The International Journal of Biochemistry & Cell Biology
Tumor suppressor SMAR1 downregulates Cytokeratin 8 expression by displacing p53 from its cognate site
Introduction
The loss of cellular differentiation is a characteristic feature of malignant transformation that leads to a whole series of structural changes, which enables the tumor cells to detach from the epithelial layer and metastasize. The role of cytoskeletal and cell adhesion molecules in this process is of paramount importance. Intermediary filaments, first described by Holtzer et al. (1976), serve as ubiquitous cytoskeletal scaffolds in both nucleus and cytoplasm of higher metazoans and maintain the structural integrity of cells (Erber et al., 1998). Cytokeratins (CK), being the predominant intermediary filaments involved in structural changes that lead to malignant transformation (Tsubura et al., 1991, Brotherick et al., 1998, Bichat et al., 1997, Willipinski-Stapelfeldt et al., 2005) and provide cellular resistance to apoptosis (Oshima, 2002). Keratins (referred to as cytokeratins here), are subdivided into type I (K9–K20) and type II (K1–K8), that form heteropolymers conferring structural integrity to cells (Kim and Coulombe, 2007). Investigations on loss of function of specific cytokeratins reveal their role in diseases related to mechanistic defects and epidermal fragility.
The CK pair 8/18 is expressed in the differentiation compartment (luminal cells) and along with several others is persistently expressed in carcinomas. Thus, they have proven to be useful cancer markers (Ferrero et al., 1990). Several lines of evidence indicate the role of CK8 in transformation to malignant phenotype, associated with invasiveness and increased metastatic potential (Iwaya et al., 2003, Raul et al., 2004, Korsching et al., 2005). Though the expression pattern and the level of Cytokeratins have been studied in detail, the mechanisms that govern their transcription are unclear. For example, transcriptional deregulation of the CK18 gene has been documented as an effect of increased acetylation of the promoter in SW613-S colon carcinoma cell line (Prochasson et al., 2002, Gunther et al., 1995). Recent reports indicate that in breast tumor cells, the transcription factor SLUG modulates the expression of CK8 and 19 (Tripathi et al., 2005). Studies have also highlighted the role of proto-oncogene AP-1 in regulation of carcinoma-associated K6 (Navarro et al., 1995). It is interesting to note that CK8, known to be an excellent prognostic marker for malignant head and neck carcinomas (Xu et al., 1995) and invasive breast carcinomas (Takei et al., 1995) can be activated by tumor suppressor protein p53 (Mukhopadhyay and Roth, 1996).
This study stems from the fact that the dysregulation of SMAR1 (a nuclear matrix-associated protein) in higher grades of cancer results in increased invasiveness and metastasis. This is attributed to modulation of the major cell migration pathways involving TGFβ and CUTL1 (Singh et al., 2007). Moreover, this protein also cross talks with p53, Cyclin and Rb pathways causing a delay of tumor onset by cell cycle arrest (Chattopadhyay et al., 2000, Kaul et al., 2003, Jalota-Badhwar et al., 2007, Rampalli et al., 2005, Pavithra et al., communicated). We have also demonstrated that the loss of mRNA stability is a major factor that leads to loss of function under hormone responsive conditions (Pavithra et al., 2007). Additionally, the knock down of SMAR1 in B16 F1 melanoma cells has been recently shown to induce phenotypic alterations including loss of cell–cell contacts and acquisition of invasive growth (Singh et al., 2007).
Since the invasiveness of tumor cells is often determined by the profile of their expressed genes, we investigated the gene expression differences upon SMAR1 expression in breast cancer-derived cells. MCF-7 cells derived from the mammary epithelia are estrogen responsive and provide an excellent model for study of adenocarcinomas. Moreover, MCF-7 cells have low levels of endogenous SMAR1 protein. Therefore, cDNA microarray analysis was performed with RNA from MCF-7 cells ectopically expressing SMAR1. Though microarray analysis showed differences in the gene expression pattern of several architectural and intermediary filament genes, the downregulation of Cytokeratin 8 was significant and of special interest. Considering that CK8 is activated by p53 and SMAR1 interacts with p53, we checked the existence of a regulatory network between these proteins in regulating the transcription of CK8 gene. Our findings here point out at the complex regulation of CK8 by cross talk between SMAR1 and p53 following genotoxic stress. Additionally, expression of CK8 and SMAR1 are negatively correlated as shown in normal-metastatic tissue microarray. Therefore, we demonstrate the role of a candidate tumor suppressor protein SMAR1 in regulating the expression of intermediary filament protein CK8, to ensure the normal phenotype of the cells.
Section snippets
Cell culture and transfections
MCF7 (human breast adenocarcinoma cell line, wild type p53 background) and H1299 (human lung cancer cell line, p53 null background) cells were grown in DMEM supplemented with 10% FBS in the presence of 5% CO2 at 37 °C. Transfections were done using 1 μg DNA of Flag-SMAR1/Flag-vector/pCDNA3.1-p53 per 35 mm dish using lipofectamine 2000 (Invitrogen, USA). In case of siRNA treatment, 100 nM of SMAR1-siRNA or scrambled siRNA (Ambion, Austin, TX, USA) were transfected 12 h before treatments. Cells were
SMAR1 alters the expression of cellular architecture maintenance genes
SMAR1 is shown be a potent tumor suppressor gene that delays the onset of B16F1 melanoma in mouse model (Kaul et al., 2003). Further, recent studies by Singh et al., 2007, demonstrated the downregulation of SMAR1 in higher grades of breast cancer associated with a malignant phenotype, related to TGF-β signaling. To assess the phenotypic changes attributed to the over-expression of SMAR1 in breast epithelia derived MCF-7 cells, we generated stable clones of GFP-SMAR1 in this cell line and
Discussion
Understanding the role of cytoskeletal components in determination of cell differentiation is increasingly becoming important in the context of breast cancer progression. To further dissect the role of individual cytokeratins in metastatic process and investigate their prognostic potential, elucidation of the gene control of each of these genes is desired. For example, the low CK18 mRNA and high protein expression is associated with an increased invasive potential in breast cancers. Studies by
Acknowledgements
We thank Dr. Avinash Pradhan, Chief Pathologist, KEM Hospital, Pune for providing the breast cancer samples. LP and SS are recipients of senior research fellowships from University Grants Commission, Govt. of India. KS is a recipient of senior research fellowship from CSIR, Govt. of India.
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