Pyrroline-5-Carboxylate Reductase-2 Promotes Colorectal Carcinogenesis by Modulating Microtubule-Associated Serine/Threonine Kinase-like/Wnt/β-Catenin Signaling

Background: Despite significant progress in clinical management, colorectal cancer (CRC) remains the third most common cause of cancer-related deaths. A positive association between PYCR2 (pyrroline-5-carboxylate reductase-2), a terminal enzyme of proline metabolism, and CRC aggressiveness was recently reported. However, how PYCR2 promotes colon carcinogenesis remains ill understood. Methods: A comprehensive analysis was performed using publicly available cancer databases and CRC patient cohorts. Proteomics and biochemical evaluations were performed along with genetic manipulations and in vivo tumor growth assays to gain a mechanistic understanding. Results: PYCR2 expression was significantly upregulated in CRC and associated with poor patient survival, specifically among PYCR isoforms (PYCR1, 2, and 3). The genetic inhibition of PYCR2 inhibited the tumorigenic abilities of CRC cells and in vivo tumor growth. Coinciding with these observations was a significant decrease in cellular proline content. PYCR2 overexpression promoted the tumorigenic abilities of CRC cells. Proteomics (LC-MS/MS) analysis further demonstrated that PYCR2 loss of expression in CRC cells inhibits survival and cell cycle pathways. A subsequent biochemical analysis supported the causal role of PYCR2 in regulating CRC cell survival and the cell cycle, potentially by regulating the expression of MASTL, a cell-cycle-regulating protein upregulated in CRC. Further studies revealed that PYCR2 regulates Wnt/β-catenin-signaling in manners dependent on the expression of MASTL and the cancer stem cell niche. Conclusions: PYCR2 promotes MASTL/Wnt/β-catenin signaling that, in turn, promotes cancer stem cell populations and, thus, colon carcinogenesis. Taken together, our data highlight the significance of PYCR2 as a novel therapeutic target for effectively treating aggressive colon cancer

P62/SQSTM1 binds with claudin-2 to target for selective autophagy in stressed intestinal epithelium

Abstract Impaired autophagy promotes Inflammatory Bowel Disease (IBD). Claudin-2 is upregulated in IBD however its role in the pathobiology remains uncertain due to its complex regulation, including by autophagy. Irrespective, claudin-2 expression protects mice from DSS colitis. This study was undertaken to examine if an interplay between autophagy and claudin-2 protects from colitis and associated epithelial injury. Crypt culture and intestinal epithelial cells (IECs) are subjected to stress, including starvation or DSS, the chemical that induces colitis in-vivo. Autophagy flux, cell survival, co-immunoprecipitation, proximity ligation assay, and gene mutational studies are performed. These studies reveal that under colitis/stress conditions, claudin-2 undergoes polyubiquitination and P62/SQSTM1-assisted degradation through autophagy. Inhibiting autophagy-mediated claudin-2 degradation promotes cell death and thus suggest that claudin-2 degradation promotes autophagy flux to promote cell survival. Overall, these data inform for the previously undescribed role for claudin-2 in facilitating IECs survival under stress conditions, which can be harnessed for therapeutic advantages

Abstract 1175: Lossof claudin-3 expression induces de-differentiation in colonic epithelial cellsto promote colon cancer malignancy and associates with poor patient survival

Abstract Dysregulation of colonocyte differentiation, imposing a crypt progenitor phenotype, characterize colorectal cancer (CRC) progression. Thus, an improved understanding of the molecular processes that regulate colonocyte differentiation can help identify novel therapeutic targets and biomarkers. In this regard, we found claudin-3, a tight junction integral protein, to be the highest expressed cell-cell adhesion moiety in the normal colonic epithelium and particularly concentrated amongst terminally differentiated colonocytes at the crypt top. We therefore postulated a key role for claudin-3 in maintaining colonocyte differentiation and negative association with colon tumorigenesis. In accordance, claudin-3 expression was low in 10 out of 14 CRC cells lines tested and undetectable in poorly differentiated and highly tumorigenic HCT116 and SW620 cell lines. In further analysis, using mRNA and protein expression, and utilizing samples from a large patient cohort (&amp;lt;250 CRC specimen), we found claudin-3 expression to be significantly suppressed (p&amp;lt;0.001 versus normal) in cancer tissues versus normal mucosa. The colon tissues from the established mouse models of colon cancer (APCmin mice and Azoxymethane (AOM)-DSS-induced colon cancer) demonstrated similar tumor specific decrease in claudin-3 expression. Interestingly, claudin-3 negative tumors retained E-cadherin expression. Most notably, we found a significant and positive correlation (p&amp;lt;0.001) of the greater levels of claudin-3 expression with patient survival. Genetic manipulation studies using colon cancer cells further supported a causal role of claudin-3 in upholding colonocyte differentiation, by modulating ZEB-1 protein synthesis and Wnt-signaling activation (Topflash promoter activity, p&amp;lt;0.05; claudin-3 siRNA versus control cells), and inhibiting cancer cell mobility and invasive ability. Importantly, homozygous deletion of claudin-3 expression in mice similarly inhibited colonocyte differentiation (down-regulated P-27 expression and up-regulated Vimentin expression), and promoted colon cancer growth and invasion when subjected to the AOM-DSS-induced mouse model of colorectal cancer (p&amp;lt;0.001 versus WT littermates). Pharmacological manipulations further revealed epigenetic regulation as potential mechanism for the inhibition of caludin-3 expression in CRC. Taken together, our data reveal a tissue-specific and inverse causal association of claudin-3 expression with CRC progression and patient survival, and highlights key importance of claudin-3 in maintaining colonocyte differentiation in Wnt-ZEB1-dependent manner. Citation Format: Rizwan Ahmad, Zhimin Chen, Balawant Kumar, Xi Chen, Mary kay Washington, Punita Dhawan, Amar B. Singh. Lossof claudin-3 expression induces de-differentiation in colonic epithelial cellsto promote colon cancer malignancy and associates with poor patient survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1175.</jats:p

Dynamics of Physical Interaction between HIV-1 Nef and ASK1: Identifying the Interacting Motif(S)

<div><p>FasL mediated preferential apoptosis of bystander CTLs while protection of infected CD4⁺T cells remains one of the hallmarks of immune evasion during HIV infection. The property of infected host cells to evade cell-autonomous apoptosis emanates from ability of HIV-1Nef -protein to physically interact with ASK-1 and thereby inhibit its enzymatic activity. The specific domains of HIV-1Nef through which it may interact with ASK1 and thereby impair the ASK1 activity remain unidentified so far and represent a major challenge towards developing clear understanding about the dynamics of this interaction. Using mammalian two hybrid screen in association with site directed mutagenesis and competitive inhibitor peptides, we identified constituent minimal essential domain (152 DEVGEANN 159) through which HIV-1Nef interacts with ASK1 and inhibits its function. Furthermore our study also unravels a novel alternate mechanism underlying HIV-1 Nef mediated ASK1 functional modulation, wherein by potentiating the inhibitory ser⁹⁶⁷ phosphorylation of ASK1, HIV-1Nef negatively modulated ASK1function.</p> </div

Identification of Nef interacting region within ASK1.

<p>(<b>A</b>) ASK1 overlapping fragments casing full length of ASK1 were designed to generate multiple ASK1 truncations viz. ASK1 (1-345 aa), ASK1 (319-670 aa), ASK1 (607-904 aa), ASK1 (861-1051 aa), ASK1 (1059-1182 aa), ASK1 (1152-1374 aa). The HEK-293 cells were co-transfected with ASK1 truncations and Nef along with pG5<i>LUC</i> reported vector. Different truncations exhibited varying degrees of interaction as measured by fold increase in luciferase/renilla expression over pACT-pBIND negative control vector viz. 2.05, 1.08, 0.59, 2.93, 0.71, 0.79 respectively. ASK1(1-345 aa) and ASK1(861-1051 aa) fragment showing significant interaction while other fragments are not significantly (ns) interact with Nef (<b>B</b>) A distinct ASK1 truncation that contained only C-terminal Nef interacting domain and lacked N-terminal Nef interacting domain i.e. ASK1 (319-1051 aa) was designed. Similarly, another distinct ASK1 truncation that contained only N-terminal Nef interacting domain and lacked C-terminal Nef interacting domain i.e. ASK1 (1-904 aa) was also designed. Using co-transfection studies in HEK-293 cells, both the truncations were individually evaluated for their interaction ability with Nef vis a vis ASK1 (1-1051 aa) truncation that harbored both N and C terminal domains and ASK1 (319-904 aa) truncation that lacked both N and C terminal domains. Co-transfection with ASK1 (1-1051 aa) caused 156.56 fold increase in luciferase/renilla expression compared to negative control pACT-pBIND while other ASK1 fragment viz. ASK1 (319-1051 aa), ASK1 (1-904 aa), ASK1 (319-904 aa) caused only 2.83, 8.75, 0.59 fold increases respectively. The data are means <u>+</u>SEM from three distinct experiments. <i>P</i> values of <0.05 were obtained (two-tailed paired <i>t</i> test) when luciferase/renilla expression from ASK1 fragments and Nef co-transfected cell were compared with negative control pACT-pBIND.</p

Inhibition of ASK1 (1-1051 aa) induced apoptosis by Nef.

<p>(<b>A</b>) <b>Expression of Nef:</b> HEK-293 cell was transfected with pEYFP-N1-Nef plasmid and vector back bone. Cells were harvested after 48 hrs and western bolting was done by anti Nef antibody. Showing stable expression of Nef protein. (<b>B</b>) HEK-293 cell was transfected with ASK1(1-1051 aa) along with/without Nef showing ASK1-Ser⁹⁶⁷ phosphorylation. ASK1-Ser⁹⁶⁷ was very low in vector control and ASK1 transfected HEK-293 cells, co-transfection with Nef significantly potentiated ASK1-Ser⁹⁶⁷ phosphorylation. (<b>C</b>) HEK-293 cells transfected with ASK1 (1-1051 aa) exhibited apoptotic cell count of 45.65% while HEK-293 cell that were co-transfected with ASK1 (1-1051 aa) and Nef (full length) exhibited 23.61% apoptotic cell count. pYFP-N1 vector control exhibited an apoptotic cell count of 11.78%. Cells that harbored vector backbone served as control. The data are means <u>+</u>SEM from three distinct experiments. <i>P</i> values of <0.05 were obtained from two-tailed paired <i>t</i> test. (<b>D</b>) p54/46JNK1 pathway activation was studied using western blot /densitometry analysis of HEK-293 cells that were transfected with ASK1 (1-1051 aa) with/without Nef. (<b>E</b>) Annexin-V/PI labeling followed by flow cytometric analysis of ASK1(1-1051 aa) transfected Jurkat T cell revealed 32.85% Annexin-V positive apoptotic cell count, While Jurkat T cell that were co-transfected with ASK1(1-1051 aa) and Nef (full length) exhibited 12.9% apoptotic cell count. Vector control (pYFP-N1) showed 12.79% Annexin-V positive apoptotic cells. (<b>F</b>) Jurkat T cell were transfected with plasmid encoding ASK1 (1-1051 aa) with and without plasmid encoding HIV-1Nef. Cells were harvested after 40 hrs of transfection and western blot analysis for JNK1 activation was done using anti phospho-p54/46JNK1 and p54/46JNK1 antibodies showing relative levels of activated p54/46JNK1 i.e. ratio between integrated density of phospho-p54/46JNK1 and p54/46JNK1 bands. All western bolting was done three times and integrated density was determined by densitometry analysis using imageJ software.</p

Loss of Claudin-3 Expression Induces IL6/gp130/Stat3 Signaling to Promote Colon Cancer Malignancy by Hyperactivating Wnt/β-Catenin signaling

The Hyperactivated Wnt/β-catenin signaling acts as a switch to induce EMT and promote colorectal cancer. However, due to its essential role in gut homeostasis, therapeutic targeting of this pathway has proven challenging. Additionally, IL-6/Stat-3 signaling, activated by microbial translocation through the dysregulated mucosal barrier in colon adenomas, facilitates the adenoma to adenocarcinomas transition. However, inter-dependence between these signaling pathways and key mucosal barrier components in regulating colon tumorigenesis and cancer progression remains unclear. In current study, we have discovered, using a comprehensive investigative regimen, a novel and tissue specific role of claudin-3, a tight junction integral protein, in inhibiting colon cancer progression by serving as the common rheostat of Stat-3 and Wnt-signaling activation. Loss of claudin-3 also predicted poor patient survival. These findings however contrasted an upregulated claudin-3 expression in other cancer types and implicated role of the epigenetic regulation. Claudin-3−/− mice revealed dedifferentiated and leaky colonic epithelium, and developed invasive adenocarcinoma when subjected to colon cancer. Wnt-signaling hyperactivation, albeit in GSK-3β independent manner, differentiated colon cancer in claudin-3−/− mice versus WT-mice. Claudin-3 loss also upregulated the gp130/IL6/Stat3 signaling in colonic epithelium potentially assisted by infiltrating immune components. Genetic and pharmacological studies confirmed that claudin-3 loss induces Wnt/β-catenin activation, which is further exacerbated by Stat-3-activation and help promote colon cancer. Overall, these novel findings identify claudin-3 as a therapeutic target for inhibiting overactivation of Wnt-signaling to prevent CRC malignancy

MASTL induces Colon Cancer progression and Chemoresistance by promoting Wnt/β-catenin signaling

Abstract Background Chemotherapeutic agents that modulate cell cycle checkpoints and/or tumor-specific pathways have shown immense promise in preclinical and clinical studies aimed at anti-cancer therapy. MASTL (Greatwall in Xenopus and Drosophila), a serine/threonine kinase controls the final G2/M checkpoint and prevents premature entry of cells into mitosis. Recent studies suggest that MASTL expression is highly upregulated in cancer and confers resistance against chemotherapy. However, the role and mechanism/s of MASTL mediated regulation of tumorigenesis remains poorly understood. Methods We utilized a large patient cohort and mouse models of colon cancer as well as colon cancer cells to determine the role of Mastl and associated mechanism in colon cancer. Results Here, we show that MASTL expression increases in colon cancer across all cancer stages compared with normal colon tissue (P < 0.001). Also, increased levels of MASTL associated with high-risk of the disease and poor prognosis. Further, the shRNA silencing of MASTL expression in colon cancer cells induced cell cycle arrest and apoptosis in vitro and inhibited xenograft-tumor growth in vivo. Mechanistic analysis revealed that MASTL expression facilitates colon cancer progression by promoting the β-catenin/Wnt signaling, the key signaling pathway implicated in colon carcinogenesis, and up-regulating anti-apoptotic proteins, Bcl-xL and Survivin. Further studies where colorectal cancer (CRC) cells were subjected to 5-fluorouracil (5FU) treatment revealed a sharp increase in MASTL expression upon chemotherapy, along with increases in Bcl-xL and Survivin expression. Most notably, inhibition of MASTL in these cells induced chemosensitivity to 5FU with downregulation of Survivin and Bcl-xL expression. Conclusion Overall, our data shed light on the heretofore-undescribed mechanistic role of MASTL in key oncogenic signaling pathway/s to regulate colon cancer progression and chemo-resistance that would tremendously help to overcome drug resistance in colon cancer treatment