use of biomarkers in treatment strategies for triple-negative breast cancer subtypes. J Pathol 2014; 232: 142–150. 23. Coleman ML, Sahai EA, Yeo M, et al. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nature Cell Biol 2001; 3: 339–345. 24. Sebbagh M, Renvoize C, Hamelin J, et al. Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nature Cell Biol 2001; 3: 346–352. 25. Shi J, Wei L. Rho kinase in the regulation of cell death and survival. Arch Immunol Ther Exp (Warsz) 2007; 55: 61–75. 26. Song MS, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nature Rev Mol Cell Biol 2012; 13: 283–296. 27. Marty B, Maire V, Gravier E, et al. Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells. Breast Cancer Res 2008; 10: R101. 28. Lopez-Knowles E, O’Toole SA, McNeil CM, et al. PI3K pathway activation in breast cancer is associated with the basal-like phenotype and cancer-specific mortality. Int J Cancer 2010; 126: 1121–1131. 29. Peddi PF, Ellis MJ, Ma C. Molecular basis of triple negative breast cancer and implications for therapy. Int J Breast Cancer 2012; 2012: 217185. 30. Iqbal J, Thike AA, Cheok PY, et al. Insulin growth factor receptor-1 expression and loss of PTEN protein predict early recurrence in triple-negative breast cancer. Histopathology 2012; 61: 652–659. 31. Laios A, O’Toole SA, Flavin R, et al. An integrative model for recurrence in ovarian cancer. Mol Cancer 2008; 7: 8. 32. Di Fiore PP. Playing both sides: nucleophosmin between tumor suppression and oncogenesis. J Cell Biol 2008; 182: 7–9. 33. Grisendi S, Bernardi R, Rossi M, et al. Role of nucleophosmin in embryonic development and tumorigenesis. Nature 2005; 437: 147–153. 34. Hollestelle A, Elstrodt F, Nagel JH, et al. Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines. Mol Cancer Res 2007; 5: 195–201.
SUPPORTING INFORMATION ON THE INTERNET The following supporting information may be found in the online version of this article: Supplementary materials and methods. Figure S1. Morgana overexpression does not alter centrosome number or asymmetric cell division. Figure S2. Morgana binds ROCK I coiled coil domain. Figure S3. PTEN down-regulation in T47D cells increases chemoresistance Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
ORIGINAL PAPER
Morgana acts as a proto-oncogene through inhibition of a ROCK–PTEN pathway Federica Fusella,1 Roberta Ferretti,1† Daniele Recupero,2 Stefania Rocca,1 Augusta Di Savino,1 Giusy Tornillo,1‡ Lorenzo Silengo,1 Emilia Turco,1 Sara Cabodi,1 Paolo Provero,1 Pier Paolo Pandolfi,1,3 Anna Sapino,2 Guido Tarone1 and Mara Brancaccio1* 1
Department of Biotechnology and Health Sciences, University of Torino, Torino, Italy Department of Medical Sciences, University of Torino, Torino, Italy 3 Cancer Genetics Program, Division of Genetics, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 2
*Correspondence to: Mara Brancaccio, Molecular Biotechnology Center, Via Nizza, 52, 10126 Torino, Italy. e-mail: [email protected] † ‡
Current address: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Current address: Cardiff School of Biosciences, The Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, UK.
Abstract Morgana/CHP-1 is a ubiquitously expressed protein able to inhibit ROCK II kinase activity. We have previously demonstrated that morgana haploinsufficiency leads to multiple centrosomes, genomic instability, and higher susceptibility to tumour development. While a large fraction of human cancers has shown morgana down-regulation, a small subset of tumours was shown to express high morgana levels. Here we demonstrate that high morgana expression in different breast cancer subtypes correlates with high tumour grade, mitosis number, and lymph node positivity. Moreover, morgana overexpression induces transformation in NIH-3T3 cells and strongly protects them from various apoptotic stimuli. From a mechanistic point of view, we demonstrate that morgana causes PTEN destabilization, by inhibiting ROCK activity, hence triggering the PI3K/AKT survival pathway. In turn, morgana down-regulation in breast cancer cells that express high morgana levels increases PTEN expression and leads to sensitization of cells to chemotherapy. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: breast cancer; chemoresistance; morgana; PTEN; ROCK
Received 15 October 2013; Revised 11 February 2014; Accepted 13 February 2014
No conflicts of interest were declared.
Introduction Morgana/CHP-1 (coded by the CHORDC1 gene) is a ubiquitous protein [1] with chaperone activity [2,3], and plays a key regulatory role in centrosome duplication and genomic stability through ROCK II inhibition [4,5]. Morgana is highly conserved during phylogenesis [1,6,7] and has an essential role during developmental processes [4]. The absence of morgana in Drosophila neuroblasts causes a strong mitotic phenotype, consisting of a high frequency of polyploid cells, with most diploid cells displaying supernumerary centrosomes. Moreover, morgana haploinsufficiency in mouse embryonic fibroblasts (MEFs) derived from morgana +/− embryos causes a higher frequency of polyploid cells, supernumerary centrosomes, and multipolar spindles [4]. Genomic instability is a typical feature of cancer cells and is known to favour cancer onset and progression [8–10]. In line with these considerations, morgana +/− MEFs are transformed by oncogenic Ras, rather than undergoing senescence; in addition, tumour Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
susceptibility in response to chemical mutagens is significantly higher in morgana +/− mice than in wild-type mice [4]. The role of morgana as a tumour suppressor in humans is supported by the fact that morgana expression is strongly reduced in a large percentage of human breast and lung cancer samples compared with normal tissues [4]. However, our tissue array analysis also indicated that morgana is overexpressed in a small fraction of human breast and lung cancers [4]. To further investigate this intriguing result, we analysed morgana levels in human breast cancer samples, and here we show that morgana is overexpressed in different breast cancer subtypes, in particular in triple-negative breast cancers (TNBCs), and that its overexpression correlates with tumour grade, mitosis number, and lymph node positivity. Furthermore, we found that morgana overexpression is able to transform NIH-3T3 cells and to protect them from different apoptotic stimuli. Accordingly, morgana down-regulation in T47D and MDA-MB-231 breast cancer cells that express high morgana levels confers J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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sensitivity to chemotherapeutic agents. On a molecular level, inhibition of ROCK by high morgana levels causes down-regulation of phosphatase and tensin homologue (PTEN), leading to enhanced AKT activation and cell survival.
Materials and methods Tissue array preparation and immunohistochemistry (IHC) This study was conducted in compliance with the ethical regulatory requirements for the handling of biological specimens after appropriate informed consent and Institutional Review Board approval. A series of 208 breast cancer samples and 51 TNBC samples was collected at the Breast Unit of the San Giovanni Battista Hospital of Torino, Italy. Tissue array preparation and morgana detection are described in the Supplementary materials and methods. In the tissue array analysed, three cores were obtained for each breast cancer sample in the areas of higher cellularity and one core from normal adjacent breast tissue. The analysis of morgana staining intensity in the normal breast epithelium of different patients indicated a comparable expression level and was used as a reference. High and low levels were defined when the staining intensity was respectively higher or lower compared with the staining intensity in normal tissue. Slides were scored independently by two pathologists.
Analysis of copy number alteration Previously generated somatic copy number alteration data [11] were used to determine the fraction of breast tumours with morgana coding gene (CHORDC1) amplification. The data were obtained from the website associated with the paper [11] and included 773 breast cancer samples, 499 of which were classified into gene expression-based molecular subtypes.
Antibodies and reagents Western blotting was performed using the following primary antibodies: anti-morgana P1/PP0 (1 μg/ml) [4], actin (sc-1615; 1 : 3000), ROCK I (sc-5560; 1 : 1000), ROCK II (sc-5561; 1 : 1000), MLC2 (sc-15370; 1 : 1000), cyclin D1 (sc-753; 1 : 1000), and p70S6K (sc-230; 1 : 1000), which were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); Bad (9292; 1 : 1000), ROCK I (4035; 1 : 1000), caspase 3 (9665; 1 : 1000), phospho-MLC2 (3671; 1 : 1000), PTEN (9559; 1 : 1000), phospho-AKT Ser473 (9271; 1 : 1000), AKT (4691; 1 : 1000), phospho-GSK3β (9336; 1 : 1000), GSK3β (9315, 1 : 1000), phospho-p70S6K (9208, 1 : 1000), phospho-4E-BP1 (2855, 1 : 1000), and 4E-BP1 (9452, 1 : 1000), which were from Cell Signaling (Danvers, MA, USA). Vinculin (SAB4200080, 1 : 5000) and α-tubulin (T5168; 1 : 8000) were from Sigma-Aldrich Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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(St Louis, MO, USA). GAPDH (MAB374, 1 : 3000), phospho-MYPT1 (07–251; 1 : 1000), and GFP were from Millipore (Billerica, MA, USA). Finally, ROCK II (07–443; 1 : 1000) and MYPT1 (07–672; 1 : 1000) were from Upstate Biotechnology (Lake Placid, NY, USA). Fetal bovine serum (FBS), fetal calf serum (FCS), penicillin–streptomycin (PS), and basic fibroblast growth factor (bFGF) were from Invitrogen (Carlsbad, CA, USA). Epidermal growth factor (EGF), heparin, insulin, hydrocortisone, Akt inhibitor GSK690693, docetaxel, epirubicin, and etoposide were from Sigma-Aldrich.
Cell culture NIH-3T3 and human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and propagated and maintained according to protocols supplied by ATCC. The experiments were performed within 6 months after resuscitation. NIH-3T3 cells were maintained in DMEM with 10% FCS and 5% PS. MDA-MB-231 and 293 cells were cultured in DMEM with 10% FBS and 5% PS. MCF-7 and T47D were cultured in RPMI with 10% FCS and 5% PS. NIH-3T3 cells stably overexpressing morgana or GFP were obtained as previously described [2]. MCF-7 cells overexpressing morgana were obtained using a pLVX lentiviral vector coding for mouse morgana. Morgana knockdown in MDA-MB-231 and T47D was performed by infecting cells with pGIPZ lentiviral particles expressing two different morgana shRNAs, together with the TurboGFP (Thermoscientific Open Biosystems, Chesterville, AL, USA). Expression of ROCK I deletion mutants fused with GFP was obtained by calcium phosphate transfection of 293 cells.
Transforming assays To assess the NIH-3T3 cell proliferation rate, 5 × 105 cells were seeded in 35 mm plates and counted at the indicated times. MDA-MB-231 and T47D cell growth curves were generated by plating 50 000 cells and staining cells with crystal violet at the indicated times, as previously described [4]. Soft agar colony formation assays were performed by resuspending cells in complete DMEM containing 0.3% low gelling agarose (Sigma-Aldrich) and seeding cells in triplicate into six-well plates containing a 2 ml layer of solidified 0.6% agar (Sigma-Aldrich). The Akt inhibitor, GSK690693, was added every 24 h with a daily change of medium at the final concentration of 2 nM. After 4 weeks, colonies were stained with nitroblue tetrazolium (Sigma-Aldrich), photographed with Zeiss microscopy, and counted using the ImageJ Software.
Assessment of tumourigenicity in nude mice The use of animals complied with the Guide for the Care and Use of Laboratory Animals published by the J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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US National Institutes of Health and was approved by the Animal Care and Use Committee of the University of Torino. A total of 106 NIH-3T3 or 106 MDA-MB-231 cells were resuspended in 200 μl of PBS and injected subcutaneously into the left flank of female nude athymic mice or SCID mice. Tumours were measured using a caliper.
Cell death treatments NIH-3T3 cells were detached and kept in suspension for 12 h, serum-starved for 24 h, or treated with 125 μM etoposide for 16 h. Apoptosis was assayed by annexin V staining and by TUNEL assay (In Situ Cell Death Detection Kit TMR red; Roche, Mannheim, Germany). Cells treated with etoposide were stained for annexin V. Apoptosis of T47D and MDA-MB-231 cells treated with etoposide was detected by staining cells with anti-cleaved caspase3 antibody (Cell Signaling). Apoptosis of T47D and MDA 231 treated with docetaxel was assayed using a TUNEL assay. Apoptosis of T47D and MDA-MB-231 cells treated with epirubicin was detected by annexin V staining (BD Biosciences, San José, CA, USA).
ROCK kinase assay Kinase assays were performed as previously described [12] and as described in the Supplementary materials and methods. Kinase activity was detected by western blotting using anti-phospho-MYPT1 and anti-MYPT1 antibodies.
Immunoprecipitation and western blot analysis For immunoprecipitation, cells were lysed in NP-40 lysis buffer [20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.5% NP-40, 10 mM NaF, 1 mM PMSF, 1 mM Na3 VO4 , and protease inhibitors]. Five micrograms of the selected antibody was added, along with protein G-coated Sepharose, to the protein extracts and processed as previously described [12]. For western blot analysis, cells were lysed in Tris-buffered saline with 1% Triton X-100, plus phosphatase and protease inhibitors. To detect phosphorylation of MLC2, cells were lysed with SDS buffer [1% SDS, 60 mM Tris–HCl (pH 6.8)]. Total protein extracts (30 μg) were analysed by western blotting and detected by the chemiluminescent reagent LiteAblot (Euroclone, Milano, Italy). Band intensities were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).
Statistical significance The data are presented as means ± SEM. For statistical analyses, significance was tested using a two-tailed Student’s t-test or, when required, one- or two-way ANOVA with Bonferroni’s correction. A minimum value of p < 0.05 was considered to be statistically significant. Statistical analyses were performed using GraphPad Prism 4. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Results Morgana overexpression is associated with aggressive phenotypes in human breast cancers We evaluated morgana expression levels in a tissue array containing 208 human breast cancer specimens. Of the tumours analysed, 63% had levels of morgana lower than the normal breast epithelium, confirming that morgana is frequently down-regulated in human breast cancers [4]. However, 17% of tumour samples had higher morgana expression levels compared with normal tissue (Figure 1A). Interestingly, morgana levels correlated positively with tumour grade (Figure 1B), mitosis number (Figure 1C), mean Ki67 labelling index (Figure 1D), and lymph node involvement (Figure 1E). Although morgana overexpression did not correlate with oestrogen (ER), progesterone (PR) or HER2 status, we noticed that seven triple-negative cancer samples out of the nine included in the tissue array expressed high levels of morgana. Triple-negative breast cancers do not express ER or PR, lack HER2 overexpression, and are associated with a shorter median time-to-relapse and increased mortality [13]. To expand the analysis to a relevant number of tumours, we analysed morgana expression levels in a tissue array containing 51 specimens from human triple-negative breast cancers. The results indicated that 36% of the samples showed strong overexpression of morgana compared with normal breast epithelium (Figure 1F). In accordance, analysis of the data within the Cancer Genome Atlas project [11] highlighted that the morgana gene (CHORDC1) locus is amplified in 19% of basal-like triple-negative breast cancers (Figure 1G).
Morgana overexpression induces transformation in NIH-3T3 cells and protects cells from apoptotic stimuli To investigate the possible role of morgana overexpression in tumourigenesis, we chose the NIH-3T3 cell line, which represents a widely used model system for testing oncogene function. We infected NIH-3T3 cells with a lentivirus coding for morgana fused to a myc epitope (NIH MORGANA) or GFP as a control (NIH GFP) (Figure 2A). NIH MORGANA showed the same growth rate as control cells during the exponential phase, but continued to proliferate once they reached confluence (Figure 2B), reaching a 3.5-fold higher saturation density compared with controls (Figure 2C). Accordingly, NIH MORGANA failed to down-regulate cyclin D1 at confluence (Figure 2D) and formed foci when subjected to a focus-forming assay. In addition, NIH MORGANA formed ten times more colonies than control cells in a soft agar assay (Figure 2E). When injected into nude mice, NIH MORGANA invariably formed tumours within 2 weeks, while mice injected with control cells did not develop tumours, even 5 weeks after injection (Figures 2F and 2G). J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 1. Morgana overexpression is associated with an aggressive phenotype in human breast cancers. (A) Morgana expression levels in breast cancer tissue arrays. Representative pictures of IHC staining of normal tissue and cancer samples that are strong, normal or low for morgana expression are shown. (B) Correlation between morgana expression and tumour grade, (C) mitosis number, (D) proliferative index, and (E) lymph node involvement in human breast cancer samples. (F) Comparison between the percentage of strong, normal, and low morgana expression levels in samples from non-triple-negative breast cancers (non-TNBCs) and TNBCs. Representative pictures of IHC staining of normal tissue and TNBC samples that are strong, normal or low for morgana expression are shown. (G) The table shows the incidence of amplifications involving the CHORDC1 locus in different breast cancer subtypes obtained from the data within the Cancer Genome Atlas project. Graph bars represent standard errors (*p < 0.05; ***p < 0.001).
Furthermore, we subjected NIH MORGANA and control cells to three distinct apoptotic stimuli, namely suspension, serum withdrawal, and DNA damage. In all cases, morgana overexpression conferred resistance to apoptosis, as assessed by cytofluorimetric analysis of annexin V and propidium iodide-stained cells (Figure 3A) and by TUNEL assay (Figure 3B). Accordingly, the apoptotic molecular markers BAD and cleaved caspase 3 were strongly reduced in NIH MORGANA compared with control cells (Figure 3C). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Morgana destabilizes the tumour suppressor PTEN by inhibiting ROCK Given that morgana haploinsufficiency leads to centrosome overduplication and genomic instability [4] and that these features have been demonstrated to critically contribute to cell transformation [8–10], we investigated the possibility that morgana overexpression affects centrosome number. Interestingly, NIH-3T3 cells and primary MEFs overexpressing morgana did J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 2. Morgana overexpression induces NIH-3T3 cell transformation. All analyses were performed on NIH-3T3 cells infected in three independent experiments and overexpressing morgana to similar levels. (A) Western blot showing that morgana levels in NIH-3T3 cells overexpressing morgana (NIH MORGANA) are approximately three times higher than in uninfected NIH-3T3 cells (NIH) or cells infected with a control vector coding for GFP (NIH GFP). (B) Growth curves of NIH, NIH GFP, and NIH MORGANA. (C) Saturation density of NIH, NIH GFP, and NIH MORGANA after 4 days of confluence. (D) Western blot analysis of cyclin D1 on NIH, NIH GFP, and NIH MORGANA protein extracts; vinculin was used as loading control. The graph shows the average intensity of the cyclin D1 bands normalized to vinculin. (E) Soft agar colony formation assay of NIH, NIH GFP, and NIH MORGANA. (F) Representative images of nude mice injected subcutaneously with 1 × 106 NIH, NIH GFP, and NIH MORGANA at day 20 post-injection. The table shows the results of the tumour growth in nude mice. (G) Tumour volume curves in nude mice. The data are the results of three independent experiments. Graph bars represent standard errors (*p < 0.05; **p < 0.01; ***p < 0.001).
not show supernumerary centrosomes (Supplementary Figures 1A and 1B) or signs of mitotic defects, suggesting that genomic instability is not involved in morgana-mediated NIH-3T3 cell transformation. The centrosome is also an essential component of the cellular machinery, which directs spindle orientation and governs asymmetric cell division. However, morgana overexpression did not cause stem cell amplification in mammosphere generation assays [14,15] (Supplementary Figures 1C–1E). In an effort to understand the molecular mechanisms by which morgana plays a role in cancer, we discovered Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
that NIH MORGANA showed a significantly higher level of AKT phosphorylation, together with its downstream targets, GSK3β, p70S6K, and 4E-BP1, compared with control cells (Figure 4A). In addition, PTEN, the phosphatase that antagonizes PI3K, was less abundant in NIH MORGANA compared with control cells (Figure 4A). Given that the PI3K–AKT pathway is well known to play a crucial role in tumourigenesis and chemoresistance [16], we decided to further investigate this aspect. It has been reported that ROCK I can phosphorylate PTEN on Ser 229 and Thr 321 [17,18], causing J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 3. Morgana protects cells from apoptosis. Percentage of apoptotic uninfected NIH-3T3 cells (MOCK) or infected with an empty vector (EMPTY) or morgana (MORGANA) subjected to suspension for 12 h or starvation for 24 h or etoposide treatment for 16 h (black bars), assessed by annexin V and propidium iodide staining (A) or TUNEL assay (B). The basal level of apoptosis in NIH-3T3 cells is also shown for all the experiments (NT, white bars). (C) Western blot analysis of extracts from NIH, NIH EMPTY, and NIH MORGANA subjected to suspension, starvation or etoposide treatment, immunostained for morgana, caspase 3, BAD, and GAPDH as loading control. The data are the results of three independent experiments. Graph bars represent standard errors (**p < 0.01; ***p < 0.001).
increased PTEN stability and activity [19]. We previously demonstrated the ability of morgana to bind and inhibit ROCK II kinase activity [4]. However, morgana also binds ROCK I [4], and co-immunoprecipitation experiments with different ROCK I domains revealed that morgana binds the coiled coil domain of ROCK I (Supplementary Figures 2A and 2B), similarly to other known regulators of ROCK activity, such as Gem, Rad, Shroom 3, and Rho [20]. In line with our previous findings [4], the total ROCK activity in NIH-3T3 cells overexpressing morgana is reduced compared with controls, as assessed by analysing MLC2 phosphorylation in total cell extracts Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
(Figure 4B). Similar results were obtained using MEFs overexpressing morgana, demonstrating that morgana also inhibits ROCK in primary cells (Figure 4C). In vitro kinase assays on ROCK I immunoprecipitated from NIH MORGANA and NIH GFP showed that ROCK I activity was significantly lower in morgana-overexpressing cells than in control cells (Figure 4D), leading us to conclude that morgana is able to inhibit both ROCK I and ROCK II kinase activity. The expression of constitutively active ROCK (amino acids 106–553) induced PTEN stabilization per se in NIH-3T3 cells (EMPTY versus EMPTY ROCK in Figure 5A). Morgana overexpression induced a J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 4. Morgana overexpression inhibits ROCK I activity and destabilizes the tumour suppressor PTEN. (A) Western blot analysis of P-AKT, AKT, PTEN, P-GSK3β, GSK3β, P-p70S6K, p70S6K, P-4E-BP1, 4E-BP1, morgana, and vinculin on protein extracts of NIH-3T3 infected with GFP (GFP) or morgana (MORGANA). The graphs show the average intensity of the P-AKT bands normalized to AKT and PTEN bands normalized to vinculin. (B) Western blot analysis of protein extracts from NIH (MOCK), NIH GFP, and NIH MORGANA immunostained for P-MLC2, MLC2, ROCK II, ROCK I, morgana, and tubulin. The graph shows the average intensity of the P-MLC2 bands on MLC2. (C) Western blot analysis of extracts from primary WT MEFs (MOCK), MEFs infected with morgana (MORGANA) or with GFP (GFP) immunostained for P-MLC2, MLC2, ROCK II, ROCK I, morgana, and tubulin as loading control. The graph shows the average intensity of the P-MLC2 bands to MLC2. (D) ROCK I was immunoprecipitated from NIH GFP and NIH MORGANA, and subjected to an in vitro kinase assay; ROCK I, phospho-MYPT1 (P-MYPT1), and MYPT1 were detected by western blot. The ROCK inhibitor, Y-27632, was added to a ROCK I immunoprecipitate from NIH GFP, as control. Immunoprecipitation with unrelated immunoglobulins (IgG) from NIH GFP total extract was performed as a control. TE, total extract from NIH GFP. The graph shows the average intensity of the P-MYPT1 bands normalized to MYPT1. The data are the results of three independent experiments. Graph bars represent standard errors (*p < 0.05; **p < 0.01).
significant reduction in PTEN levels (EMPTY versus MORGANA). Notably, the expression of constitutively active ROCK in morgana-overexpressing cells restored PTEN levels (EMPTY versus MORGANA ROCK), as well as MLC-2 phosphorylation (Figure 5A) and apoptosis sensitivity (Figure 5B), to the levels of control cells. The results indicate that morgana acts via ROCK to decrease PTEN expression and to protect cells from apoptosis. Furthermore, NIH MORGANA treated with the Akt inhibitor, GSK690693, lose their ability to form colonies in soft agar (Figure 5C), demonstrating that the activation of the Akt pathway is crucial for morgana-dependent transformation. In summary, high morgana levels, resulting in decreased ROCK I activity, cause reduced PTEN stability that in turn, by counteracting PI3K, leads to increased AKT activation. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
Morgana confers chemoresistance to breast cancer cells by destabilizing PTEN We analysed morgana expression in normal breast epithelium-derived cell lines (MCF-10) and in some breast carcinoma cells (MCF-7, T47D, and MDA-MB-231). Interestingly, morgana is expressed at low levels in MCF-10 and MCF-7, and at higher levels in the more aggressive T47D and MDA-MB-231 cell lines (Figure 6A). We first investigated if morgana overexpression in the less aggressive MCF-7 breast cancer cells can increase their tumourigenic potential (Figure 6B). Morgana overexpression did not influence MCF-7 growth rate in the exponential phase (not shown), but it strongly enhanced the ability of MCF-7 to form colonies in soft agar (Figure 6C) and to survive to apoptosis (Figure 6D). J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 5. Morgana provides protection from apoptosis via the ROCK–PTEN axis. (A) Western blot analysis of PTEN, P-MLC2, MLC-2, morgana, and vinculin on NIH EMPTY and NIH MORGANA, uninfected or infected with a lentivirus coding for a constitutively active ROCK I (amino acids 106–553, ROCK). The graph shows the average intensity of the PTEN bands normalized to vinculin. (B) Percentage of apoptotic NIH EMPTY, NIH MORGANA, and NIH MORGANA ROCK treated with 125 μM etoposide for 16 h (black bars). The data were normalized by setting the percentage of NIH EMPTY apoptotic cells to 100%. The basal level of apoptosis in NIH EMPTY cells is also shown (NT, white bars). (C) Western blot analysis of P-GSK3β, GSK3β, morgana, and vinculin on untreated NIH MORGANA and NIH EMPTY or treated with 2 nM of the Akt inhibitor, GSK690693. The graph shows the number of colonies grown in a soft agar assay in the presence or absence of 2 nM of the Akt inhibitor, GSK690693. The data are the results of three independent experiments. Graph bars represent standard errors (*p < 0.05; **p < 0.01; ***p < 0.001).
On the other hand, by using shRNAs, morgana expression was reduced in T47D and MDA-MB-231 cell lines (Figure 6E). Notably, while morgana down-regulation did not alter the growth ability of these cells, neither in vitro (Figure 6F) nor in vivo (Figure 6G), it strongly influenced their susceptibility to apoptosis. In fact, silencing of morgana caused increased apoptosis of both T47D and MDA-MB-231 cells in response to different chemotherapeutic agents (Figures 6H–6M), among them docetaxel and epirubicin, two drugs used in breast cancer neoadjuvant chemotherapy. We next evaluated the relevance of morgana-dependent PTEN regulation in T47D and MDA-MB-231 cancer cells, which carry an intact PTEN gene [21]. In accordance with our previous results, in both T47D and MDA-MB-231 cells, morgana down-regulation caused an increase in ROCK activity detected as a higher level of phosphorylated MLC2 in the absence of changes in ROCK I and II expression (Figures 7A and 7B). Moreover, both breast cancer cells, in which morgana was down-regulated by shRNA, showed increased PTEN expression levels Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
and decreased AKT phosphorylation when compared with control cells (Figures 7A and B). Given that T47D harbours a constitutively activating PI3K mutation, we demonstrated that PTEN modulation was still able to impact on T47D chemoresistance (Supplementary Figures 3A and 3B). In accordance with a role of PTEN in morganadependent chemoresistance, PTEN down-regulation in MDA-MB-231 cells expressing shRNA targeting morgana (Figure 7C) restored apoptosis resistance to the level of MDA-MB-231 control cells, both with docetaxel and with epirubicin (Figure 7D). Overall, these results demonstrated that morgana acts via ROCK to decrease PTEN expression levels and confers resistance to chemotherapeutic agents in cancer cells.
Discussion We have previously characterized morgana as a protein involved in the regulation of the centrosome cycle and therefore responsible for genomic stability maintenance. J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Figure 6. Morgana confers chemoresistance to breast cancer cells. (A) Western blot analysis of MCF-10, MCF-7, T47D, and MDA-MB-231 protein extracts immunostained for morgana and vinculin. (B) Western blot analysis of protein extracts from uninfected MCF-7 (MOCK) or infected with a vector coding for morgana (MORGANA) or with an empty vector (EMPTY) immunostained for morgana and vinculin. (C) Soft agar colony formation assay of MCF-7 MOCK, EMPTY, and MORGANA. (D) Percentage of apoptotic MCF-7 EMPTY or MORGANA subjected to starvation for 24 h (black bars). The basal level of apoptosis in MCF-7 cells is also shown (NT, white bar). (E) Western blot analysis on uninfected T47D and MDA-MB-231 protein extracts (MOCK) or infected with an empty vector (EMPTY) or shRNAs against morgana (shMORG1 and shMORG2) immunostained for morgana and vinculin. (F) Growth curves of T47D and MDA-MB-231 MOCK, EMPTY, shMORG1, and shMORG2. (G) Tumour weight graph after 1 month from subcutaneous injection of MDA-MB-231 MOCK, EMPTY, shMORG1, and shMORG2 in SCID mice (n = 4 mice per group). (H) Percentage of apoptotic T47D EMPTY and shMORG1 treated with 500 μM etoposide for 24 h (black bars). (I) Percentage of apoptotic T47D EMPTY and shMORG1 treated with docetaxel 100 μM for 24 h (black bars). (J) Percentage of apoptotic T47D EMPTY and shMORG1 treated with epirubicin 50 μM for 24 h (black bars). The basal level of apoptotic T47D cells is shown (NT, white bars). (K) Percentage of apoptotic MDA-MB-231 EMPTY, shMORG1, and shMORG2 treated with 500 μM etoposide for 24 h (black bars). (L) Percentage of apoptotic MDA-MB-231 EMPTY, shMORG1, and shMORG2 treated with 50 μM docetaxel for 24 h (black bars). (M) Percentage of apoptotic MDA-MB-231 EMPTY, shMORG1, and shMORG2 treated with 50 μM epirubicin for 24 h (black bars). The basal level of apoptotic MDA-MB-231 cells is shown (NT, white bar). The data are the results of three independent experiments. Graph bars represent standard errors (**p < 0.01; ***p < 0.001). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Figure 7. Morgana confers chemoresistance to breast cancer cells by destabilizing PTEN. (A) Western blot analysis of extracts from T47D infected with an empty vector (EMPTY) or shRNA targeting morgana (shMORG) immunostained with phosphorylated and total MLC2, PTEN, phosphorylated and total AKT, ROCK II, ROCK I, morgana, and vinculin. The graphs show the average intensity of the P-AKT bands normalized to AKT and of PTEN normalized to vinculin. (B) Western blot analysis of MDA-MB-231 EMPTY or shMORG protein extracts immunostained with phosphorylated and total MLC2, PTEN, phosphorylated and total AKT, ROCK II, ROCK I, morgana, and vinculin. The graphs show the average intensity of the P-AKT bands normalized to AKT and of PTEN normalized to vinculin. (C) Western blot analysis of extracts from MDA-MB-231 EMPTY or shMORG infected with an empty vector (EMPTY) or shRNA against PTEN (shPTEN) immunostained with PTEN, morgana, and vinculin. (D) Percentage of apoptotic MDA-MB-231 EMPTY EMPTY, shMORG EMPTY, and shMORG shPTEN treated with 50 μM docetaxel for 24 h or treated with 50 μM epirubicin for 24 h. (E) Morgana overexpression, via ROCK I inhibition, leads to PTEN destabilization and consequently to AKT overactivation, inducing cell survival and chemoresistance. The data are the results of three independent experiments. Graph bars represent standard errors (*p < 0.05; **p < 0.01; ***p < 0.001).
Morgana haploinsufficiency leads to multiple centrosomes and a higher tumourigenicity in response to chemical carcinogen exposure in mice [4]. The relevance of morgana in human carcinogenesis is highlighted by the fact that morgana expression is strongly down-regulated in a large proportion of human Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
lung and breast tumours [4]. All of these lines of evidence qualify morgana as a bona fide tumour suppressor. However, a small percentage of human tumours have shown strong and homogeneous expression of morgana. Here we show that high morgana expression levels in human breast cancers correlate with tumour J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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grade, proliferative capacity, and lymph node involvement. Morgana overexpression does not correlate with ER, PR, and HER2 status; however, triple-negative breast cancers (TNBCs) overexpress morgana with a higher incidence. Notably, TNBCs are very aggressive tumours associated with metastases, high recurrence after chemotherapy, and high mortality rates [13,22]. In accordance, the morgana coding gene was found to be amplified in 19% of basal-like triple-negative breast cancers present in the Cancer Genome Atlas [11]. Overall, these data strongly suggest that high morgana levels enhance cancer aggressiveness. Indeed, three-fold overexpression of morgana induced transformation of NIH-3T3 cells, resulting in loss of contact inhibition and acquisition of substrate-independent growth and formation of tumours in nude mice. The oncogenic features of morgana-overexpressing NIH-3T3 cells were not related to alterations in the centrosome cycle or function. In fact, morgana-overexpressing cells showed a normal centrosome number, correct formation of bipolar spindles during mitosis, and no difference in symmetric cell division. However, morgana overexpression clearly provides protection from apoptosis and confers chemoresistance to fibroblasts and breast cancer cells. We have demonstrated that morgana is able to bind and inhibit ROCK I and II kinase activity. A specific role for ROCK I is well established in apoptosis. In fact, the expression of a constitutively active form of ROCK I induces apoptotic membrane blebbing responsible for cell contraction and nuclear disintegration [23,24]. Increasing evidence shows that ROCK I is also involved in the induction of the first apoptotic phase, upstream of caspase 3 activation. In particular, it has been demonstrated that expression of a constitutively active ROCK I mutant is sufficient to induce caspase 3 cleavage and activation in neonatal rat cardiomyocytes [19,25]. Moreover, it has been shown that in haematopoietic and cardiac cells, ROCK I can phosphorylate PTEN on Ser 229 and Thr 321 [17,18], increasing PTEN stabilization and activity [19]. We have demonstrated that morgana can inhibit ROCK I kinase activity, and that high morgana expression results in reduced PTEN levels, both in NIH-3T3 and in breast cancer cell lines. The ability of morgana to regulate PTEN levels depends on ROCK I, since constitutively active ROCK I restores normal PTEN levels and susceptibility to apoptotic stimuli in morgana-overexpressing cells. PTEN is a potent tumour suppressor that acts as a lipid phosphatase of phosphatidylinositol (3,4,5)-triphosphate (PIP3), thus opposing PI3K function. PTEN loss leads to PIP3 accumulation and activation of AKT and mammalian target of rapamycin (mTOR) signalling. Interestingly, PTEN is a haploinsufficient tumour suppressor and subtle down-regulation of PTEN has been demonstrated to strongly impact on cancer susceptibility and progression [26]. Moreover, sustained AKT activation is a potent mechanism for enhancing cell survival and chemotherapy resistance in cancer [16]. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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Notably, it has been reported that a high percentage of TNBCs show low PTEN expression [22,27–29], and that PTEN loss predicts early recurrence in TNBCs [30]. It is also known that PTEN expression and function are modulated by different mechanisms, such as genetic mutations, epigenetic silencing, microRNA regulation, competitive endogenous RNA, post-translational modification, and alteration in localization [26]. Here we describe a new mechanism adopted by cancer cells to destabilize PTEN by post-translational modification. Increased morgana expression levels result in ROCK I inhibition, which in turn leads to PTEN destabilization and enhancement of the AKT pro-survival pathway, conferring resistance to chemotherapeutic agents. In accordance with our results, morgana transcript levels have been found to be up-regulated in a microarray analysis of ovarian cancers that recur after chemotherapy, compared with primary tumours [31]. Overall, our findings indicate that both underexpression of morgana [4] and overexpression of morgana sustain tumourigenesis. Very few other proteins have been described to play both tumour suppressive and oncogenic functions, the most famous example being nucleophosmin (NPM) [32,33]. On the one hand, morgana acts as a tumour suppressor, the down-regulation of which results in centrosome amplification and genomic instability through ROCK II activation. On the other hand, morgana overexpression, through ROCK I inhibition, destabilizes PTEN and induces cell survival, thus acting as an oncogene (Figure 7E). Neoadjuvant chemotherapy, based on docetaxel and epirubicin, is widely used in breast cancer to increase tumour resectability, decrease the risk of recurrence, and increase overall survival. However, neoadjuvant treatment is ineffective in half of the patients treated and often results in toxicity. For this reason, identification of markers able to predict tumour resistance to neoadjuvant chemotherapy is of great clinical interest. To this end, high morgana expression levels, conferring resistance to both epirubicin and docetaxel, represent a novel indicator of cancer resistance to neoadjuvant chemotherapy. Finally, the presence of high morgana expression levels, unveiling PTEN down-regulation and overactivation of the PI3K downstream pathways, will provide valuable information for directing targeted therapy.
Acknowledgments We would like to thank Flavio Cristofani and Antonellisa Sgarra for assistance with animal experiments; and Shuh Narumiya, Claudio Brancolini, Ferdinando Di Cunto, and Enzo Calautti for sharing DNA constructs. We also thank Radhika Srinivasan for manuscript revision. This work was supported by an AIRC 2011 (IG 11456) grant to MB and by an AIRC 2011 (IG 11346) grant to SC. AD was supported by a fellowship from AIRC (annual fellowship ‘Marco Fabio Sartori’). J Pathol 2014; 234: 152–163 www.thejournalofpathology.com
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Author contribution statement FF designed the study and performed the experiments. RF, SR, GT, and AD performed the experiments. DR performed tissue array analysis. DR and AS scored breast cancer slides. PP performed bioinformatics analysis. LS, ET, SC, PPP, AS, and GT planned the experiments. MB supervised the project and planned the experiments. FF and MB wrote the article.
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SUPPORTING INFORMATION ON THE INTERNET The following supporting information may be found in the online version of this article: Supplementary materials and methods. Figure S1. Morgana overexpression does not alter centrosome number or asymmetric cell division. Figure S2. Morgana binds ROCK I coiled coil domain. Figure S3. PTEN down-regulation in T47D cells increases chemoresistance Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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