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. 2009 Sep 1;106(35):14884-9.
doi: 10.1073/pnas.0902042106. Epub 2009 Aug 17.

SIP1 protein protects cells from DNA damage-induced apoptosis and has independent prognostic value in bladder cancer

A Emre Sayan  1 , Thomas R Griffiths, Raj Pal, Gareth J Browne, Andrew Ruddick, Tamer Yagci, Richard Edwards, Nick J Mayer, Hasan Qazi, Sandeep Goyal, Serena Fernandez, Kees Straatman, George D D Jones, Karen J Bowman, Alexandra Colquhoun, J Kilian Mellon, Marina Kriajevska, Eugene Tulchinsky
Affiliations

SIP1 protein protects cells from DNA damage-induced apoptosis and has independent prognostic value in bladder cancer

A Emre Sayan et al. Proc Natl Acad Sci U S A. .

Abstract

The epithelial-mesenchymal transition (EMT) contributes to cancer metastasis. Two ZEB family members, ZEB1 and ZEB2(SIP1), inhibit transcription of the E-cadherin gene and induce EMT in vitro. However, their relevance to human cancer is insufficiently studied. Here, we performed a comparative study of SIP1 and ZEB1 proteins in cancer cell lines and in one form of human malignancy, carcinoma of the bladder. Whereas ZEB1 protein was expressed in all E-cadherin-negative carcinoma cell lines, being in part responsible for the high motility of bladder cancer cells, SIP1 was hardly ever detectable in carcinoma cells in culture. However, SIP1 represented an independent factor of poor prognosis (P = 0.005) in a series of bladder cancer specimens obtained from patients treated with radiotherapy. In contrast, ZEB1 was rarely expressed in tumor tissues; and E-cadherin status did not correlate with the patients' survival. SIP1 protected cells from UV- and cisplatin-induced apoptosis in vitro but had no effect on the level of DNA damage. The anti-apoptotic effect of SIP1 was independent of either cell cycle arrest or loss of cell-cell adhesion and was associated with reduced phosphorylation of ATM/ATR targets in UV-treated cells. The prognostic value of SIP1 and its role in DNA damage response establish a link between genetic instability and metastasis and suggest a potential importance for this protein as a therapeutic target. In addition, we conclude that the nature of an EMT pathway rather than the deregulation of E-cadherin per se is critical for the progression of the disease and patients' survival.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Cancer-specific survival of patients with muscle invasive TCC treated with radical radiotherapy according to immuno-positivity of (A) E-cadherin; (B) ZEB1; (C) SIP1. Note that SIP1 positivity, but not E-cadherin status or ZEB1 levels predict the outcome of the disease. (D) Proportion of tumors with a lack of response to radiotherapy divided into groups according to SIP1 or E-cadherin immunopositivity. SIP(-), no SIP1-positive cells detected in the sample; SIP(+/−), samples mostly negative, but with a few areas with faintly stained cells; SIP1(+), samples with 20–50% cells having strong nuclear staining; SIP1(++), >50% cells with strong nuclear staining. E-cad(+), membranous localization of E-cadherin throughout the whole section; E-cad(-, -/+), E-cadherin staining is absent either completely or focally. Examples of staining patterns are available as SI Methods (Fig. S2). Dotted line shows a percentage of nonresponding tumors in the whole series (n = 56). The data demonstrate a trend toward a lack of response to radiotherapy in patients with SIP1-positive tumors.
Fig. 2.
Fig. 2.
ZEB family members are differentially expressed in cultured cancer cells. (A) Transcription of genes coding for cadherins and ZEB family members was analyzed in bladder cancer cell lines by semi quantitative RT-PCR. GAPDH was used as an internal control. (B) Western blot analysis of the expression of ZEB proteins and cadherins in bladder cancer cell lines. A H1299 lung adenocarcinoma lysate was used as a positive control. To control for equal loading blots were probed with anti-α-tubulin antibody. (C) Protein expression of SIP1, ZEB1, and E-cadherin in a panel of human cancer cell lines. Note the correlation of the presence of ZEB1 and absence of E-cadherin. SIP1 expression was only detected in H1299, HOS, and SaOS-2 cell lines.
Fig. 3.
Fig. 3.
SIP1, but not a dominant negative E-cadherin mutant (Ec1WVM), protects RT112 cells from UV-induced apoptosis. (A) Ectopic expression of SIP1 and Ec1WVM in RT112 cells. RT112/SIP1 cells were maintained with or without DOX for 48 h. Cells were immunostained with an anti-SIP1 antibody. RT112/Ec1WVM cells were stained either with anti-myc antibody detecting mutant E-cadherin or with the anti-Ecadherin antibody recognising endogenous, but not mutant, E-cadherin (clone C20820; BD Biosciences). Cells were counterstained with DAPI; and merged images are presented. (B) SIP1, but not Ec1WVM reduced the extent of DNA fragmentation in UV-treated cells. RT112/SIP1, parental RT112/rtTA, and RT112/Ec1WVM were UV-treated (80 mJ/cm2) to induce apoptosis. RT112/SIP1 and RT112/rtTA cells were maintained with or without DOX for 48 h before UV treatment. Cells were harvested, stained for DNA and analyzed by flow cytometry for subG1 DNA content; the percentage of subG1 cells in each population is shown in the bar chart. The results show mean ± SD of triplicate experiments. In some experiments DOX-treated RT112/SIP1 cells were stained for SIP1, the fraction of SIP1-positive cells was gated and compared with the untreated and ungated cells. Flow cytometry profiles show an example of decreased apoptosis in UV-treated cells gated for SIP1. (C and D) Analysis of PARP and pro-caspase 3 cleavage in RT112/SIP1, RT112/rtTA and RT112/Ec1WVM cells. Cells were treated as described in B; and expression of caspase 3 and PARP was analyzed by western blotting (C) or immunofluorescence (D). Blots were probed with an anti-α-Tubulin antibody to control for equal loading. Note decreased cleavage of caspase 3 and PARP in cells expressing SIP1.
Fig. 4.
Fig. 4.
SIP1 has a G1 arrest-independent antiapoptotic activity in A431 cells. (A) Flow cytometry analyses of A431 cells with DOX-regulated expression of SIP1, SIP1+cyclin D1 (clone A431/SIP1/cyclD1–2), SIP1 mutant (SIP1ZFmut), or Ec1WVM. Cells with DOX-regulated expression of indicated proteins were maintained with or without DOX for 48 h before UV treatment (60 mJ/cm2). 6 h after irradiation, the cells were harvested and analyzed by flow cytometry. The diagram shows the results (mean ± SD) of four independent experiments. (B) UV-induced PARP and caspase 3 cleavage in different A431 clones. DOX-treated and untreated cells were exposed to UV and, at the indicated time points, cells were harvested and lysed. The expression of PARP, pro-caspase 3, SIP1, SIP1ZFmut, and Ec1WVM was analyzed by western blotting. Anti-α-tubulin antibody was used to confirm equal loading.
Fig. 5.
Fig. 5.
SIP1 inhibits phosphorylation of ATM/ATR substrates without affecting the degree of DNA damage. (A) RT112/SIP1 and A431/SIP1 cells were maintained with or without DOX for 48 h, irradiated with 80 or 60 mJ/cm2 UV respectively, incubated for 2.5 h, fixed and double-stained for SIP1 and γ-H2AX. (B) Western blot analysis of phosphorylated ATM/ATR substrates in SIP1-expressing or nonexpressing A431 cells before and 2 h after UV treatment. (C) A431/SIP1 or RT112 cells were cultured with or without DOX for 48 h and treated with different UV doses as indicated. A denaturing comet assay was performed and DNA was stained and examined by fluorescence microscopy as described in SI Methods section. The percentage of DNA in the tail of the comet (% tail DNA) was calculated for each cell by the Komet Analysis software. Results are means ± SD of the four gels. Images show examples of UV-treated and -untreated nuclei.
Fig. 6.
Fig. 6.
Scheme depicting effects of SIP1 and an E-cadherin dominant-negative mutant on cell fate-regulating pathways. SIP1 and Ec1WVM activate cell motility, morphological EMT, and expression of vimentin. SIP1, but not Ec1WVM, affects cell fate by regulating cell proliferation, senescence, and DNA damage-induced apoptosis.
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