AUTOPHAGY 2016, VOL. 12, NO. 10, 1960–1961 http://dx.doi.org/10.1080/15548627.2016.1213928
AUTOPHAGIC PUNCTUM
Hierarchical heterogeneity in mammary tumors and its regulation by autophagy Syn Kok Yeo and Jun-Lin Guan Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
ABSTRACT
ARTICLE HISTORY
Intra-tumor heterogeneity can be attributed in part to the ability of tumor cells to acquire traits associated with less differentiated cells. In MMTV-PyMT mammary tumors, this hierarchical heterogeneity can be illustrated with the use of ITGB1/CD29hi ITGB3/CD61C markers to enrich for mammary stem-like cells and ALDHC to identify luminal progenitor-like cells. Macroautophagy/autophagy appears to be important for maintaining the cancer stem-like traits of both these populations. Interestingly, the regulation of these distinct cancer stem-like cells by autophagy occurs through EGFR-STAT3 and TGFB/TGF-b-SMAD pathways, respectively. These findings indicate that autophagy plays a significant role in cancer stem-like cells, and distinct cancer stem-like cells within a tumor may require different treatment modalities.
Received 24 June 2016 Revised 1 July 2016 Accepted 13 July 2016
Breast cancer is a heterogeneous disease and can be molecularly classified into at least 6 distinct subtypes which include Luminal A, Luminal B, Her2-enriched, Basal A, Basal B and normal-like breast cancers. For that reason, the stratification of patients for treatment based on these ‘molecular portraits’ is vital for achieving effective therapeutic outcomes. However, this complexity is not limited to variations between tumors from different patients (inter-tumor heterogeneity). In fact, there can be a large degree of intra-tumor heterogeneity both at the genetic level and epigenetically. The genetic heterogeneity within tumors is apparent from a multitude of sequencing studies. Furthermore, epigenetic heterogeneity within tumors has been eloquently depicted in studies of cancer stem-like cells (CSCs). Cells within a tumor behave in a hierarchical manner, where CSCs occupy a less differentiated state at the apex of the hierarchy and possess the ability to produce progeny of a more differentiated state. Importantly, CSCs exhibit increased tumor initiating potential relative to bulk tumor cells and this may be associated with the intrinsic characteristics of stem/progenitor cells. Autophagy is a process that enables cells to recycle cytoplasmic entities and organelles, which are sequestered within autophagosomes. This allows for sustenance under nutrientlimiting conditions and the elimination of defective organelles or proteins, which may perturb cellular homeostasis. As such, it may not be too surprising that some of the functions of autophagy in different cell types will depend on the material that is predominantly available for recycling and the need for clearance in that particular cell. Accordingly, it has been observed by Karantza and colleagues in normal mammary epithelial cells (MECs) that the predominant cargo in autophagosomes for basal (mammary stem cell-enriched) MECs is mitochondria
KEYWORDS
autophagy; breast cancer; cancer stem cell; epigenetic; heterogeneity
whereas that in luminal MECs is more diverse. Basal MECs also contain diminished numbers of autophagosomes relative to luminal MECs, illustrating the heterogeneity in terms of autophagic activity and diversity in autophagic cargo in different cell types within the mammary hierarchy. The role of autophagy in breast cancers appears to be dependent on the context of the tumor subtype and this is conceivable, considering different MECs utilize autophagy dissimilarly. Previous studies from our lab have demonstrated a protumorigenic role for the essential autophagy gene Rb1cc1/ Fip200 in MMTV-PyMT-driven mammary tumors, although there are other studies which show tumor suppressive functions of Becn1/Beclin1, another essential autophagy gene in MMTVWNT1-driven mammary tumors and lymphoma. Apart from the differential utilization of autophagy in distinct breast cancer subtypes, these contextual outcomes may also be due to nonautophagy functions of the genes examined. The latter may be addressed by specific disruption of autophagy functions in these genes rather than complete ablation of the entire protein. To this end, we have recently generated a RB1CC1 knockin mouse model that is defective in ATG13 binding and this will allow us to address the raised concerns in the near future. In our recent study, we showed that 2 distinct CSC populations in MMTV-PyMT tumors can be enriched and isolated using the markers ITGB1/CD29hi ITGB3/CD61C and ALDHC, respectively. While both these CSC populations exhibit increased tumor initiating frequencies, the ITGB1hi ITGB3C CSC are more mesenchymal and invasive. Further interrogation of these 2 CSC populations revealed that the ITGB1hi ITGB3C CSCs have a gene signature more associated with normal mammary stem cells (MaSC), whereas the ALDHC CSC expressed genes are reminiscent of luminal progenitors (LP).
CONTACT Jun-Lin Guan [email protected] Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kaup. Punctum to: SK Yeo, et al. Differential Regulation of Distinct Breast Cancer Stem Cells by Autophagy through Egfr/Stat3 and Tgfb/Smad Signaling. Cancer Res. 2016; 76: 3397-3410. http://dx.doi.org/10.1158/0008-5472.CAN-15-2946 © 2016 Taylor & Francis
AUTOPHAGY
1961
Figure 1. Illustration of heterogeneity within mammary tumors with ITGB1hi ITGB3C CSCs (red), ALDHC CSCs (green) and bulk tumor cells (white). The model shows that ITGB1hi ITGB3C CSCs are more mammary stem cell-like and at the top of the hierarchy within the epigenetic landscape (adapted from Conrad Waddington). ALDHC CSCs, which are luminal progenitor-like, occupy an intermediate epigenetic state followed by bulk tumor cells at the bottom. ITGB1hi ITGB3C CSCs are more dependent on TGFB-SMAD signaling, whereas ALDHC CSCs are reliant on EGFR-STAT3. Both these pathways are regulated by autophagy and RB1CC1, highlighting an important role for this process in CSCs.
The tumors that arise from transplantation of respective CSC populations can recapitulate the expression of these putative markers as in parental tumors, indicating that there is conversion and plasticity between these different CSC populations. Interestingly, the association of the 2 distinct CSC populations with MaSC and LP, respectively, suggest that there are different levels of differentiation within mammary tumors. In other words, these 2 distinct CSC populations along with bulk PyMT tumor cells are arranged in a hierarchical manner, where ITGB1hi ITGB3C CSCs (MaSC-like) occupy a least differentiated epigenetic state, ALDHC CSCs (LP-like) in an intermediate differentiation state, and bulk tumor cells are at the bottom of the hierarchy (Fig. 1). This may have important clinical implications because the ITGB1hi ITGB3C CSCs are more MaSC-like and basal, whereas the ALDHC and bulk tumor cells are luminal, and this means that these populations may each represent a different subtype of breast cancer. Importantly, that would imply the presence of multiple disease subtypes within the same tumor that may require different therapies. Consequently, we investigated the effects of RB1CC1 depletion on these distinct populations in PyMT tumors and found decreased levels of ITGB1hi ITGB3C CSCs and Aldh1a3 transcript levels, indicating a reduction in both CSC populations. This was accompanied by an impairment of tumor initia-
tion for both CSC populations. Mechanistically, we found that loss of RB1CC1 reduces Tgfb2 and Tgfb3 transcript levels along with a concomitant decrease in phosphorylated SMAD2. We further showed that perturbation of the TGFB/TGF-b-SMAD pathway either chemically or genetically, affects ITGB1hi ITGB3C CSCs but not ALDHC CSCs. Conversely, Rb1cc1 deletion also decreases EGFR levels, leading to decreased phosphorylated STAT3, and the EGFR-STAT3 pathway was shown to be important for the maintenance and tumorigenicity of ALDHC CSCs specifically. Altogether, these results suggest that autophagy is important for maintaining the CSC traits of both populations, albeit through the regulation of distinct signaling pathways. We also went on to show the importance of targeting both distinct CSC populations, with the combination of Stattic (a STAT3 inhibitor) and LY2157299 (a TGFBR1 inhibitor) and found that effective reduction of both CSC populations was key for therapeutic efficacy both in vitro and in vivo. In summary, work from our recent study indicates a hierarchical organization of distinct CSCs along with bulk tumor cells in mammary tumors. Autophagy appears to impinge on the traits of these distinct CSC populations through the regulation of TGFB-SMAD and EGFR-STAT3 pathways, respectively, and this extends on our previous findings for a pro-tumorigenic function of RB1CC1 in PyMT tumors.
AUTOPHAGIC PUNCTUM
Hierarchical heterogeneity in mammary tumors and its regulation by autophagy Syn Kok Yeo and Jun-Lin Guan Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
ABSTRACT
ARTICLE HISTORY
Intra-tumor heterogeneity can be attributed in part to the ability of tumor cells to acquire traits associated with less differentiated cells. In MMTV-PyMT mammary tumors, this hierarchical heterogeneity can be illustrated with the use of ITGB1/CD29hi ITGB3/CD61C markers to enrich for mammary stem-like cells and ALDHC to identify luminal progenitor-like cells. Macroautophagy/autophagy appears to be important for maintaining the cancer stem-like traits of both these populations. Interestingly, the regulation of these distinct cancer stem-like cells by autophagy occurs through EGFR-STAT3 and TGFB/TGF-b-SMAD pathways, respectively. These findings indicate that autophagy plays a significant role in cancer stem-like cells, and distinct cancer stem-like cells within a tumor may require different treatment modalities.
Received 24 June 2016 Revised 1 July 2016 Accepted 13 July 2016
Breast cancer is a heterogeneous disease and can be molecularly classified into at least 6 distinct subtypes which include Luminal A, Luminal B, Her2-enriched, Basal A, Basal B and normal-like breast cancers. For that reason, the stratification of patients for treatment based on these ‘molecular portraits’ is vital for achieving effective therapeutic outcomes. However, this complexity is not limited to variations between tumors from different patients (inter-tumor heterogeneity). In fact, there can be a large degree of intra-tumor heterogeneity both at the genetic level and epigenetically. The genetic heterogeneity within tumors is apparent from a multitude of sequencing studies. Furthermore, epigenetic heterogeneity within tumors has been eloquently depicted in studies of cancer stem-like cells (CSCs). Cells within a tumor behave in a hierarchical manner, where CSCs occupy a less differentiated state at the apex of the hierarchy and possess the ability to produce progeny of a more differentiated state. Importantly, CSCs exhibit increased tumor initiating potential relative to bulk tumor cells and this may be associated with the intrinsic characteristics of stem/progenitor cells. Autophagy is a process that enables cells to recycle cytoplasmic entities and organelles, which are sequestered within autophagosomes. This allows for sustenance under nutrientlimiting conditions and the elimination of defective organelles or proteins, which may perturb cellular homeostasis. As such, it may not be too surprising that some of the functions of autophagy in different cell types will depend on the material that is predominantly available for recycling and the need for clearance in that particular cell. Accordingly, it has been observed by Karantza and colleagues in normal mammary epithelial cells (MECs) that the predominant cargo in autophagosomes for basal (mammary stem cell-enriched) MECs is mitochondria
KEYWORDS
autophagy; breast cancer; cancer stem cell; epigenetic; heterogeneity
whereas that in luminal MECs is more diverse. Basal MECs also contain diminished numbers of autophagosomes relative to luminal MECs, illustrating the heterogeneity in terms of autophagic activity and diversity in autophagic cargo in different cell types within the mammary hierarchy. The role of autophagy in breast cancers appears to be dependent on the context of the tumor subtype and this is conceivable, considering different MECs utilize autophagy dissimilarly. Previous studies from our lab have demonstrated a protumorigenic role for the essential autophagy gene Rb1cc1/ Fip200 in MMTV-PyMT-driven mammary tumors, although there are other studies which show tumor suppressive functions of Becn1/Beclin1, another essential autophagy gene in MMTVWNT1-driven mammary tumors and lymphoma. Apart from the differential utilization of autophagy in distinct breast cancer subtypes, these contextual outcomes may also be due to nonautophagy functions of the genes examined. The latter may be addressed by specific disruption of autophagy functions in these genes rather than complete ablation of the entire protein. To this end, we have recently generated a RB1CC1 knockin mouse model that is defective in ATG13 binding and this will allow us to address the raised concerns in the near future. In our recent study, we showed that 2 distinct CSC populations in MMTV-PyMT tumors can be enriched and isolated using the markers ITGB1/CD29hi ITGB3/CD61C and ALDHC, respectively. While both these CSC populations exhibit increased tumor initiating frequencies, the ITGB1hi ITGB3C CSC are more mesenchymal and invasive. Further interrogation of these 2 CSC populations revealed that the ITGB1hi ITGB3C CSCs have a gene signature more associated with normal mammary stem cells (MaSC), whereas the ALDHC CSC expressed genes are reminiscent of luminal progenitors (LP).
CONTACT Jun-Lin Guan [email protected] Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kaup. Punctum to: SK Yeo, et al. Differential Regulation of Distinct Breast Cancer Stem Cells by Autophagy through Egfr/Stat3 and Tgfb/Smad Signaling. Cancer Res. 2016; 76: 3397-3410. http://dx.doi.org/10.1158/0008-5472.CAN-15-2946 © 2016 Taylor & Francis
AUTOPHAGY
1961
Figure 1. Illustration of heterogeneity within mammary tumors with ITGB1hi ITGB3C CSCs (red), ALDHC CSCs (green) and bulk tumor cells (white). The model shows that ITGB1hi ITGB3C CSCs are more mammary stem cell-like and at the top of the hierarchy within the epigenetic landscape (adapted from Conrad Waddington). ALDHC CSCs, which are luminal progenitor-like, occupy an intermediate epigenetic state followed by bulk tumor cells at the bottom. ITGB1hi ITGB3C CSCs are more dependent on TGFB-SMAD signaling, whereas ALDHC CSCs are reliant on EGFR-STAT3. Both these pathways are regulated by autophagy and RB1CC1, highlighting an important role for this process in CSCs.
The tumors that arise from transplantation of respective CSC populations can recapitulate the expression of these putative markers as in parental tumors, indicating that there is conversion and plasticity between these different CSC populations. Interestingly, the association of the 2 distinct CSC populations with MaSC and LP, respectively, suggest that there are different levels of differentiation within mammary tumors. In other words, these 2 distinct CSC populations along with bulk PyMT tumor cells are arranged in a hierarchical manner, where ITGB1hi ITGB3C CSCs (MaSC-like) occupy a least differentiated epigenetic state, ALDHC CSCs (LP-like) in an intermediate differentiation state, and bulk tumor cells are at the bottom of the hierarchy (Fig. 1). This may have important clinical implications because the ITGB1hi ITGB3C CSCs are more MaSC-like and basal, whereas the ALDHC and bulk tumor cells are luminal, and this means that these populations may each represent a different subtype of breast cancer. Importantly, that would imply the presence of multiple disease subtypes within the same tumor that may require different therapies. Consequently, we investigated the effects of RB1CC1 depletion on these distinct populations in PyMT tumors and found decreased levels of ITGB1hi ITGB3C CSCs and Aldh1a3 transcript levels, indicating a reduction in both CSC populations. This was accompanied by an impairment of tumor initia-
tion for both CSC populations. Mechanistically, we found that loss of RB1CC1 reduces Tgfb2 and Tgfb3 transcript levels along with a concomitant decrease in phosphorylated SMAD2. We further showed that perturbation of the TGFB/TGF-b-SMAD pathway either chemically or genetically, affects ITGB1hi ITGB3C CSCs but not ALDHC CSCs. Conversely, Rb1cc1 deletion also decreases EGFR levels, leading to decreased phosphorylated STAT3, and the EGFR-STAT3 pathway was shown to be important for the maintenance and tumorigenicity of ALDHC CSCs specifically. Altogether, these results suggest that autophagy is important for maintaining the CSC traits of both populations, albeit through the regulation of distinct signaling pathways. We also went on to show the importance of targeting both distinct CSC populations, with the combination of Stattic (a STAT3 inhibitor) and LY2157299 (a TGFBR1 inhibitor) and found that effective reduction of both CSC populations was key for therapeutic efficacy both in vitro and in vivo. In summary, work from our recent study indicates a hierarchical organization of distinct CSCs along with bulk tumor cells in mammary tumors. Autophagy appears to impinge on the traits of these distinct CSC populations through the regulation of TGFB-SMAD and EGFR-STAT3 pathways, respectively, and this extends on our previous findings for a pro-tumorigenic function of RB1CC1 in PyMT tumors.
