Importantly, in human tumor cell line xenograft models, siRNA treatment reduced the growth of colorectal cancers and synergized with 5-fluorouracil (5-FU) to induce regression of established tumors
Importantly, in human tumor cell line xenograft models, siRNA treatment reduced the growth of colorectal cancers and synergized with 5-fluorouracil (5-FU) to induce regression of established tumors. Results MUC13 is required for survival and growth of colorectal cancer cells To assess the effects of endogenous MUC13 on the sensitivity of human cancer cells to death, we used three colorectal cancer cell linesLS513, LIM2463 and HT29. and SUMOylation of NF-B essential modulator. Moreover, elevated expression of cytoplasmic MUC13 and NF-B correlated with colorectal cancer progression and metastases. Our demonstration that MUC13 enhances NF-B signaling in response to both TNF and DNA-damaging agents provides a new molecular target for specific inhibition of NF-B activation. As proof of principle, silencing MUC13 sensitized colorectal cancer cells to killing by cytotoxic drugs and inflammatory signals and abolished chemotherapy-induced enrichment of CD133+ CD44+ cancer stem cells, slowed xenograft growth in mice, and synergized with 5-fluourouracil to induce tumor regression. Therefore, these data indicate that combining chemotherapy and MUC13 antagonism could improve the treatment of metastatic cancers. Introduction Colorectal cancers are the third most common cause of cancer in men and women. Mortality has been decreasing due to polyp detectionCcancer prevention programs, but mortality remains high when colorectal cancer is metastatic. One of the hallmark features of cancers is resistance to apoptotic cell death. Most metastatic cancer therapies act either directly or indirectly via induction of apoptosis in cancer cells,1 but such therapies are not selective for neoplastic cells.2 Thus, enhancing selectivity of cancer treatments remains an important chemotherapeutic goal. Mucins are complex cell surface and secreted glycoproteins that provide protection and lubrication to the epithelial surface of mucosal tissues.3, 4, 5 Aberrant expression of cell surface mucins occurs in many cancers and has been linked to the initiation, progression and poor prognosis of multiple types of adenocarcinoma.6, 7 The advantage of expression in these cancers is likely linked to the normal functions of mucins related to epithelial resistance and resilience to toxic challenges at mucosal surfaces.4, 5 Consequently, mucins are now recognized as potential diagnostic markers and therapeutic targets in many cancers.8, 9, 10, 11, 12, 13, 14, 15 The MUC13 cell surface mucin is over produced in gastric,16 colorectal,17, 18, 19 pancreatic20, 21 and ovarian22 cancers. Normally this protein is synthesized on the apical borders of epithelial cells, including the luminal surface glycocalyx of enterocytes and goblet cells in the small and large intestine, 23 with increased cytoplasmic expression seen in response to infection24 and inflammation.25 MUC13 has a 69 amino-acid cytoplasmic domain that includes eight serine and two tyrosine residues for potential phosphorylation, TSU-68 (Orantinib, SU6668) and a protein kinase C consensus phosphorylation motif23 that could play a critical role in tumorigenesis via cell signaling pathways that regulate apoptosis Rabbit Polyclonal to SRPK3 and proliferation.18, 22, 23, 25 We have previously shown that MUC13 protects colonic epithelial cells from apoptosis25 and, therefore, targeting MUC13 and MUC13-regulated pathways to sensitize cancer cells to killing may present an attractive target for cancer treatment. The intrinsic cell death pathway involves cellular stresses including DNA damage, whereas the extrinsic cell death pathway responds to immune-mediated signals.26 The nuclear factor-kappa-B (NF-B) family of transcription factors play a key role in the transcription of several genes involved in the suppression of both cell death pathways.27 NF-B signaling networks can be induced by both inflammatory signals (such as tumor necrosis factor- (TNF-) and chemotherapy agents). Thus, activation of NF-B by chemotherapeutic compounds can contribute substantially to the acquired chemo-resistance that hinders effective cancer therapy28 and promotes recurrence.29 In this study, we demonstrate that MUC13 protects human colorectal cancer cells from cell death in response to activation of both intrinsic and extrinsic pathways via NF-B activation and subsequent upregulation of the critical regulator of apoptosis, BCL-XL. These data are supported by analysis of patient colorectal cancers which showed TSU-68 (Orantinib, SU6668) a correlation between cytoplasmic MUC13 expression, tumor grade, and expression of NF-B proteins and BCL-XL. Importantly, in human tumor cell line xenograft models, siRNA treatment reduced the growth of colorectal cancers and synergized with 5-fluorouracil (5-FU) to induce regression of established tumors. Results MUC13 is required for survival and growth of colorectal cancer cells To assess the effects of endogenous MUC13 on the sensitivity of human cancer cells to death, we used three colorectal cancer cell linesLS513, LIM2463 and HT29. LS513 and LIM2463 cells have high MUC13 expression and harbor inactivating mutations in the tumor suppressors and with siRNA in these cell lines, and then treated them TSU-68 (Orantinib, SU6668) with TNF and cycloheximide (which sensitizes cells to TNF-induced apoptosis by blocking synthesis of antiapoptotic proteins) and cell survival TSU-68 (Orantinib, SU6668) was determined by measuring ATP levels. siRNA reduced MUC13 protein expression by ~80% in these cell lines (Supplementary Figure S1A) and resulted in a significant decrease.