Overexpression Of Prostate Apoptosis Response Protein-4 In Colon Cancer Cells Can Inhibit Metastasis By Upregulating E-cadherin Expression

Colon cancer has a five-year survival of 64.7%, and about 50,000 people are expected to die from colon cancer this year. Patients with metastatic colorectal cancer have a significantly worse prognosis, a 12.9% five -year survival. This emphasizes the need for strategies to inhibit the growth and metastases of colorectal cancer. Prostate apoptosis response protein 4 (Par-4) is a pro-apoptotic protein that has been shown to mediate apoptosis in response to stimuli, such as chemotherapeutics and radiation. Recombinant Par-4 protein has been shown to reduce the occurrence of Lewis lung carcinoma metastases in-vivo; however, the mechanism by which Par-4 can inhibit metastasis has not been elucidated. In this study, human colon cancer cell lines SW480 and SW620 were transfected with Par-4 plasmid or anti-Par-4 shRNA, and the effect on metastasis was examined. Par-4 overexpression inhibited cell migration and invasion, while Par-4 knockdown promoted it. Moreover, the morphology of SW620 cells was altered when Par-4 levels were increased. The change was characteristic of a mesenchymal-to-epithelial transition (MET) in these cells. MET can be induced by upregulation of E-cadherin expression, and RT-PCR and Western blot analyses showed that E-cadherin mRNA and protein levels, respectively, were increased in the Par-4 overexpressing cells concomitant with a decrease in vimentin. The results of this study demonstrate the potential of Par-4 in colon cancer therapy, not only in primary tumors but also in metastatic cells. DOI : 10.14302/issn.2471-7061.jcrc-14-574 Corresponding Author: Rosalyn B. Irby, Ph.D., 500 University Drive, Hershey, PA 17033, Tel: 717-5315035, Email: rirby@hmc.psu.edu


Introduction
Although the 5-year survival rates of patients having early stages of colon cancer are higher than 60%, the 5-year survival rate of patients with metastatic colorectal cancer is only 12.9% [1] . Despite increases in length of survival with the combination of targeted agents, including anti-epidermal growth factor receptor and anti-vascular endothelial growth factor agents, with a 5-fluorouracil based regimen, patients with metastases are expected to survive for 2 years. This underscores the need for strategies to inhibit colorectal cancer metastases.
Prostate apoptosis response protein -4 (Par- 4) was first identified in prostate cancer cells that were induced to undergo cell death. Since the report of its identification, it has been shown to play a role in apoptosis in a cell-type-specific manner. Par-4 overexpression is sufficient to induce apoptosis in vitro and in vivo in a myriad of cancer cell types: breast cancer [2,3] , androgen-independent and androgendependent prostate cancer cell line TRAMP, lung cancer, cervical cancer, nasopharyngeal cancer, and melanoma [4] . In other cell types -Jurkat T lymphocytes [5] , androgen-dependent prostate cancer cell line, LNCaP [6,7] , melanoma cells [8] , and renal carcinoma -Par-4 increases susceptibility to pro-apoptotic stimuli, including UV irradiation, serum-withdrawal, ionizing radiation, doxorubicin, and camptothecin. In colon cancer cells, Par-4 overexpression increases apoptosis in response to the chemotherapeutic agent 5-fluorouracil [9] .
Par-4 not only induces cell death in cancer cells, but it may also inhibit their metastasis. This was suggested in a previous study, where mRNA and microRNA microarray analyses on Par-4 overexpressing HT-29 colorectal cancer cells showed that Par-4 altered the expression of genes involved in cell movement, including cell migration and invasion [16] . In addition, Par -4 induces the upregulation of 13 and downregulation of 9 microRNA's. Among the predicted target mRNAs of these dysregulated microRNAs, a significant number are involved in the WNT/β-catenin pathway, a pathway that has been strongly implicated in colon cancer metastasis.
In vivo, recombinant Par-4 protein inhibits the formation of lung nodules by mouse Lewis lung carcinoma cells in a tail vein metastasis model [17] . The goal of this study is to uncover the mechanisms by which Par-4 inhibits metastasis.

Materials and Methods
Cell Culture and Transfection

Results
Par-4 increases Susceptibility of Metastatic SW620 Cells to 5-FU Par-4 expression was increased in the SW620 colorectal cancer cell line by stably transfecting cells with a plasmid vector encoding human Par-4 (Fig 1a and b).

SW620 Cells
The effects of Par-4 overexpression on two key steps of metastasis, migration and invasion, were examined. We performed scratch assays on SW620 cells and quantified the number of cells that migrated into the area of the scratch after 24 hours to examine migration.
Fewer Par-4 overexpressing cells than mock-transfected cells migrated into the scratch area (Fig 2a and b) It is possible that more of the mock-transfected cells appeared to invade through the Matrigel, because increased Par-4 expression reduced cell proliferation. To assess this, we monitored the cell growth of mock-and Par-4 transfected SW620 cells (Fig 2d).

Par-4 induces a Mesenchymal-to-Epithelial Transition in SW620 Cells
Given the novel observations that Par-4 inhibits migration and invasion of metastatic colon cancer cells, the mechanisms behind these effects were investigated.
Par-4 overexpression altered the morphology of SW620 cells (Fig 4a). This is significant, since the SW620 cell line is one that was vimentin levels were reduced (Fig 4b). Par-4 overexpression in SW480 cells also increased the expression of E-cadherin (Fig 4c). These data further support the observation of an MET phenotypic change.    and ZO-1 were all found to be upregulated by Par-4 ( Fig   5b).

Discussion
The potential of Par-4 in cancer therapy has been increasingly appreciated given its ability to induce cell death by itself or in combination with chemotherapeutics and radiation [19] . In this study, we have shown that SW620 cells are sensitized to 5-FU upon ectopic Par-4 expression. This sensitization corroborates our prior findings in HT-29 cells in response to 5-FU and ISC-4 treatment, respectively [9,16,20,21] .
Although it has been shown that recombinant Par-4 can inhibit metastasis [17] , the mechanisms behind this effect have been scarcely elucidated. Furthermore, ectopic Par-4 expression in HT-29 cells was shown to deregulate mRNA's and microRNA's involved in cell migration and motility [16] . Specifically, several target genes of these deregulated miRNA's are part of the Wnt/β-catenin and PI3K/Akt pathways, both of which have been implicated in colon cancer progression and metastasis [22,23] . We have shown in this study that Par-4 can inhibit the ability of colon cancer cell lines, SW480 and SW620, to migrate and invade.
The ability of SW620 cells to migrate and invade was reduced with increased expression of Par-4.
Moreover, the ability to SW480 cells to migrate was also inhibited by Par-4 overexpression, while downregulation of Par-4 resulted in increased migration. One of the mechanisms behind this reduction in migration and invasion may be the Par-4-induced upregulation of Ecadherin. Increased expression of E-cadherin in mammary and prostate epithelial carcinoma cells has been shown to inhibit migration and invasion [24,25] .
Small molecules that can restore E-cadherin expression in SW620 cells have been shown to reduce invasion [26] .
The ability of E-cadherin to inhibit cell migration and invasion has been shown to be independent of its role in mediating cell-cell adhesions, and instead is due to its role in downregulating the β-catenin/TCF pathway [25] . E -cadherin sequesters β-catenin, keeping β-catenin from interacting with actinin-4. On the other hand, in the absence of E-cadherin or when E-cadherin is downregulated, β-catenin has been found to colocalize with actinin-4 in bleb-like membrane protrusions in colorectal cancer cells [27] . Overexpression of actinin-4 has been shown to increase motility of colorectal cancer cells [28] . The mechanism underlying the β-catenin/ actinin-4 complex-induced migration remains to be elucidated. Another mechanism by which E-cadherin can inhibit migration and invasion is due to downregulation of hNanos1. Overexpression of hNanos1 is sufficient to induce invasion in collagen type I gels and increase migration [29] . This may be due in part to the hNanos1induced expression of the matrix metalloproteinase MT1-MMP (membrane type1-matrix metalloproteinase) [30] .
The loss or heterogenous expression of Ecadherin in colorectal cancer tissue samples has been correlated to an advanced clinical stage and liver metastasis [31] . This makes the upregulation of E-  cadherin induced by Par-4 overexpression in SW480 and SW620 cells a significant finding. E-cadherin expression is regulated via different mechanisms [32] . Although the CpG island on the E-cadherin promoter is methylated in a number of cancers [33] , including colorectal cancer [34,35] , this may not be the dominant mechanism of E-cadherin downregulation in colorectal cancer [36] . E-cadherin expression is also inhibited by a number of transcriptional repressors, including members of the ZEB and basic helix-loop-helix (bHLH) families [37] . In this study, we reported the transcriptional upregulation of E-cadherin as a result of Par-4 overexpression. In addition, the expression levels of other tight junction proteins -claudin-1, occludin, and ZO-1 -were upregulated. The mechanisms by which Par-4 regulates the expression of these genes is an area for further study.
Increasing Par-4 levels in SW620 cells was sufficient to alter the cell morphology from a rounded phenotype with minimal cell-to-cell contacts to one with many cell-to-cell contacts, which is characteristic of epithelial cells. Par-4-overexpressing cells had a decreased expression of mesenchymal markers, such as vimentin, and upregulation of the epithelial cell protein, E-cadherin. This is significant, because it suggests an MET transition, the opposite of the epithelial-to-mesenchymal transition (EMT) that primary tumor cells must undergo in order to metastasize. In colorectal cancer, EMT has been observed in cells at the invasive front of primary tumors and lymph node metastases [38] . Characteristics of cells undergoing EMT include the decreased expression of epithelial markers, in particular E-cadherin, and upregulation of mesenchymal markers, like vimentin. A further indication of EMT is the increased nuclear localization of β-catenin. In this study, we report that by increasing the levels of Par-4 in the metastatic cell line, SW620, MET was induced, which is significant in that it helps to resolve a discrepancy in