Downregulation of Sef, an inhibitor of receptor tyrosine kinase ... - Nature

Oncogene (2007) 26, 6093–6098

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Downregulation of Sef, an inhibitor of receptor tyrosine kinase signaling, is common to a variety of human carcinomas S Zisman-Rozen1,6, D Fink1,6, O Ben-Izhak2, Y Fuchs1, A Brodski3, MH Kraus4, J Bejar5 and D Ron1 1 Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel; 2Department of Pathology, Rambam Medical Center, Haifa, Israel; 3Department of General Surgery, Bnai-Zion Medical Center, Haifa, Israel; 4Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA and 5Department of Pathology, Bnai-Zion Medical Center, Haifa, Israel

Carcinomas are tumors of epithelial origin accounting for over 80% of all human malignancies. A substantial body of evidence implicates oncogenic signaling by receptor tyrosine kinases (RTKs) in carcinoma development. Here we investigated the expression of Sef, a novel inhibitor of RTK signaling, in normal human epithelial tissues and derived malignancies. Human Sef (hSef) was highly expressed in normal epithelial cells of breast, prostate, thyroid gland and the ovarian surface. By comparison, substantial downregulation of hSef expression was observed in the majority of tumors originating from these epithelia. Among 186 primary carcinomas surveyed by RNA in situ hybridization, hSef expression was undetectable in 116 cases including 72/99 (73%) breast, 11/16 (69%) thyroid, 16/31 (52%) prostate and 17/40 (43%) ovarian carcinomas. Moderate reduction of expression was observed in 17/186, and marked reduction in 40/186 tumors. Only 13/186 cases including 12 low-grade and one intermediate grade tumor retained high hSef expression. The association of hSef downregulation and tumor progression was statistically significant (Po0.001). Functionally, ectopic expression of hSef suppressed proliferation of breast carcinoma cells, whereas inhibition of endogenous hSef expression accelerated fibroblast growth factor and epidermal growth factor-dependent proliferation of cervical carcinoma cells. The inhibitory effect of hSef on cell proliferation combined with consistent downregulation in human carcinoma indicates a tumor suppressor-like role for hSef, and implicates loss of hSef expression as a common mechanism in epithelial neoplasia. Oncogene (2007) 26, 6093–6098; doi:10.1038/sj.onc.1210424; published online 9 April 2007 Keywords: Sef; RTK; cancer

Introduction Growth factor-mediated signaling by receptor tyrosine kinases (RTKs) is essential in development and multicellular communication of metazoans. Physiologically, RTK activity is tightly controlled, whereas deregulation results in sustained signaling and malignant transformation (Blume-Jensen and Hunter, 2001). Although extensive evidence implicates oncogenic RTK activation by overexpression, genetic alterations and autocrine ligand expression in human cancer, less is known about abrogation of negative regulatory constraints. Sef (similar expression to FGF genes), originally identified in zebrafish, is a novel inhibitor of RTKmediated signaling. It encodes a type I transmembrane protein (prototypical Sef) that is conserved throughout vertebrate evolution (Furthauer et al., 2002; Tsang et al., 2002; Harduf et al., 2005). Previous studies indicated that human Sef (hSef) encodes different isoforms including the prototypical and a cytosolic isoform, designated hSef-a and hSef-b, respectively (Preger et al., 2004). Both isoforms inhibited the Ras/MAPK pathway, and hSef-a also inhibited PI-3K signaling (Xiong et al., 2003; Yang et al., 2003; Preger et al., 2004; Torii et al., 2004, Ziv et al., 2006). Proteins in these pathways are known to be subverted by oncogenic mutations in a variety of human cancers (Blume-Jensen and Hunter, 2001; Thompson and Lyons, 2005). Given the inhibitory effect of hSef on RTK signaling, it is conceivable that its loss of function leads to unchecked cell proliferation in malignancy. Here we investigated whether loss of hSef expression is common to human carcinomas, and determined the biological effect of hSef upregulation and downregulation in tumor cells. Our results provide compelling evidence that hSef may function as a tumor suppressor.

Results and discussion Correspondence: Dr D Ron, Department of Biology, Technion-Israel Institute of Technology, Technion city, Haifa 32000, Israel. E-mail: dinar@ tx.technion.ac.il 6 These two authors contributed equally. Received 14 July 2006; revised 1 February 2007; accepted 5 February 2007; published online 9 April 2007

Breast cancer is the most common malignancy and the second leading cause of cancer deaths in women (Jemal et al., 2006). To assess hSef levels in breast cancer, we initially investigated its expression in normal breast

hSef downregulation in human carcinomas S Zisman-Rozen et al

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E

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G

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Figure 1 hSef expression in normal human breast tissues (A–C). RT–PCR analysis of hSef transcripts and glyceraldehyde-3phosphate dehydrogenase (GAPDH) control in normal human breast tissues (A). RNA extraction, PCR conditions and primers were described elsewhere (Preger et al., 2004). PC: positive control for amplification using hSef-a, hSef-b or GAPDH vectors as templates; NT: no template. (B and C) hSef expression pattern. Probe preparation and RNA in-situ hybridization conditions have previously been described (Sher et al., 2006). The hybridization probe spanned nucleotides 1606-2208 of the hSef common coding region (accession # AY489047). Normal lobule (B); higher magnification of intra-lobular ducts (C). Summary of hSef expression levels in breast carcinomas relative to normal ductal epithelium (D). Sixty-two carcinomas from tissue microarrays (Cybrdi, Gaithersburg, MD, USA), 37 carcinoma and nine normal breast paraffin-embedded tissue specimens were analysed by ISH. Tissue morphology was confirmed by hematoxylin and eosin staining. Thirty-two tumors contained morphologically normal tissue adjacent to the tumor region, and remaining tumors contained stromal fibroblasts/endothelial cells with hSef levels comparable with those of normal breast tissue: Ten of 13 noninvasive carcinoma cases were DCIS, Three of 10 low-grade noninvasive tumors were papillary carcinoma, one with negative and two with moderate hSef signal. Tumor grading was according to World Health Organization criteria (Tavassoli and Devilee, 2003). Signal intensity was examined microscopically, and further quantified using ‘Tina’ software. Staining intensity as compared with normal epithelium (scored very strong) was strong (B50–75% positive), moderate (B20–50%), weak or low (o20%) and negative (o5%). (a) Two cases displayed very strong hSef signal; (b) three cases with heterogeneous expression (F). hSef expression in clinical breast biopsies (E–I). Coexisting breast hyperplasia (red arrow) and low-grade DCIS (black arrow) exhibiting nearly normal hSef levels (scored very strong, E); heterogeneous hSef expression in low-grade, well-differentiated invasive carcinoma. Arrows point to areas of solid growth (red) or preserved (black arrow/inset) ductal structure (F); invasive breast carcinomas with low hSef Expression in infiltrating ductal carcinoma grade I (G); negative hSef signal in grade II (H) and grade III (I) infiltrating ductal carcinoma. Inset depicts normal ducts within the same tumor. Arrow in (H) points to hSef-positive normal duct surrounded by negative cancer tissue. Counterstaining with Meyer’s hematoxylin. Bars, 100 mm.

Oncogene


6095 Table 1 hSef expression in ovarian, prostate and thyroid tumors Expression level Tumors histological type Ovarian Serous papillary adenocarcinoma/grade I Grade II Grade III

N

Strong (%) 4 10

Othersb Summary of primary ovarian carcinomas Metastasis from other organs

18 8 40 (100%) 10 (100%)

Thyroid Papillary carcinoma Follicular carcinoma Summary of thyroid carcinomas

11 5 16 (100%)

Prostate BPH Prostatic adenocarcinoma grade GG6 GG7 GG8–10 Summary of adenocarcinoma cases

Moderate

Low

Negative

3

3 4

3

1 1 5 (12.5 %) 1 (10%)

5 4 16 (40 %) 1 (10%)

12 2 17 (42.5 %) 8 (80%)

1 1 (6%)

7 4 11 (69%)

4

1

2 2 4 8 (26%)

1 5 10 16 (52%)

1

1 2 (5%)

3 3 (19 %)

1 1 (6%)

5 4 10 17 31 (100%)

2 2 (6%)

Heteroa

1 1 3 5 (16%)

Expression levels in tumors were compared with hSef expression in epithelial cells of the corresponding normal tissues which scored very strong. For ovarian tumors, levels are relative to those observed in the normal OSE and fallopian tube epithelium (four cases); for thyroid, relative to expression in follicular cells (seven cases), and for prostate relative to the level observed in morphologically normal glandular epithelium (four cases). Signal intensity score was determined as described in Figure 1 (legend). All tumors except three high-grade prostate carcinoma included in this survey contained normal cells (glandular epithelium, endothelial cells and stromal fibroblasts) with hSef expression levels comparable to those observed in corresponding tissues from healthy donors. The secondary ovarian tumors positive for hSef expression were 2/6 Krukenberg tumors, and the remaining metastatic lesions were three colon and one breast carcinoma. aHeterogonies. bOther ovarian tumors include two cases of lowgrade transitional cell and endometrioid carcinoma (with strong and low staining intensity, respectively), and five cases of high-grade tumors (mucinous with moderate signal, one case each of squamous and clear cell tumors negative for hSef expression and three cases with low signal including two undifferentiated and one small cell carcinoma). Examples for the different staining intensities are shown in Figure 2 and Supplementary data.

tissues. Reverse transcriptase-polymerase chain reaction (RT–PCR) with isoform-specific primer sets indicated that hSef-a is the predominant isoform in normal human breast (Figure 1A). RNA in situ hybridization (ISH) revealed the strongest hSef signal in ductal and lobular epithelial cells, a weaker signal in stromal fibroblasts and endothelial cells, whereas vascular smooth muscle cells, myoepithelium and adipocytes were negative. A sense probe yielded no signal (Figure 1B and C, data not shown). hSef expression in 101 breast tumors (two benign lesions, 13 noninvasive and 86 invasive carcinomas) was compared with expression in normal breast tissues (nine healthy donors and 32 matched normal epithelia) using ISH (Figure 1). hSef expression levels were retained in ductal epithelial cells of two benign lesions and 6/13 noninvasive carcinomas (Figure 1D and E). Only 2/13 noninvasive carcinoma cases [high-grade ductal carcinoma in situ (DCIS); low-grade papillary carcinoma] lacked hSef expression. By contrast, hSef expression was lost in 70/86 invasive carcinomas including 3/13 grade I, 46/50 grade II and 21/23 grade III tumors (Figure 1D, H and I). Uniformly weak expression was observed in 10/86 cases (Figure 1G) and heterogeneous expression in 3/86 cases of grade I well-differentiated invasive carcinoma. In these tumors, very low hSef signal was associated with areas of solid growth and strong signal

in intraductal cancer cells (Figure 1F). Altogether, these results indicated a strong association between loss of hSef expression and tumor invasion. To determine whether deregulated hSef expression is common in epithelial neoplasia, we subsequently analysed hSef mRNA levels in ovarian, prostate and thyroid carcinomas. Eighty-seven primary carcinomas (40 ovarian, 31 prostate and 16 thyroid), five cases of benign prostatic hyperplasia (BPH) and 10 secondary ovarian tumors (nine gastrointestinal and one breast) were analysed for hSef expression by ISH (Table 1). Epithelial ovarian cancer (EOC) represents the majority of malignant ovarian tumors in adult women, with the highest mortality from gynecologic tumors. Ninety-five percent of EOC originate from the ovarian surface epithelium (OSE), whereas 5% are metastases from other organs (Bell, 2005). hSef transcripts were readily detected in normal OSE, and the fallopian tube epithelium (n ¼ 4), identifying the source of hSef-a detection in normal ovary by RT–PCR (Preger et al., 2004). Among 40 primary tumors analysed, 32 were serous carcinomas, the most common ovarian malignancy, and eight were EOC of lower incidence (Kosary, 1994; Bell, 2005). hSef was downregulated in 33/40 primary EOC including 16 with low expression and 17 negative cases. Moderate expression was observed in five EOC (three grade II and two grade III) and strong in Oncogene


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A a

c

e

b

d

f

B a

c

e

b

d

f

Figure 2 hSef Expression in normal ovarian epithelium and ovarian carcinoma (A). hSef expression in normal (OSE (a)) and epithelial lining of the fallopian tube (FT (b)). hSef Expression in ovarian serous papillary cystic adenocarcinoma with strong hSef signals in grade I (c); moderate and low in grade II (d and e); and negative in grade III (f). Inset shows stromal fibroblasts strongly stained for hSef transcripts. Bars, 25 mm (a) and 100 mm (b–f). Ovarian carcinoma was graded according to Federation of Gynecology and Obstetrics (FIGO) grading system (Kosary, 1994). Tissue microarrays containing 50 cancer cases and normal ovaries from four individuals were screened. hSef expression in normal thyroid gland and thyroid carcinoma (B). High hSef expression in follicular cells of normal thyroid gland (a and b). Strong or moderate hSef levels in two cases of low-grade papillary carcinoma (c and d). In (d), note negative staining on the bottom right and moderate hSef signal in areas of higher differentiation (top left). A high-grade papillary carcinoma negative for hSef expression (f) with a small region containing a mixture of glands with well-preserved structure stained very strongly (e, black arrow) and papillary structures stained moderately for hSef transcripts (e, red arrow). Bars ¼ 25 mm (a and b); 100 mm (c–f). Counterstaining with hematoxylin (e and f). Staining intensities were scored as described in the legend to Figure 1.

two low-grade tumors (Figure 2A and Table 1). Eight out of 10 metastases were negative for hSef expression (Table 1). Analogous to the observations in breast and ovarian cancer, hSef was downregulated in thyroid and prostate cancer. Thyroid carcinoma is the most common malignancy of the endocrine system, and the majority of tumors originate from the follicular cells of the thyroid gland. These cells give rise to papillary and follicular carcinoma (Gimm, 2001). hSef signal was detected in the follicular cells of normal thyroid (n ¼ 7). By contrast, hSef expression was reduced in 13/16 Oncogene

papillary and follicular carcinomas with 11 being negative. Strong hSef expression was detected in the remaining 3/16 cases (two low grade and one intermediate grade papillary carcinoma; Figure 2B, and Table 1). In normal prostate (n ¼ 4), hSef transcripts were readily detected in the peripheral zone epithelium from which the majority of prostatic cancers originate (De Marzo et al., 1999). This cancer is the second leading cause of cancer-related death in men (Jemal et al., 2006). hSef expression was reduced in 26/31 and heterogeneous in 5/31 primary prostate adenocarcinomas. Half of intermediate grade and 59% of high-grade


6097 Table 2 hSef expression in primary carcinomas Carcinoma grade Expression level Very strong/strong Moderate Low Negative

Low (%) N ¼ 41 12 6 16 7

(29) (15) (39) (17)

Intermediate (%) N ¼ 81 1 8 10 62

(1) (10) (12) (77)

High (%) N ¼ 64

Total cases (%) N ¼ 186 N ¼ 13 N ¼ 17 N ¼ 40 N ¼ 116

None 3 (5) 14 (22) 47 (73)

(7) (9) (21.5) (62.5)

Low grade are tumors classified as grade I carcinoma and prostate adenocarcinoma GG6. Intermediate grade includes all grade II tumors, 4/5 cases of follicular carcinoma and prostate adenocarcinoma GG7. High grade are grade III carcinomas and prostate adenocarcinoma GG8-10. Prostate adenocarcinomas with heterogeneous hSef expression were included in the moderate expression level group. w2 test indicated that association of hSef downregulation and tumor progression was statistically significant (Po0.001).

tumors lacked hSef expression, corroborating a recent report by Darby et al. (2006). Markedly reduced expression was observed in 26% of the tumors (8/31) and moderate in 6% (2/31 cases). Among five BPH, four displayed low hSef signal and one was negative (Table 1 and Supplementary data). Overall, hSef expression was downregulated in 93% (173/186) of malignancies originating from breast, ovary, prostate and thyroid. Among these, hSef expression was lost in 62.5% and substantially reduced in 21.5%. The majority of malignancies devoid of hSef expression (96%) were intermediate or high-grade tumors. By contrast, strong hSef expression was almost exclusively associated with low-grade carcinomas, affecting about one-third of these tumors ((12/41 cases), Table 2). The association of hSef loss of expression with tumor grade indicates that hSef downregulation correlates with aggressiveness of the tumor cells in vivo. Extent and kinetics of hSef loss of expression, however, varied among the different tumor types, which may reflect differences in tissue-specific mechanisms for silencing hSef expression. To examine the role of hSef in human tumor cells, we tested the effect of up- and downmodulation of hSef expression on tumor cell growth. Ectopic expression of hSef-a or hSef-b isoforms suppressed clonal expansion of MDA-MB-435 breast cancer cells by 95% relative to empty control vector (Figure 3a). In light of hSef reduction in various human carcinomas and high expression in the corresponding normal epithelial cells, these results indicated a role for endogenous hSef in constraining proliferation. To corroborate this finding, hSef expression in Hela cervical carcinoma cells was knocked down using the pSUPER system for RNA interference. hSef RNA levels were substantially reduced in cells stably expressing hSef short hairpin (shRNA), but not in cells expressing the control shRNA (Ct-sh, Figure 3b). As hSef isoforms inhibit cellular responses to fibroblast growth factor (FGF) and epidermal growth factor (EGF) (Preger et al., 2004; Torii et al., 2004; Ziv et al., 2006), we examined the consequence of its downregulation on the response of Hela cells to these two RTK ligands. As shown in Figure 3c, hSef knock down accelerated Hela cell proliferation by four-fold following exogenous ligand stimulation, and by about two-fold under serum-free conditions (SFM). These results combined with the reported capacity of hSef to inhibit two major pathways for transduction of onco-

a

c

b ng/ml

Figure 3 Ectopic expression of hSef isoforms inhibited the growth of cancer cells (a). MDA-MB- 435 cells were stably transfected with hSef-a and hSef-b expression vectors using DreamFect reagent according to the OZ Biosciences instructions. Colony suppression assay was performed as described previously (Preger et al., 2004). Results are representative of at least three independent experiments. Suppression of hSef expression enhanced cell proliferation (b and c). Three hSef siRNAs (Sef-sh) and control siRNA (Ct-sh) were individually cloned into the pSUPER vector (for sequences see Supplementary data). Hela cells stably expressing a combination of the three Sef-sh siRNA vectors (1.6 mg each) or control Ctsh (5 mg) were generated by co-transfection with pCDNA 3.1 vector for selection in 0.5 mg/ml G418. RT–PCR analysis of hSef in total RNA from transfected cells (b). Amplification was performed with primers common to hSef isoforms as described previously (Preger et al., 2004). PC: positive control for amplification using hSef expression vector as template. NT: RNA template without reverse transcription. Hela cells stably expressing hSef shRNA (shaded bars) or control shRNA (solid bars) were seeded at a density of 25 000 cell/35 mm plate, and subjected to a proliferation assay (c) in SFM alone or along with the indicated growth factors concentrations. The assay was performed as described previously (Shaoul et al., 1995). Error bars indicate standard deviation of three independent experiments.

genic signals (Xiong et al., 2003; Preger et al., 2004; Torii et al., 2004; Ziv et al., 2006), strongly support a role for hSef in negatively regulating cellular growth, and for its loss of function in the neoplastic process. We and others have demonstrated that FGF induce hSef expression in vivo (Furthauer et al., 2002; Tsang et al., 2002; Harduf et al., 2005), and our unpublished results indicate induction of hSef expression by EGF as well. The present findings that hSef is downregulated in tumor types where EGF and FGF signaling is known to be upregulated (Grose and Dickson, 2005; Normanno et al., 2005), suggest that specific mechanisms must abrogate hSef induction by these RTK pathways in cancer cells. Such mechanisms may involve transcriptional repression, DNA methylation or genetic alterations. It is noteworthy that hSef maps to chromosome 3p14, where defined genetic and epigenetic alterations have been Oncogene


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detected in a number of human malignancies including breast cancer (Matsumoto et al., 1997). In summary, the present results provide the first evidence for hSef downregulation as a general characteristic of human cancer, and suggest a role for hSef loss of function in the neoplastic process. Further insights into specific mechanisms underlying hSef loss of expression in human malignancy will advance our understanding of tumor pathogenesis, and facilitate the design of novel therapeutic strategies based on upregulating hSef in human carcinomas.

Abbreviations EOC, epithelial ovarian cancers; GG, Gleason grade; OSE, ovarian surface epithelium; RTK, receptor tyrosine kinase; Sef, similar expression to FGF genes. Acknowledgements The authors thank Sharon Lubinsky-Mink and Dr Orit Goldsmidt for excellent technical assistance. This work was supported by grants from the ICRF (# 2004973), and the Israel Ministry of Health (# 1005314) to Dina Ron.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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