Tumor Suppressive Functions Of Leucine Zipper Transcription Factor Like 1

Background:Chromosomal abnormalities at the 3p21.3 region are frequent and early events in the formation of human tumors of the lung, kidney, head and neck, breast, cervix, and gastrointestinal tract. These abnormalities range from homozygous deletions and loss of heterozygosity to loss of protein expression, suggesting the presence of tumor suppressor genes (TSG) in this region. As TSGs offer opportunities for molecular cancer therapy, there have been intense efforts during the past decades to identify candidate 3p21.3 TSGs and to characterize their biological functions. A critical region of～120 kb in the 3p21.3 centromeric border (also called LUCA region) has been identified and contains nine candidate TSGs:HYAL2, HYAL1, FUS1, RASSF1, BLU, NPRL1,101F6, PL6 and CACNA2DA. The functions of many of these gene products are being elucidated and they seem to be involved in a wide spectrum of biological processes, including cell proliferation, cell cycle kinetics, signaling transduction, ion exchange and transportation, and cell death. The 3p21.3 region also contains other candidate TSGs outside the LUCA region, but they are less well studied. In an effort to discover potential TSGs in the 3p21.3 region, Kiss and colleagues identified and cloned leucine zipper transcription factor-like 1 (LZTFLl) through an elimination test and subsequent genomic sequencing and cDNA cloning. LZTFL1 is located-5 Mb from the LUCA region on the telomeric end of the 3p21.3 region. Northern blot analysis indicates that LZTFL1 mRNA is expressed ubiquitously in both human and mouse. The open reading frame from human and mouse cDNAs revealed a protein of 299 amino acids with a molecular weight of 34.6 kDa. The sequence analysis suggested that LZTFL1 shares 90.6% sequence identity between human and mouse. LZTFL1 contains a basic region, a coilcoil domain, and a leucine zipper domain, it was suggested that LZTFL1 may function as a tumor suppressor. However, the biochemical and genetic evidence of LZTFL1 as a tumor suppressor gene is lacking.The loss of differentiation in cancer cells is often associated with tumor progression, but the underlying causes and mechanisms remain poorly understood. The majority of human solid tumors are carcinomas that originated from various epithelial cell types. Differentiated carcinomas are composed of cohesive polarized epithelial cells connected to one another by intercellular adherens junctions. E-cadherin is the core molecule of adherens junctions. The cytoplasmic tail of E-cadherin is indirectly linked to the actin cytoskeleton through catenins, includingα-andβ-catenin, and other associated proteins. The attachments of E-cadherin to the cytoskeleton, hence associated proteins in the adherens junction, are essential for maintaining the differentiated state of epithelial cells and the apico-basal polarity of the epithelium. Disruption of the adherens junction can generate invasive mesenchymal cells through a process called epithelial-mesenchymal transition (EMT), which converts polarized, immotile epithelial cells to motile invasive mesenchymal cells. EMT has been proposed to be a potential mechanism for carcinoma metastases. Loss of membranous E-cadherin can also increase the cytoplasmic pool ofβ-catenin, which can then translocate to the nucleus and activate genes that promote cell proliferation and EMT.In the present study, we sought to test whether LZTFL1 functions as a tumor suppressor. We asked three experimental questions. First, is LZTFL1 expression downregulated in tumors and does the loss of LZTFL1 expression have any clinical significance? Second, can LZTFL1 gain-of function inhibit tumor growth? Finally, what is the potential mechanism(s) behind LZTFL1 inhibition of tumor cell growth?Methods and Findings:1. The expression of LZTFL1 in human tumors and its clinical significanceIn order to study the biological function of LZTFL1, we first generated and affinity-purified a rabbit polyclonal antibody against LZTFL1. We next surveyed the expression of LZTFL1 in various normal human tissues and their corresponding cancer samples by immunohistochemical analysis of tissue microarrays. Intense LZTFL1 staining was visible in epithelial cells of normal tissues of breast, esophagus, pancreas, stomach, ovary, prostate, lung, colon, thyroid, kidney, bladder, and liver. In almost all the corresponding invasive carcinoma samples, only diffused, low levels of LZTFL1 staining were observed. Analysis of multiple cases in each individual type of normal and matched cancer samples in the tissue microarray showed that LZTFL1 was significantly downregulated in the aforementioned human tumors.To address the clinical significance of downregulation of LZTFL1 in cancers, tissue samples from a cohort of 84 patients diagnosed with the stomach cancer were screened by immunohistochemistry for LZTFL1 expression. Loss of LZTFL1 expression was found to have significant inverse correlations with TNM stages of the tumor and with the number of metastasized lymph nodes, and LZTFL1 expression level correlated significantly with the survival time as well, The overall survival was significantly better for patients with tumors demonstrating moderate or strong LZTFL1 expression than those whose tumors showed negligible or weak expression.2. The biological function of LZTFL1 in vitro and in vivoIn order to determine whether LZTFL1 plays a role in the tumorigenesis, we performed gain-of-function studies to test whether increased level of LZTFL1 expression in tumor cells can inhibit tumor cell growth. We used an inducible expression system to induce LZTFL1 expression through addition of doxycycline (Dox) in cultured Hela-Tet-on cells that constitutively produce the reverse tetracycline transactivator. We found that LZTFL1 has effect on tumor cell growth under anchorage-independent conditions in soft agar assays. A large number of colonies of Hela-Tet-on cells were visible within 4 weeks, The LZTFL1-expressing cells upon addition of Dox showed dramatically reduced number of colonies. As controls, the numbers of colonies in Hela-Tet-on cells with and without Dox, Hela-Tet-on-EGFP cells with and without Dox, and in Hela-LZTFL1-EGFP cells without Dox were similar, suggesting that LZTFL1 indeed specifically inhibited anchorage-independent growth of tumor cells. We also tested whether overexpression of LZTFL1 inhibits the colony formation ability of intestinal epithelial carcinoma cell HT-29 and observed similar inhibitory effect of LZTFL1 in this cell.As downregulation of LZTFL1 in human gastric tumors correlated with tumor metastases, we next investigated a role for LZTFL1 in cell migration. Upregulation of LZTFL1 in Hela-Tet-on-LZTFL-EGFP upon Dox induction significantly reduced the migration properties of Hela cells in Transwell assays, In negative controls, Dox had no effect on migration of Hela-Tet-on cells. The number of cells migrated through the Transwells are similar among Hela-Tet-on cells, Hela-Tet-on-EGFP cells and Hela-Tet-on-LZTFL1-EGFP cell without Dox.To further test whether overexpression of LZTFL1 results in suppression of tumor growth in vivo, we injected subcutaneously Hela-Tet-on and Hela-Tet-on-LZTFL1-EGFP cells into the flank of nude mice. As expected, at the end of 5 weeks, mice injected with Hela-Tet-on cells developed large tumors. The tumor size in mice with Hela-Tet-on-LZTFLl-EGFP cells in the presence of Dox in the drinking water for induction of LZTFL1 were significantly reduced compared to those in the absence of Dox. Our results suggest that LZTFLl inhibited tumor growth significantly in vivo.3. The mechanism of LZTFL1 inhibit tumor cell growthIt has been demonstrated that many TSGs are inactivated in cancer by epigenetic silence induced by aberrant methylation of CpG island in the promoter region of the TSG or by overexpression of histone deacetylases(HDACs). To understand the mechanism of LZTFL1 inactivation in tumor cells, we treated HT-29 cells with 5'-aza-2'-deoxycytidine, a DNA methylation inhibitor, and Sodium butyrate (NaB), a HDAC inhibitor, respectively. No difference of LZTFL1 expression was observed between 5'-aza-2'-deoxycytidine treated and untreated cells whereas NaB treatment increased the level of LZTFL1 expression. Other HDAC inhibitors had similar effects on the upregulation of LZTFL1 expression in HT-29 cells, These results suggest that LZTFL1 is inactivated in HT-29 cells by alterations in chromatin structure.NaB is a naturally-occurring compound in the intestine and induces differentiation of epithelial cells in culture, Upregulation of LZTFL1 in NaB treated HT-29 cells suggests that the expression level of LZTFL1 may be correlated with the differentiation status of the cell. To test this hypothesis in vivo, we stained the mouse small intestine with anti-LZTFL1 antibody along the crypt-villus axis. The intestinal epithelium undergoes constant self-renewing processes. A graded expression of LZTFL1 along the crypt-villus axis was observed with a minimal staining of LZTFLl in the crypt and maximum staining at the apex of the villus. Next, we performed co-localization studies of LZTFL1 with E-cadherin/β-catenin using confocal immunofluorescence microscopy. Expression of LZTFLl overlaps with that of E-cadherin at the plasma membrane in differentiated normal colonic epithelial cells, This co-localization was absent in colorectal carcinomas due to a loss of LZTFL1 protein expression.LZTFL1 and E-cadherin co-localization suggest LZTFL1 may be able to stabilize E-cadherin-mediated adhesion junctions. Indeed, we observed that, when treated with phorbol 12-myristate13-acetate(PMA), a known scatter factor to break down the epithelial tight junction, the LZTFL1 expressing HT-29 cells are more resistant to PMA-induced cell scattering than EGFP-expressing or parental HT-29 cells.Conclusions:Our results indicate that LZTFL1 is a tumor suppressor and loss of LZTFL1 expression has significant clinical outcomes.LZTFL1 expression may serve as an independent prognostic marker for survival outcome of gastric cancer patients. We hypothesize that LZTFL1 may inhibit tumor cell growth by promoting tumor cell differentiation and tumor metastasis through suppression of epithelail-mesenchymal transition. Upregulation of LZTFL1 expression in tumors may have therapeutic value in differentiation therapy against cancer.