Cell Differentiation and Deoelopment, 32 (1990) 401-408 © 1990 Elsevier Scientific Publishers Ireland, Ltd. 0922-3371/90/$03.50
401
CELDIF 99925
Expression of tenascin and of the ED-B containing oncofetal fibronectin isoform in human cancer Guido Nicolb *, Sandra Salvi *, Gianbattista Oliveri *, L a u r a Borsi * *, Patrizia Castellani * * and Luciano Zardi * * Laboratories of 1 Anatomic Pathology and 2 Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy
Tenascin (TN) and the oncofetal ED-B containing fibronectin isoform (B-FN) have been reported to be stromal markers of a number of malignancies. Here we report on studies of the distribution of TN and B-FN in normal adult tissues and in benign and malignant tumors, as well as on the levels of the B-FN mRNA in cultured fetal and non-fetal human fibroblasts originating from different tissues. B-FN has an extremely restricted distribution in normal adult tissues, is not expressed in benign tumors, but is greatly expressed in a high percentage of malignant tumors. On the contrary, human TN in normal adult tissues is less restricted than what has previously been reported and it is largely expressed in a number of both benign and malignant tumors. Moreover, we observed a great variability in the relative amount of B-FN mRNA among the 17 normal human fibroblast cell lines tested. We found very low levels in non-fetal skin fibroblasts and higher levels in fetal lung fibroblasts. We also found differences in the relative amounts of B-FN mRNA between fibroblast cell lines originating from the skin and the lung of the same subject. Tenascin; Oncofetal fibronectin
Introduction
The components of the extraceUular matrix of tumor stroma are different with respect to those of normal tissues from which tumors originate. A number of studies have indicated that these stroma components may play an active role in sustaining tumor growth and progression (van den Hooff, 1988, and references therein). In particular, recent studies have indicated two oncofetal fibronectin (FN) isoforms and tenascin (TN) as specific constituents of the extracellular matrix of malignant
Correspondence address: L. Zardi, Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16132 Genoa, Italy.
tumor stroma (Matsuura and Hakomori, 1985; Chiquet-Ehrismann et al., 1986; Carnemolla et al., 1989; Loridon-Rosa et al., 1990). TN is a polymorphic, high molecular mass extracellular matrix glycoprotein composed of six similar subunits joined together at their amino terminal by disulfide bonds (for reviews see Erickson and Lightner, 1988; Erickson and Bourdon, 1989; Natali and Zardi, 1989). TN is rather unusual in its molecular organization, being made up of epidermal growth factor-like (EGF-L) repeats, fibronectin type III repeats and a fibrinogen-like region. TN has been shown to be a stromal marker of malignant mammary carcinoma, glioma and of other tumors (Bourdon et al., 1983; Mackie et al., 1987; Natali and Zardi, 1989), thus raising the question of whether TN neo-expression may be a
402 marker of malignant transformation. Since TN 1) stimulates the growth of mammary tumor cells in vitro, 2) is highly expressed in the stroma of malignant tumors and 3) contains EGF-L sequences, it has been suggested that TN may also play a role in supporting the growth of epithelial tumors in vivo (Chiquet-Ehrismann et al., 1986; Mackie et al., 1987; Chiquet-Ehrismann et al., 1989). FN is the blend of structurally and functionally different isoforms resulting from the alternative splicing at three regions (IIICS, ED-A, ED-B) of the FN primary transcript. The make-up of the different FN isoforms depends on the FN source (Hynes, 1985; Owens et al., 1986; Zardi et al., 1987). The alternative splicing of the FN premRNA is regulated in a cell, tissue and developmentally specific manner (Kornblihtt et al., 1984; Schwarzbauer et al., 1985; Zardi et al., 1987; ffrench-Constant and Hynes, 1989; Carnemolla et al., 1989; Oyama et al., 1989a). Furthermore, it has recently been demonstrated that the splicing pattern of FN pre-mRNA is deregulated in transformed cells and in malignancies (Castellani et al., 1986; Borsi et al., 1987; Vartio et al., 1987; Zardi et al., 1987; CarnemoUa et al., 1989; Oyama et al., 1989b; Oyama et al., 1990). In fact the FN isoform containing the ED-B sequence (B-FN), which is absent in normal adult tissues with some very rare exceptions, has a much greater expression in fetal and tumor tissues (Carnemolla et al., 1989). The ED-B sequence (oncofetal domain) of human FN is a complete type III homology repeat of 91 amino acids which is coded for by a single exon and is either included or excluded from the mature mRNA depending on the splicing pattern of the primary transcript. Here we report on studies of the distribution of TN and B-FN in normal adult tissues and in benign and malignant tumors, as well as on the levels of the B-FN mRNA in cultured fetal and non fetal human fibroblasts originating from different tissues. Materials and Methods
Cultured normal human non-fetal skin fibroblasts (GM-5757, GM-3652, GM-4390, GM-3651,
GM-5659, GM-3377, GM-3440), fetal skin fibroblasts (GM-5386, GM-6113, GM-1603, GM5388), fetal lung fibroblasts (GM-5387, GM-6114, GM-5389) and the SV-40 transformed cell line AG-280, were purchased from NIGMS Human Genetic Mutant Cell Repository (New Jersey, U.S.A.). Fetal lung fibroblast cell lines, MRC-5, IMR-90, WI-38, were obtained from American Type Culture Collection (Rockville, MD, U.S.A.) as well as the SV-40 transformed WI-38VA13 cell lines. The cells were grown in Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Flow Laboratories, Irvine, Scotland, U.K.). Normal human fibroblast cell lines were utilized from the 11th to the 25 th passage. RNA S1 nuclease protection analysis for the determination of the ED-B containing FN mRNA was carried out as described by Borsi et al. (1990). Monoclonal antibodies to TN and to B-FN were obtained and characterized as described by Natali et al. (1990) and Carnemolla et al. (1989), respectively. Normal and neoplastic tissues were obtained from surgical samples. Immunohistochemical analysis were carried out using the APAAP kit System 40 (DAKO Corporation, Carpinteria, CA, U.S.A.) as described by Carnemolla et al. (1989).
Results
Levels of ED-B containing FN mRNA in cultured normal and SV-40 transformed human fibroblasts Through S1 nuclease analysis we have determined the relative amount of ED-B containing FN mRNA in a large panel of cultured normal human fibroblasts from non-fetal skin, fetal skin and fetal lung. The results, reported in Table I, show a great variability in the % of B-FN mRNA. In non-fetal skin fibroblast cell lines we obtained the lowest levels of B-FN mRNA (from 0.55 to 2.1%) while we found the highest levels in fetal lung fibroblasts (from 6.6 to 30.6%). We have also analyzed three pairs of cell lines (GM-5386, GM5387; GM-5388, GM-5389; GM-6113, GM6114), coming from the skin and the lung of the same subject. In all three cases we observed that the percentage of B-FN mRNA in lung fibroblasts
403 was a b o u t 2.5 times higher t h a n that of skin fibroblasts (see T a b l e I). W e also tested the percentage of B - F N m R N A i n W I - 3 8 V A 1 3 a n d A G 280 cell lines, which are the SV-40 t r a n s f o r m e d c o u n t e r p a r t of the W I - 3 8 a n d I M R - 9 0 fetal lung fibroblast cell lines. I n b o t h cases we o b t a i n e d a two-three fold increase i n the relative a m o u n t s of B - F N m R N A i n SV-40 t r a n s f o r m e d cells comp a r e d to that of the u n t r a n s f o r m e d counterparts.
TABLE I Relative abundance of ED-B containing FN mRNA in cultured normal human fibroblasts of different origin Cell lines
Age of donor
Tissue of origin
B-FNmRNA (%) ( 5: S.E.)
GM-5757 7 yr skin GM-3652 24 yr skin GM-4390 23 yr skin GM-3651 25 yr skin GM-5659 14 mo skin GM-3377 19 yr skin GM-3440 20 yr skin GM-5386 20 fw skin GM-6113 20 fw skin GM-1603 12 fw skin GM-5388 20 fw skin GM-5387 20 fw lung GM-6114 20 fw lung GM-5389 20 fw lung MRC-5 14 fw lung IMR-90 16 fw lung WI-38 12 fw lung AG-280 (SV-40transformed IMR-90) WI-38VA13(SV-40 transformed WI-38)
0.55 + 0.1 0.8 + 0.04 0.8 +0.08 1.2 +0.05 1.5 +0.15 2.0 + 0.1 2.1 5:0.4 2.5 5:0.3 2.6 + 1.1 4.4 5:0.1 6.5 5:2.3 6.6 + 0.1 6.8 5:1.3 17.2 5:1.9 17.5 + 2.4 19.2 5:1.9 30.6 5-1.4 42.5 5:3.4 92.1 5:2.4
Expression of B-FN in human normal adult tissues and tumors W e have previously reported that the B - F N isoform, while b e i n g restricted o n l y to synovial cells a n d to some vessels of the ovary i n n o r m a l adult h u m a n tissues, has, o n the contrary, a m u c h greater expression i n fetal a n d m a l i g n a n t t u m o r tissues ( C a r n e m o l l a et al., 1989). S u b s e q u e n t exp e r i m e n t s have c o n f i r m e d a n d furthered these resuits. Fig. 2 shows that while the B - F N isoform is present i n the s t r o m a of a l u n g s q u a m o u s
HOMOLOGY: typel D typell 0 typelll [ ]
I,~DFORMS :
ONCOFETAL DOMAIN (BC- I )
ONCOFETAL EPITOPE (FDC-6)
EDI-B
ED-A
~nTCS
NH~
COOH .
DOMAINS :
.
1
INTIBRACTION$ : heparin DNA fibrin
.
.
2
.
3
gelatin heparin DNA
.
.
.
.
.
.
4
5
cell heparin
heparin DNA
6
fibrin
DNA
Fig. 1. Model of the domain structure of a subunit of human fibronectin. The ED-B oncofetal domain recognized by the Mab BC-1 (Carnemolla et al., 1989) and the oncofetal epitope recognized by the Mab FDC-6 (Matsuura et al., 1985) are indicated. The figure also indicates the internal homologies, the major macromoleculesinteracting with the various FN domains and the possible isoforms generated by alternative splicing of the sequences IIICS, ED-A and ED-B.
4{)4
14
I
i/2
EGF-like repeats
FIBRINOGEN LIKE SEQUENCE,
FN-LIKEREPEATS
II
I
~I~
!{~i:!!~ ~. :!~! :.~!'~!~i~]~:.l~:~[]]:.(~J
Fig. 3. Model of the domain structure of a human TN subunit. The expression of the FN-like repeats 6-12 (dotted) is regulated by alternative splicing of the pre-mRNA.
c a r c i n o m a , it is u n d e t e c t a b l e in n o r m a l lung. F u r t h e r m o r e Fig. 2 shows a histological section o f a lung alveolar a d e n o c a r c i n o m a , which c o n t a i n s b o t h a t u m o r a l a n d a n o r m a l p a r t o f the lung. It is p o s s i b l e to see that the B - F N i s o f o r m is p r e s e n t o n l y in the s t r o m a of the t u m o r while, in the s t r o m a of the n o r m a l lung, it is u n d e t e c t a b l e . I n a b r e a s t f i b r o a d e n o m a section, even t h o u g h b o t h the epithelial a n d the s t r o m a cells are in active proliferation, n o B - F N staining is d e t e c t a b l e (Fig. 2).
Distribution of TN in normal adult tissues and in benign and malignant tumors Fig. 3 shows a schematic r e p r e s e n t a t i o n o f the d o m a i n structure of h u m a n T N , showing the Epid e r m a l G r o w t h F a c t o r - l i k e , the F i b r o n e c t i n - l i k e a n d the F i b r i n o g e n - l i k e sequences. Fig. 4 shows the presence of T N in n o r m a l h u m a n skin. It is p o s s i b l e to observe a massive p r e s e n c e of T N in the p a p i l l a r y d e r m a a n d it is f u r t h e r m o r e e v i d e n t that the vessels p r e s e n t in the d e r m a are also
intensively stained. I n n o r m a l b r e a s t tissue we f o u n d o n l y small a m o u n t s of T N , a n d m a i n l y in the vessels. M a c k i e et al. (1987) have suggested that T N c o u l d b e a m a r k e r for b r e a s t m a l i g n a n c y : we have s t u d i e d a n e l e v a t e d n u m b e r of m a l i g n a n t b r e a s t t u m o r s and, i n d e e d , we have f o u n d elevated a m o u n t s of T N in a l m o s t all m a l i g n a n t t u m o r s ( N a t a l i a n d Z a r d i , 1989; N a t a l i et al., 1990). In fact, in Fig. 4, showing a histological section of an invasive b r e a s t c a r c i n o m a which c o n t a i n s b o t h t u m o r a l a n d n o r m a l b r e a s t structures, it is possib l e to observe t h a t o n l y the t u m o r s t r o m a c o n t a i n s T N while the s t r o m a o f n o r m a l b r e a s t tissue does n o t show a n y d e t e c t a b l e staining. However, we f o u n d elevated a m o u n t s of T N even in benign t u m o r s ( N a t a l i et al., 1990; N a t a l i et al., s u b m i t t e d for p u b l i c a t i o n ) . I n fact, in a histological section o f a b r e a s t f i b r o a d e n o m a - a localized n o d u l a r h y p e r p l a s i a of g l a n d u l a r a n d s t r o m a tissues - it is p o s s i b l e to observe a c o n s i d e r a b l e presence of T N in the s t r o m a of the f i b r o a d e n o m a . O n the contrary, in a n a t y p i c a l d u c t a l b r e a s t h y p e r p l a s i a (a
Fig. 2. Study of distribution of the B-FN isoform by indirect immunoenzyrnatic staining using the Mab BC-1. The Mab BC-1 does not react with normal lung structures, but shows a very strong reaction with the stroma of a squamous lung carcinoma. In a lung histological section including both a portion of normal tissue and a portion of an alveolar adenocarcinoma, it is possible to see, on the right, that the stroma of the tumor shows presence of B-FN while, on the left, the stroma of the normal tissue does not show any detectable B-FN. In a breast fibroadenoma histological section, even though the stromal elements are in active proliferation, no B-FN is detectable. Bar, 10 ram. Fig. 4. Study of the distribution of TN by indirect immunoenzymatic staining. In a normal human skin histological section it is possible to see an intensive staining in the papillary derma and in the vessels. In a breast histological section including both a portion of normal tissue and a portion of an invasive breast carcinoma it is possible to see, on the left, that only the tumor stroma shows presence ofTN while the stroma of the normal tissue, on the right, does not show any detectable TN. In a histological section of a breast fibroadenoma it is possible to observe an intense staining of the stroma of the lesion, while the pseudo-capsula (on the fight), made up of mature connective tissue, shows a very light staining. In a histological section of an atypical ductal breast hyperplasia the stroma does not show any staining while some staining is visible within the epithelial cells. Bar, 10 mm.
Lung s q u a m o u s c a r c i n o m a
Normal lung
L u n g alveolar a d e n o c a r c i n o m a
Fig. 2.
Normal skin
Breast flbroadenoma
Breast flbroadenoma
Invasive b r e a s t c a r c i n o m a
Fig. 4.
Atypical ductal b r e a s t hyperplasia
406 pre-neoplastic lesion, defined as such since a high percentage develops into invasive carcinoma) it is possible to see that the stroma does not present any detectable TN, while a light staining within the epithelial elements is visible (Fig. 4). In these lesions, while there is a proliferation of atypical epithelial elements, there is no proliferation of stromal cells.
Discussion
Through $1 nuclease protection analysis we have determined the relative amount of B-FN mRNA in a panel of human fibroblast cell lines purchased directly from the American Type Culture Collection and the NIGMS Human Genetic Mutant Cell Repository. Among the 17 normal human fibroblast cell lines, we found a great variability in the relative amount of B-FN mRNA. We found very low levels in non-fetal skin fibroblasts (average: 1.28) and higher levels in fetal lung fibroblasts (average: 16.32). We also found differences between skin and lung fibroblasts obtained from the same subjects. These data are in agreement with in vivo studies. In fact, B-FN is undetectable in adult skin while it is present in large amounts in fetal lung (Carnemolla et al., 1989). Furthermore, SV-40 transformation induces a considerable increase in the expression of the B-FN. We have previously reported that the WI-38 cell line produces very low amounts of B-FN. The discrepancies with our present data (see Table I) may possibly be due to the fact that in our previous experiments (Zardi et al., 1987; Carnemolla et al., 1989) this cell line was not directly obtained from the American Tissue Culture Collection. Although establishing the splicing pattern of the ED-B sequence in different cell lines could be of some relevance, the study of the expression of this sequence in vivo in different normal and pathological tissues is of greater importance. By using the Mab BC-1, which recognizes an epitope within the ED-B sequence (Carnemolla et al., 1989), we have confirmed that the B-FN isoform could be considered a tumor marker. In particular, we observed large amounts of B-FN in a higla percentage of lung and intestinal tumors (Zardi et al.,
in preparation). Another Mab, FDC-6, has been reported to recognize an oncofetal isoform of FN (Matsuura and Hakomori, 1985). This epitope is localized within the IIICS sequence (see Fig. 1) and is composed of an oligosaccharide linked to a hexapeptide by O-glycosilation (Matsuura et al., 1989). Thus, the oncofetal epitope recognized by the Mab FDC-6 originates from an altered glycosilation of FN in fetal tissues and tumors and it is not related to the ED-B sequence. Loridon-Rosa et al. (1990) reported that this oncofetal FN isoform is undetectable in normal breast and in benign breast tumors, while it is present in 60% of breast carcinomas and is hnked with intermediary or high malignancy grades. It would thus be interesting to compare the patterns of distribution of the oncofetal B-FN and of the oncofetal FN isoform recognized by the Mab FDC-6 in different tissues. Our studies on TN have demonstrated that the distribution of this glycoprotein in normal adult tissues is less restricted than what has previously been reported (Natali et al., in press; Natali et al., submitted for publication). Furthermore, in the case of breast tumors, our studies seem to indicate that TN, more than a marker for malignant tumors, is a marker of connective element proliferation. In fact, we did not detect any TN in the stroma of pre-neoplastic lesions in which only atypical epithelial elements proliferate. We did find, however, large amounts of TN in fibroadenoma, a benign tumor in which there is proliferation of stromal cells in addition to epithelial cells. It was previously demonstrated that, in cultured human fibroblasts, TGF-fl modulates the splicing of the FN primary transcript, increasing the relative amounts of B-FN (Borsi et al., 1990), and increases the production of TN as well (Pearson et al., 1988). These data suggest that the presence of B-FN and TN in tumor stroma may be a consequence of a paracrine effect by which tumor cells, by releasing growth factors, induce the surrounding cells to organize a stroma more adequate to sustain tumor growth. In fact, a number of studies have suggested that the tumor stroma represents a more "permissive environment" for tumor growth compared to the stroma of normal tissues (van den Hooff, 1988, and references
407
therein), and that TGF-fl may trigger its formation (Sieweke et al., 1990). Future studies should be directed towards establishing whether TN and the tumor-associated FN isoforms functionally contribute to the generation of this permissive environment.
Acknowledgements This study has been partially supported by AIRC funds. We are indebted to Mr Thomas Wiley for manuscript revision and to Prof. L. Santi for his support and encouragement.
References Borsi, L., P. Castellani, A.M. Risso, A. Lepftni and L. Zardi: Transforming growth factor-fl regulates the splicing pattern of fibronectin messenger RNA precursors. FEBLett. 261, 175-178 (1990). Borsi, L., B. Carnemolla, P. Castellani, C. Rosellini, D. Vecchio, G. Allemanni, S.E. Chang, J. Taylor-Papadimitriou, H. Pande and L. Zardi: Monoclonal antibodies in the analysis of fibroneetin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells. J. Cell Biol. 104, 595-600 (1987). Bourdon, M.A., C.J. Wikstrand, H. Furthmayr, T.J. Matthews and D.D. Bigner: Human glioma-mesenchymal extracellulax matrix antigen defined by monoclonal antibody. Cancer Rev. 43, 2796-2805 (1983). Carnemolla, B., E. Baiza, A. Sift, L. Zardi, M.R. Nicotra, A. Bigotti and P.G. Natali: A tumor-associated fibronectin isoform generated by alternative splicing of messenger RNA precursors. J. Cell Biol. 108, 1139-1148 (1989). Castellani, P., A. Sift, C. Rosellini, E. Infusini, L. Borsi and L. Zardi: Transformed human cells release different fibronectin variants than do normal cells. J. Cell Biol. 103, 16711677 (1986). Chiquet-Ehrismann, R., E.J. Mackie, C.A. Pearson and T. Sakakura: Tenascin: an extracellular matrix protein involved in tissue interactions during fetal development and oncogenesis. Cell 47, 131-139 (1986). Chiquet-Ehrismarm, R., P. KaUa and C.A. Pearson: Partecipitation of tenascin and TGF-fl in reciprocal epithelialmesenchymal interactions of MCF7 cells and fibroblasts. Cancer Res. 49, 4322-4325 (1989). Erickson, H.P. and M.A. Bourdon: Tenascin: an extraceUular matrix protein prominent in specialized embryonic tissues and tumors. Annu. Rev. Cell Biol. 5, 71-92 (1989). Erickson, H.P. and V.A. Lightner: Hexabrachion protein (tenascin, cytotactin, brachionectin) in connective tissues,
embryonic brain, and tumors. Advanc. Cell Biol., 2, 55-90 (1988). ffrench-Constant, C. and R.O. Hynes: Alternative splicing of fibronectin is temporally and spatially regulated in the chicken embryo. Development. 106, 375-388 (1989). Hynes, R.O.: Molecular biology of fibronectin. Annu. Rev. Cell Biol. 1, 67-90 (1985). Kornblihtt, A.R., K. Vibe-Pedersen and F.E. Baralle: Human fibronectin: cell specific alternative mRNA splicing generates polypeptide chains differing in a number of internal repeats. Nucleic Acids Res. 12, 5853-5868 (1984). Loridon-Rosa, B., P. Vielh, H. Matsuura, H. Clausen, C. Cuadrado and P. Burtin: Distribution of oncofetal fibronectin in human mammary tumors: immunofluorescence study on histological sections. Cancer Res. 50, 16081613 (1990). Mackie, E.J., R. Chiquet-Ehrismann, C.A. Pearson, Y. Inagnma, K. Taya, Y. Kawarada and T. Sakakura: Tenascin is a stromal marker for epithelial malignancy in the mammary gland. Proc. Natl. Acad. Sci. (Wash.) 84, 46214625 (1987). Matsuura, H., T. Greene and S. Hakomori: An a-N-acetylgalactosaminylation at the threonine residue of a defined peptide sequence creates the oncofetal peptide epitope in human fibronectin. J. Biol. Chem. 18, 10472-10476 (1989). Matsuura, H. and S. Hakomori: The oncofetal domain of fibronectin defined by monoclonal antibody FDC-6: its presence in fibronectin from fetal and tumor tissues and its absence in those from normal adult tissues and plasma. Proc. Natl. Acad. Sci. USA, 82, 6517-6521 (1985). Natali, P.G. and L. Zardi: Tenascin: a hexameric adhesive glycoprotein. Int. J. Cancer, Suppl. 4, 66-68 (1989). Natali, P.G., M.R. Nicotra, A. Bartolazzii, N. Coscia, F. Di Filippo, A. Bigotti and L. Zarcli: Expression and production of tenascin in benign and malignant lesions of melanocyte lineage. Int. J. Cancer, 46, 586-590 (1990). Owens, R.J., A.R. Kornblihtt and F.E Baralle: Fibronectin, the generation of multiple polypeptides from a single gene. Oxf. Surv. Eucaryot. Genes. 3, 141-160 (1986). Oyama, F., S. Hirohashi, Y. Shimosato, K. Titani and K. Sekiguchi: Deregulation of alternative splicing of fibronectin pre-mRNA in malignant human liver tumors. J. Biol. Chem. 264, 10331-10334 (1989). Oyama, F., Y. Murata, N. Suganuma, T. Kimura, K. Titani and K. Sekignchi: Patterns of alternative splicing of fibronectin pre-mRNA in human adult and fetal tissues. Biochemistry 28, 1428-1434 (1989). Oyama, F., S. Hirohashi, Y. Shimosato, K. Titani and K. Sekiguchi: Oncodevelopmental regulation of the alternative splicing of fibronectin pre-messenger RNA in human lung tissues. Cancer Res. 50, 1075-1078 (1990). Pearson, C.A., D. Pearson, S. Schibahara, J. Hofsteenge and R. Chiquet-Ehrismarm: Tenascin: cDNA cloning and induction by TGF-fl. EMBO J, 7, 2977-2981 (1988). Schwarzbaner, J.E., J.I. Patti and R.O. Hynes: On the origin of species of fibronectin. Proc. Natl. Acad. Sci. USA. 82, 1424-1428 (1985).
408 Sieweke, M.H., N.L. Thompson, M.B. Sporn and M.J. Bissel: Mediation of wound-related Rous Sarcoma virus tumorigenesis by TGF-fl. Science, 248, 1656-1660 (1990). van den Hoof, A.: Stromal involvement in malignant growth. Adv. Cancer Res. 50, 159-196 (1988). Vartio, T., L. Laitinen, O. Narvanen, M. Cutolo, L. Thomell, L. Zardi and I. Virtanen: Differential expression of the ED
sequence-containing form of cellular fibronectin in embryonic and adult human tissues. J. Cell. Sci. 88, 419-430 (1987). Zardi, L., B. Camemolla, A. Siri, T.E. Petersen, G. Paolella, G. Sebastio and F.E. Baralle: Transformed human cells produce a new fibronectin isoform by preferential splicing of a previously unobserved exon. EMBO J. 6, 2337-2342 (1987).
401
CELDIF 99925
Expression of tenascin and of the ED-B containing oncofetal fibronectin isoform in human cancer Guido Nicolb *, Sandra Salvi *, Gianbattista Oliveri *, L a u r a Borsi * *, Patrizia Castellani * * and Luciano Zardi * * Laboratories of 1 Anatomic Pathology and 2 Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy
Tenascin (TN) and the oncofetal ED-B containing fibronectin isoform (B-FN) have been reported to be stromal markers of a number of malignancies. Here we report on studies of the distribution of TN and B-FN in normal adult tissues and in benign and malignant tumors, as well as on the levels of the B-FN mRNA in cultured fetal and non-fetal human fibroblasts originating from different tissues. B-FN has an extremely restricted distribution in normal adult tissues, is not expressed in benign tumors, but is greatly expressed in a high percentage of malignant tumors. On the contrary, human TN in normal adult tissues is less restricted than what has previously been reported and it is largely expressed in a number of both benign and malignant tumors. Moreover, we observed a great variability in the relative amount of B-FN mRNA among the 17 normal human fibroblast cell lines tested. We found very low levels in non-fetal skin fibroblasts and higher levels in fetal lung fibroblasts. We also found differences in the relative amounts of B-FN mRNA between fibroblast cell lines originating from the skin and the lung of the same subject. Tenascin; Oncofetal fibronectin
Introduction
The components of the extraceUular matrix of tumor stroma are different with respect to those of normal tissues from which tumors originate. A number of studies have indicated that these stroma components may play an active role in sustaining tumor growth and progression (van den Hooff, 1988, and references therein). In particular, recent studies have indicated two oncofetal fibronectin (FN) isoforms and tenascin (TN) as specific constituents of the extracellular matrix of malignant
Correspondence address: L. Zardi, Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16132 Genoa, Italy.
tumor stroma (Matsuura and Hakomori, 1985; Chiquet-Ehrismann et al., 1986; Carnemolla et al., 1989; Loridon-Rosa et al., 1990). TN is a polymorphic, high molecular mass extracellular matrix glycoprotein composed of six similar subunits joined together at their amino terminal by disulfide bonds (for reviews see Erickson and Lightner, 1988; Erickson and Bourdon, 1989; Natali and Zardi, 1989). TN is rather unusual in its molecular organization, being made up of epidermal growth factor-like (EGF-L) repeats, fibronectin type III repeats and a fibrinogen-like region. TN has been shown to be a stromal marker of malignant mammary carcinoma, glioma and of other tumors (Bourdon et al., 1983; Mackie et al., 1987; Natali and Zardi, 1989), thus raising the question of whether TN neo-expression may be a
402 marker of malignant transformation. Since TN 1) stimulates the growth of mammary tumor cells in vitro, 2) is highly expressed in the stroma of malignant tumors and 3) contains EGF-L sequences, it has been suggested that TN may also play a role in supporting the growth of epithelial tumors in vivo (Chiquet-Ehrismann et al., 1986; Mackie et al., 1987; Chiquet-Ehrismann et al., 1989). FN is the blend of structurally and functionally different isoforms resulting from the alternative splicing at three regions (IIICS, ED-A, ED-B) of the FN primary transcript. The make-up of the different FN isoforms depends on the FN source (Hynes, 1985; Owens et al., 1986; Zardi et al., 1987). The alternative splicing of the FN premRNA is regulated in a cell, tissue and developmentally specific manner (Kornblihtt et al., 1984; Schwarzbauer et al., 1985; Zardi et al., 1987; ffrench-Constant and Hynes, 1989; Carnemolla et al., 1989; Oyama et al., 1989a). Furthermore, it has recently been demonstrated that the splicing pattern of FN pre-mRNA is deregulated in transformed cells and in malignancies (Castellani et al., 1986; Borsi et al., 1987; Vartio et al., 1987; Zardi et al., 1987; CarnemoUa et al., 1989; Oyama et al., 1989b; Oyama et al., 1990). In fact the FN isoform containing the ED-B sequence (B-FN), which is absent in normal adult tissues with some very rare exceptions, has a much greater expression in fetal and tumor tissues (Carnemolla et al., 1989). The ED-B sequence (oncofetal domain) of human FN is a complete type III homology repeat of 91 amino acids which is coded for by a single exon and is either included or excluded from the mature mRNA depending on the splicing pattern of the primary transcript. Here we report on studies of the distribution of TN and B-FN in normal adult tissues and in benign and malignant tumors, as well as on the levels of the B-FN mRNA in cultured fetal and non fetal human fibroblasts originating from different tissues. Materials and Methods
Cultured normal human non-fetal skin fibroblasts (GM-5757, GM-3652, GM-4390, GM-3651,
GM-5659, GM-3377, GM-3440), fetal skin fibroblasts (GM-5386, GM-6113, GM-1603, GM5388), fetal lung fibroblasts (GM-5387, GM-6114, GM-5389) and the SV-40 transformed cell line AG-280, were purchased from NIGMS Human Genetic Mutant Cell Repository (New Jersey, U.S.A.). Fetal lung fibroblast cell lines, MRC-5, IMR-90, WI-38, were obtained from American Type Culture Collection (Rockville, MD, U.S.A.) as well as the SV-40 transformed WI-38VA13 cell lines. The cells were grown in Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Flow Laboratories, Irvine, Scotland, U.K.). Normal human fibroblast cell lines were utilized from the 11th to the 25 th passage. RNA S1 nuclease protection analysis for the determination of the ED-B containing FN mRNA was carried out as described by Borsi et al. (1990). Monoclonal antibodies to TN and to B-FN were obtained and characterized as described by Natali et al. (1990) and Carnemolla et al. (1989), respectively. Normal and neoplastic tissues were obtained from surgical samples. Immunohistochemical analysis were carried out using the APAAP kit System 40 (DAKO Corporation, Carpinteria, CA, U.S.A.) as described by Carnemolla et al. (1989).
Results
Levels of ED-B containing FN mRNA in cultured normal and SV-40 transformed human fibroblasts Through S1 nuclease analysis we have determined the relative amount of ED-B containing FN mRNA in a large panel of cultured normal human fibroblasts from non-fetal skin, fetal skin and fetal lung. The results, reported in Table I, show a great variability in the % of B-FN mRNA. In non-fetal skin fibroblast cell lines we obtained the lowest levels of B-FN mRNA (from 0.55 to 2.1%) while we found the highest levels in fetal lung fibroblasts (from 6.6 to 30.6%). We have also analyzed three pairs of cell lines (GM-5386, GM5387; GM-5388, GM-5389; GM-6113, GM6114), coming from the skin and the lung of the same subject. In all three cases we observed that the percentage of B-FN mRNA in lung fibroblasts
403 was a b o u t 2.5 times higher t h a n that of skin fibroblasts (see T a b l e I). W e also tested the percentage of B - F N m R N A i n W I - 3 8 V A 1 3 a n d A G 280 cell lines, which are the SV-40 t r a n s f o r m e d c o u n t e r p a r t of the W I - 3 8 a n d I M R - 9 0 fetal lung fibroblast cell lines. I n b o t h cases we o b t a i n e d a two-three fold increase i n the relative a m o u n t s of B - F N m R N A i n SV-40 t r a n s f o r m e d cells comp a r e d to that of the u n t r a n s f o r m e d counterparts.
TABLE I Relative abundance of ED-B containing FN mRNA in cultured normal human fibroblasts of different origin Cell lines
Age of donor
Tissue of origin
B-FNmRNA (%) ( 5: S.E.)
GM-5757 7 yr skin GM-3652 24 yr skin GM-4390 23 yr skin GM-3651 25 yr skin GM-5659 14 mo skin GM-3377 19 yr skin GM-3440 20 yr skin GM-5386 20 fw skin GM-6113 20 fw skin GM-1603 12 fw skin GM-5388 20 fw skin GM-5387 20 fw lung GM-6114 20 fw lung GM-5389 20 fw lung MRC-5 14 fw lung IMR-90 16 fw lung WI-38 12 fw lung AG-280 (SV-40transformed IMR-90) WI-38VA13(SV-40 transformed WI-38)
0.55 + 0.1 0.8 + 0.04 0.8 +0.08 1.2 +0.05 1.5 +0.15 2.0 + 0.1 2.1 5:0.4 2.5 5:0.3 2.6 + 1.1 4.4 5:0.1 6.5 5:2.3 6.6 + 0.1 6.8 5:1.3 17.2 5:1.9 17.5 + 2.4 19.2 5:1.9 30.6 5-1.4 42.5 5:3.4 92.1 5:2.4
Expression of B-FN in human normal adult tissues and tumors W e have previously reported that the B - F N isoform, while b e i n g restricted o n l y to synovial cells a n d to some vessels of the ovary i n n o r m a l adult h u m a n tissues, has, o n the contrary, a m u c h greater expression i n fetal a n d m a l i g n a n t t u m o r tissues ( C a r n e m o l l a et al., 1989). S u b s e q u e n t exp e r i m e n t s have c o n f i r m e d a n d furthered these resuits. Fig. 2 shows that while the B - F N isoform is present i n the s t r o m a of a l u n g s q u a m o u s
HOMOLOGY: typel D typell 0 typelll [ ]
I,~DFORMS :
ONCOFETAL DOMAIN (BC- I )
ONCOFETAL EPITOPE (FDC-6)
EDI-B
ED-A
~nTCS
NH~
COOH .
DOMAINS :
.
1
INTIBRACTION$ : heparin DNA fibrin
.
.
2
.
3
gelatin heparin DNA
.
.
.
.
.
.
4
5
cell heparin
heparin DNA
6
fibrin
DNA
Fig. 1. Model of the domain structure of a subunit of human fibronectin. The ED-B oncofetal domain recognized by the Mab BC-1 (Carnemolla et al., 1989) and the oncofetal epitope recognized by the Mab FDC-6 (Matsuura et al., 1985) are indicated. The figure also indicates the internal homologies, the major macromoleculesinteracting with the various FN domains and the possible isoforms generated by alternative splicing of the sequences IIICS, ED-A and ED-B.
4{)4
14
I
i/2
EGF-like repeats
FIBRINOGEN LIKE SEQUENCE,
FN-LIKEREPEATS
II
I
~I~
!{~i:!!~ ~. :!~! :.~!'~!~i~]~:.l~:~[]]:.(~J
Fig. 3. Model of the domain structure of a human TN subunit. The expression of the FN-like repeats 6-12 (dotted) is regulated by alternative splicing of the pre-mRNA.
c a r c i n o m a , it is u n d e t e c t a b l e in n o r m a l lung. F u r t h e r m o r e Fig. 2 shows a histological section o f a lung alveolar a d e n o c a r c i n o m a , which c o n t a i n s b o t h a t u m o r a l a n d a n o r m a l p a r t o f the lung. It is p o s s i b l e to see that the B - F N i s o f o r m is p r e s e n t o n l y in the s t r o m a of the t u m o r while, in the s t r o m a of the n o r m a l lung, it is u n d e t e c t a b l e . I n a b r e a s t f i b r o a d e n o m a section, even t h o u g h b o t h the epithelial a n d the s t r o m a cells are in active proliferation, n o B - F N staining is d e t e c t a b l e (Fig. 2).
Distribution of TN in normal adult tissues and in benign and malignant tumors Fig. 3 shows a schematic r e p r e s e n t a t i o n o f the d o m a i n structure of h u m a n T N , showing the Epid e r m a l G r o w t h F a c t o r - l i k e , the F i b r o n e c t i n - l i k e a n d the F i b r i n o g e n - l i k e sequences. Fig. 4 shows the presence of T N in n o r m a l h u m a n skin. It is p o s s i b l e to observe a massive p r e s e n c e of T N in the p a p i l l a r y d e r m a a n d it is f u r t h e r m o r e e v i d e n t that the vessels p r e s e n t in the d e r m a are also
intensively stained. I n n o r m a l b r e a s t tissue we f o u n d o n l y small a m o u n t s of T N , a n d m a i n l y in the vessels. M a c k i e et al. (1987) have suggested that T N c o u l d b e a m a r k e r for b r e a s t m a l i g n a n c y : we have s t u d i e d a n e l e v a t e d n u m b e r of m a l i g n a n t b r e a s t t u m o r s and, i n d e e d , we have f o u n d elevated a m o u n t s of T N in a l m o s t all m a l i g n a n t t u m o r s ( N a t a l i a n d Z a r d i , 1989; N a t a l i et al., 1990). In fact, in Fig. 4, showing a histological section of an invasive b r e a s t c a r c i n o m a which c o n t a i n s b o t h t u m o r a l a n d n o r m a l b r e a s t structures, it is possib l e to observe t h a t o n l y the t u m o r s t r o m a c o n t a i n s T N while the s t r o m a o f n o r m a l b r e a s t tissue does n o t show a n y d e t e c t a b l e staining. However, we f o u n d elevated a m o u n t s of T N even in benign t u m o r s ( N a t a l i et al., 1990; N a t a l i et al., s u b m i t t e d for p u b l i c a t i o n ) . I n fact, in a histological section o f a b r e a s t f i b r o a d e n o m a - a localized n o d u l a r h y p e r p l a s i a of g l a n d u l a r a n d s t r o m a tissues - it is p o s s i b l e to observe a c o n s i d e r a b l e presence of T N in the s t r o m a of the f i b r o a d e n o m a . O n the contrary, in a n a t y p i c a l d u c t a l b r e a s t h y p e r p l a s i a (a
Fig. 2. Study of distribution of the B-FN isoform by indirect immunoenzyrnatic staining using the Mab BC-1. The Mab BC-1 does not react with normal lung structures, but shows a very strong reaction with the stroma of a squamous lung carcinoma. In a lung histological section including both a portion of normal tissue and a portion of an alveolar adenocarcinoma, it is possible to see, on the right, that the stroma of the tumor shows presence of B-FN while, on the left, the stroma of the normal tissue does not show any detectable B-FN. In a breast fibroadenoma histological section, even though the stromal elements are in active proliferation, no B-FN is detectable. Bar, 10 ram. Fig. 4. Study of the distribution of TN by indirect immunoenzymatic staining. In a normal human skin histological section it is possible to see an intensive staining in the papillary derma and in the vessels. In a breast histological section including both a portion of normal tissue and a portion of an invasive breast carcinoma it is possible to see, on the left, that only the tumor stroma shows presence ofTN while the stroma of the normal tissue, on the right, does not show any detectable TN. In a histological section of a breast fibroadenoma it is possible to observe an intense staining of the stroma of the lesion, while the pseudo-capsula (on the fight), made up of mature connective tissue, shows a very light staining. In a histological section of an atypical ductal breast hyperplasia the stroma does not show any staining while some staining is visible within the epithelial cells. Bar, 10 mm.
Lung s q u a m o u s c a r c i n o m a
Normal lung
L u n g alveolar a d e n o c a r c i n o m a
Fig. 2.
Normal skin
Breast flbroadenoma
Breast flbroadenoma
Invasive b r e a s t c a r c i n o m a
Fig. 4.
Atypical ductal b r e a s t hyperplasia
406 pre-neoplastic lesion, defined as such since a high percentage develops into invasive carcinoma) it is possible to see that the stroma does not present any detectable TN, while a light staining within the epithelial elements is visible (Fig. 4). In these lesions, while there is a proliferation of atypical epithelial elements, there is no proliferation of stromal cells.
Discussion
Through $1 nuclease protection analysis we have determined the relative amount of B-FN mRNA in a panel of human fibroblast cell lines purchased directly from the American Type Culture Collection and the NIGMS Human Genetic Mutant Cell Repository. Among the 17 normal human fibroblast cell lines, we found a great variability in the relative amount of B-FN mRNA. We found very low levels in non-fetal skin fibroblasts (average: 1.28) and higher levels in fetal lung fibroblasts (average: 16.32). We also found differences between skin and lung fibroblasts obtained from the same subjects. These data are in agreement with in vivo studies. In fact, B-FN is undetectable in adult skin while it is present in large amounts in fetal lung (Carnemolla et al., 1989). Furthermore, SV-40 transformation induces a considerable increase in the expression of the B-FN. We have previously reported that the WI-38 cell line produces very low amounts of B-FN. The discrepancies with our present data (see Table I) may possibly be due to the fact that in our previous experiments (Zardi et al., 1987; Carnemolla et al., 1989) this cell line was not directly obtained from the American Tissue Culture Collection. Although establishing the splicing pattern of the ED-B sequence in different cell lines could be of some relevance, the study of the expression of this sequence in vivo in different normal and pathological tissues is of greater importance. By using the Mab BC-1, which recognizes an epitope within the ED-B sequence (Carnemolla et al., 1989), we have confirmed that the B-FN isoform could be considered a tumor marker. In particular, we observed large amounts of B-FN in a higla percentage of lung and intestinal tumors (Zardi et al.,
in preparation). Another Mab, FDC-6, has been reported to recognize an oncofetal isoform of FN (Matsuura and Hakomori, 1985). This epitope is localized within the IIICS sequence (see Fig. 1) and is composed of an oligosaccharide linked to a hexapeptide by O-glycosilation (Matsuura et al., 1989). Thus, the oncofetal epitope recognized by the Mab FDC-6 originates from an altered glycosilation of FN in fetal tissues and tumors and it is not related to the ED-B sequence. Loridon-Rosa et al. (1990) reported that this oncofetal FN isoform is undetectable in normal breast and in benign breast tumors, while it is present in 60% of breast carcinomas and is hnked with intermediary or high malignancy grades. It would thus be interesting to compare the patterns of distribution of the oncofetal B-FN and of the oncofetal FN isoform recognized by the Mab FDC-6 in different tissues. Our studies on TN have demonstrated that the distribution of this glycoprotein in normal adult tissues is less restricted than what has previously been reported (Natali et al., in press; Natali et al., submitted for publication). Furthermore, in the case of breast tumors, our studies seem to indicate that TN, more than a marker for malignant tumors, is a marker of connective element proliferation. In fact, we did not detect any TN in the stroma of pre-neoplastic lesions in which only atypical epithelial elements proliferate. We did find, however, large amounts of TN in fibroadenoma, a benign tumor in which there is proliferation of stromal cells in addition to epithelial cells. It was previously demonstrated that, in cultured human fibroblasts, TGF-fl modulates the splicing of the FN primary transcript, increasing the relative amounts of B-FN (Borsi et al., 1990), and increases the production of TN as well (Pearson et al., 1988). These data suggest that the presence of B-FN and TN in tumor stroma may be a consequence of a paracrine effect by which tumor cells, by releasing growth factors, induce the surrounding cells to organize a stroma more adequate to sustain tumor growth. In fact, a number of studies have suggested that the tumor stroma represents a more "permissive environment" for tumor growth compared to the stroma of normal tissues (van den Hooff, 1988, and references
407
therein), and that TGF-fl may trigger its formation (Sieweke et al., 1990). Future studies should be directed towards establishing whether TN and the tumor-associated FN isoforms functionally contribute to the generation of this permissive environment.
Acknowledgements This study has been partially supported by AIRC funds. We are indebted to Mr Thomas Wiley for manuscript revision and to Prof. L. Santi for his support and encouragement.
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