Functional And Mechanistic Studies Of Tumor Suppressor Gene NDRG2in Response To TGF-β During Colon Cancer Progression

Colorectal cancer is a commonly diagnosed cancer in both men and women, and is thethird most common fatal malignancy and the second leading cause of cancer-related deathin the western world. The etiology of colorectal cancer is multifactorial, and it is widelyaccepted that genetic factors play key roles in the predisposition to cancer developmentand progression, such as Ras/Raf, transforming growth factor (TGF)-β, β-catenin/T-cellfactor (TCF) and p53pathway. Although genetic changes above have been identified atmultiple levels along the multistage carcinogenesis process, the initial factorscorresponding to these changes are still needed to be further elucidated.Transforming growth factor β (TGF-β) is a secreted multifunctional cytokine that regulates a variety of cellular processes. The TGF-β receptor complex activates both theSmad signaling pathway, which includes Smad2, Smad3, and Smad4, and non-Smadsignaling cascades (e.g. PI3K/AKT, p38MAPK, MAPK-ERK, JNK, etc.) to produce thefull spectrum of TGF-β responses. The role of TGF-β signaling in carcinogenesis iscomplicated. Originally named for their transforming activities in in vitro assays, TGF-βnow unequivocally demonstrates both tumor suppressor and oncogenic activities. Thetumor suppressor activities dominate in normal tissue, which are mainly through directlyregulating cell cycle inhibitors such as, p21Cip1and p15INK4B, and cell cycle activatorsc-Myc via transcriptional and post-transcriptional mechanisms. But during tumorigenesis,changes in TGF-β expression and cellular responses tip the balance in favor of itsoncogenic activities, via inducing epithelial-mesenchymal transition (EMT), whichmediated by Fibronectin, Twist, Snail, etc., and finally accelerating tumor invasion andmetastasis. There is considerable genetic evidence that loss of sensitivity to growthinhibition by TGF-β is an important event in colorectal carcinogenesis. Much of theevidence is derived from studies in human colorectal cancers demonstrating inactivatingmutations in genes encoding proteins involved in TGF-β signal transduction, includingSMAD4, SMAD2, and TGFBR2. However, it has also been defined that even therestoration of the impaired TGF-β pathway can not recover the anti-proliferative responseto TGF-β in colorectal cancer cells. Therefore, in order to fully understand the paradoxicaleffect of TGF-β in carcinogenesis, other factors and mechanisms are needed to beuncovered and elucidated.In recent years, a new tumor suppressor gene, NDRG2(N-Mycdownstream-regulated gene2), which belonged to the NDRG family, comprised of fouridentified members (NDRG1-4), has been implicated in the regulation of celldifferentiation and proliferation. The role of NDRG2in cancer was supported by the initialobservation that NDRG2inhibited the proliferation of glioblastoma cells and promoted thedifferentiation of various cell types, including dendritic cells, PC12cells and colorectalcancer cells. Consistent with its potential function as a tumor suppressor, downregulationof NDRG2expression was found in multiple human cancer cell lines and tumors, especially in colorectal carcinomas and the high-risk adenoma precursor lesions. Mostinterestingly, our recent data showed that the decrease of NDRG2expression in primaryhuman CRC was a powerful and independent poor prognosis predictor of colorectalcancers, especially with advanced TNM stage and lymph node metastasis. It is thusexpected to investigate the relevance of NDRG2with the paradoxical roles of TGF-β incolorectal cancer.【Objectives】1. To investigate the paradoxical role of TGF-β during colon cancer progression.2. To explore the underlying mechanisms of NDRG2involved in TGF-β signaling.3. To analyze the epigenetic alterations of NDRG2for the balance of TGF-β function incolon cancer.4. Functional study of NDRG2contributed to EMT and cell proliferation.【Methods and Results】1. Positive correlation of NDRG2expression and Smad2/3phosphorylation levels incolon cancer samples.Immunohistochemistry (IHC) analysis of50pairs of human adjacent normal colonsamples and colorectal tumors revealed that both NDRG2and phosphorylated Smad2/3were highly expressed in the adjacent normal colon tissues in contrast with the colorectaltumor samples. Furthermore, a correlation study determined that NDRG2expression waspositively correlated with phosphorylated Smad2/3expression in these tissue samples,which implicated that NDRG2might be involved in TGF-β downstream signaling.2. TGF-β fluences Sp1, c-Myc, Miz-1and Smad complex to control NDRG2expression.NDRG2was found as one of the intact TGF-β/Smad signaling target genes which wereinduced in TGF-β treated keratinocytes. A cluster of sites located in the core region ofNDRG2promoter was further confirmed the contribution to inducibility by TGF-β inreporter assays. Moreover, we found that Sp1can also play a key role in the positiveregulation of NDRG2in response to TGF-β signals. However, deletion of these Sp1sitesor removal of Sp1regulation by Sp1shRNA or Mithramycin in our studies did not completely abrogate the TGF-β response of the NDRG2promoter, which leads usconsidering other factors. We found that TGF-β induces a rapid reduction in c-Myc levels,depleting the pool of c-Myc that is bound to Miz-1. As a result, TGF-β preventsrecruitment of c-Myc to the NDRG2promoter. Interfering with the c-Myc–Miz-1interaction with a dominant negative Miz-1construct mimics the activating effect ofTGF-β on NDRG2promoter. In combination, we observed that, on one hand, therequirement of c-Myc removal for TGF-β activation of NDRG2and, on the other, theability of Sp1responding to TGF-β to provide a second input for NDRG2activation.3. NDRG2inhibits TGF-β induced EMT in colon cancer cells.To determine the whether NDRG2plays a role in TGF-β inducing EMT in coloncancer cells, we established stable NDRG2over-expression or knockdown transfectants ofHT-29cells respectively. While after TGF-β treatment, HT-29cells transfected withNDRG2prevented cells toward mesenchymal-like change. Moreover, the molecularchanges of NDRG2over-expression against TGF-β stimulation were also observed.Decrease of E-cadherin and induction of vimentin were observed in TGF-β treated controlcells. Furthermore, NDRG2over-expression inhibited the changes of EMT markers(E-cadherin and vimentin) after incubation with TGF-β. Consistent with the protein levels,changes of EMT markers were also confirmed in mRNA level. Properties of metastasiswere then evaluated by migration and invasion assays. The results revealed that NDRG2over-expression not only significantly decreased migratory and invasive potential forHT-29cells in the absence of TGF-β, but also significantly reduced the TGF-β-stimulatedmigration and invasion. Whereas NDRG2knock-down cells mimics the TGF-β-inducedEMT. These studies indicate NDRG2acts to impair migration and invasion and alsoinhibits TGF-β-induced tumor progression by preventing the EMT.4. Reduced NDRG2expression caused by promoter hypermethylation fostered theprogression and metastasis of colorectal cancer.NDRG2exhibited dramatically reduced expression at both the mRNA and proteinlevels in the majority of colon cance cells relative to normal HIEC cells. Moreover,hypermethylation of the NDRG2promoter was detected in most colon cancer cell lines and in75%of the clinical colorectal tumors. In addition, Sp1binding activity was blockedby the hypermethylation of the CpG dinucleotide within its binding site or the adjacentCpG sites. As expected, DNA methyltransferase inhibitor5-aza-2’-deoxycytidine restoredNDRG2expression and promoted the induction of NDRG2by Sp1, which indicates thatNDRG2promoter methylation abrogated the Sp1induction of NDRG2. In the clinicalcolorectal tumors, reduced NDRG2expression was of a relatively higher invasion stagethan preserved NDRG2expression group. Most importantly, reduced NDRG2wassignificantly correlated with both frequent lymph node and distant metastasis, whichstrongly confirms NDRG2as a potent tumor suppressor not only through anti-proliferationeffects but also via the inhibition of invasion and metastasis.5. NDRG2inhibits colon cancer proliferation by regulating Skp2, p27and p21.We firstly used different colon cancer differentiation models (high confluence orNaBT induction) to investigate the inverse expression correlation between NDRG2andSkp2. High level of NDRG2expression during colon cancer differentiation indicates thatNDRG2might influence cell differentiation by regulating Skp2expression. We then foundNDRG2over-expression significantly inhibited Skp2expression in HT-29cells. Furtherstudies showed that NDRG2expression stabilized p27via Skp2repression and increasedp21expression in mRNA levels. Finally, Knock-down p27or p21partly abolishedNDRG2mediated cell growth inhibition in xenograft tumors, which indicates that bothp27and p21are necessary for NDRG2anti-proliferation effects.【Conclusion】Our current study demonstrated for the first time that NDRG2, as a new TGF-βresponsive gene, abrogated TGF-β-induced EMT in colorectal cancer cells. TGF-βupregulated NDRG2through the transcriptional activation mediated by transcription factorSp1and abrogation of repressive c-Myc/Miz-1complex in epithelial cells. Furthermore,aberrant hypermethylation of NDRG2, which could respond to TGF-β growth inhibitionsignaling, abrogated the inhibition effect of NDRG2in TGF-β-induced EMT in colorectalcancers. In addition, NDRG2can also mediate the growth inhibition signaling byregulating Skp2, p27and p21. Together, our data showed that NDRG2, as a TGF-β responsive tumor suppressor gene, tipped the balance of TGF-β role towards theoncogenesis in late stage colorectal cancers.