Description: Cathelicidins are a family of bacteriocidal polypeptides secreted by macrophages and polymorphonuclear leukocytes (PMN). LL-37, the only human cathelicidin, has been implicated in tumorigenesis, but there has been limited investigation of its expression and function in cancer. Here, we report that LL-37 activates a p53-mediated, caspase-independent apoptotic cascade that contributes to suppression of colon cancer. LL-37 was expressed strongly in normal colon mucosa but downregulated in colon cancer tissues, where in both settings its expression correlated with terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling-positive apoptotic cells. Exposure of colon cancer cells to LL-37 induced phosphatidylserine externalization and DNA fragmentation in a manner independent of caspase activation. Apoptogenic function was mediated by nuclear translocation of the proapoptotic factors, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), through p53-dependent upregulation of Bax and Bak and downregulation of Bcl-2 via a pertussis toxin–sensitive G-protein–coupled receptor (GPCR) pathway. Correspondingly, colonic mucosa of cathelicidin-deficient mice exhibited reduced expression of p53, Bax, and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Cathelicidin-deficient mice exhibited an increased susceptibility to azoxymethane-induced colon tumorigenesis, establishing pathophysiologic relevance in colon cancer. Collectively, our findings show that LL-37 activates a GPCR-p53-Bax/Bak/Bcl-2 signaling cascade that triggers AIF/EndoG–mediated apoptosis in colon cancer cells. Cancer Res; 72(24); 6512–23. ©2012 AACR.

Description: Purpose: The principal goals were to identify and validate targetable metabolic drivers relevant to myxofibrosarcoma pathogenesis using a published transcriptome. Experimental Design: As the most significantly downregulated gene regulating amino acid metabolism, argininosuccinate synthetase ( ASS1) was selected for further analysis by methylation-specific PCR, pyrosequencing, and immunohistochemistry of myxofibrosarcoma samples. The roles of ASS1 in tumorigenesis and the therapeutic relevance of the arginine-depriving agent pegylated arginine deiminase (ADI-PEG20) were elucidated in ASS1-deficient myxofibrosarcoma cell lines and xenografts with and without stable ASS1 reexpression. Results: ASS1 promoter hypermethylation was detected in myxofibrosarcoma samples and cell lines and was strongly linked to ASS1 protein deficiency. The latter correlated with increased tumor grade and stage and independently predicted a worse survival. ASS1-deficient cell lines were auxotrophic for arginine and susceptible to ADI-PEG20 treatment, with dose-dependent reductions in cell viability and tumor growth attributable to cell-cycle arrest in the S-phase. ASS1 expression was restored in 2 of 3 ASS1-deficient myxofibrosarcoma cell lines by 5-aza-2'-deoxycytidine, abrogating the inhibitory effect of ADI-PEG20. Conditioned media following ASS1 reexpression attenuated HUVEC tube-forming capability, which was associated with suppression of MMP-9 and an antiangiogenic effect in corresponding myxofibrosarcoma xenografts. In addition to delayed wound closure and fewer invading cells in a Matrigel assay, ASS1 reexpression reduced tumor cell proliferation, induced G 1 -phase arrest, and downregulated cyclin E with corresponding growth inhibition in soft agar and xenograft assays. Conclusions: Our findings highlight ASS1 as a novel tumor suppressor in myxofibrosarcomas, with loss of expression linked to promoter methylation, clinical aggressiveness, and sensitivity to ADI-PEG20. Clin Cancer Res; 19(11); 2861–72. ©2013 AACR .

Description: Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; however, the precise molecular mechanisms underlying this phenomenon remain elusive. Herein, we identified the circadian clock PER1 mRNA as a novel substrate of the endoribonuclease activity of the UPR sensor IRE1α. Analysis of the mechanism shows that IRE1α endoribonuclease activity decreased PER1 mRNA in tumor cells without affecting PER1 gene transcription. Inhibition of IRE1α signaling using either siRNA-mediated silencing or a dominant-negative strategy prevented PER1 mRNA decay, reduced tumorigenesis, and increased survival, features that were reversed upon PER1 silencing. Clinically, patients showing reduced survival have lower levels of PER1 mRNA expression and increased splicing of XBP1, a known IRE-α substrate, thereby pointing toward an increased IRE1α activity in these patients. Hence, we describe a novel mechanism connecting the UPR and circadian clock components in tumor cells, thereby highlighting the importance of this interplay in tumor development. Cancer Res; 73(15); 4732–43. ©2013 AACR.

Description: Purpose: The therapeutic benefit of temozolomide in glioblastoma multiforme (GBM) is limited by resistance. The goal of this study was to elucidate mechanisms of temozolomide resistance in GBM. Experimental Design: We developed an in vivo GBM model of temozolomide resistance and used paired parental and temozolomide-resistant tumors to define the mechanisms underlying the development of resistance and the influence of histone deacetylation (HDAC) inhibition. Results: Analysis of paired parental and resistant lines showed upregulation of O 6-methylguanine-DNA methyltransferase (MGMT) expression in 3 of the 5 resistant xenografts. While no significant change was detected in MGMT promoter methylation between parental and derivative-resistant samples, chromatin immunoprecipitation showed an association between MGMT upregulation and elevated acetylation of lysine 9 of histone H3 (H3K9-ac) and decreased dimethylation (H3K9-me2) in GBM12 and GBM14. In contrast, temozolomide resistance development in GBM22 was not linked to MGMT expression, and both parental and resistant lines had low H3K9-ac and high H3K9-me2 within the MGMT promoter. In the GBM12TMZ-resistant line, MGMT reexpression was accompanied by increased recruitment of SP1, C-JUN, NF-B, and p300 within the MGMT promoter. Interestingly, combined treatment of GBM12 flank xenografts with temozolomide and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) favored the evolution of temozolomide resistance by MGMT overexpression as compared with treatment with temozolomide alone. Conclusion: This study shows, for the first time, a unique mechanism of temozolomide resistance development driven by chromatin-mediated MGMT upregulation and highlights the potential for epigenetically directed therapies to influence the mechanisms of resistance development in GBM. Clin Cancer Res; 18(15); 4070–9. ©2012 AACR .

Description: Expression of heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) has been reported to be dysregulated in tumors, but its specific contributions to tumor formation and progression are not fully understood. Here, we demonstrate that HNRNPAB is overexpressed in highly metastatic cells and tumor tissues from patients with hepatocellular carcinoma (HCC) with recurrence. We found that HNRNPAB overexpression promoted epithelial–mesenchymal transition (EMT) in a manner associated with HCC metastasis in vitro and in vivo. RNA interference-mediated silencing of the EMT factor SNAIL attenuated HNRNPAB-enhanced cell invasion in vitro and lung metastasis in vivo. Mechanistically, HNRNPAB acted to transactivate SNAIL1 transcription, which in turn inhibited transcription of the pivotal SNAIL target gene E-cadherin. Overexpression of HNRNPAB in HCC samples correlated with higher SNAIL levels, shorter overall survival, and higher tumor recurrence. HNRNPAB overexpression, alone or in combination with SNAIL, was found to be a significant independent risk factor for recurrence and survival after curative resection. In conclusion, our findings define HNRNPAB as an activator of EMT and metastasis in HCC that predicts poor clinical outcomes. Cancer Res; 74(10); 2750–62. ©2014 AACR.

Description: Purpose: The circadian clock gene Bmal1 is involved in cancer cell proliferation and DNA damage sensitivity. The aim of this study was to explore the effect of Bmal1 on oxaliplatin sensitivity and to determine its clinical significance in colorectal cancer. Experimental Design: Three colorectal cancer cell lines, HCT116, THC8307 and HT29, were used. The Bmal1-mediated control of colorectal cancer cell proliferation was tested in vitro and in vivo . MTT and colony formation assays were performed to determine the sensitivity of colorectal cancer cells to oxaliplatin. Flow cytometry was used to examine changes in the cell-cycle distribution and apoptosis rate. Proteins expressed downstream of Bmal1 upon its overexpression were determined by Western blotting. Immunohistochemistry was used to analyze Bmal1 expression in 82 archived colorectal cancer tumors from patients treated with oxaliplatin-based regimens. Results: Bmal1 overexpression inhibited colorectal cancer cell proliferation and increased colorectal cancer sensitivity to oxaliplatin in three colorectal cancer cell lines and HCT116 cells model in vivo . Furthermore, the overall survival of patients with colorectal cancer with high Bmal1 levels in their primary tumors was significantly longer than that of patients with low Bmal1 levels (27 vs. 19 months; P = 0.043). The progression-free survival of patients with high Bmal1 expression was also significantly longer than that of patients with low Bmal1 expression (11 vs. 5 months; P = 0.015). Mechanistically, the effect of Bmal1 was associated with its ability to regulate G2–M arrest by activating the ATM pathway. Conclusion: Bmal1 shows the potential as a novel prognostic biomarker and may represent a new therapeutic target in colorectal cancer. Clin Cancer Res; 20(4); 1042–52. ©2013 AACR .

Description: Purpose: Lung cancer is the leading cause of cancer-related death worldwide. Sustained activation, overexpression, or mutation of the MET pathway is associated with a poor prognosis in a variety of tumors, including non–small cell lung cancer (NSCLC), implicating the MET pathway as a potential therapeutic target for lung cancer. Previously, we reported on the development of LY2801653: a novel, orally bioavailable oncokinase inhibitor with MET as one of its targets. Here, we discuss the evaluation of LY2801653 in both preclinical in vitro and in vivo NSCLC models. Experimental Design/Results: Treatment with LY2801653 showed tumor growth inhibition in tumor cell lines and patient-derived tumor xenograft models as a single agent (37.4%–90.0% inhibition) or when used in combination with cisplatin, gemcitabine, or erlotinib (66.5%–86.3% inhibition). Mechanistic studies showed that treatment with LY2801653 inhibited the constitutive activation of MET pathway signaling and resulted in inhibition of NCI-H441 cell proliferation, anchorage-independent growth, migration, and invasion. These in vitro findings were confirmed in the H441 orthotopic model where LY2801653 treatment significantly inhibited both primary tumor growth (87.9% inhibition) and metastasis (64.5% inhibition of lymph node and 67.7% inhibition of chest wall). Tumor-bearing animals treated with LY2801653 had a significantly greater survival time (87% increase compared with the vehicle-treated mice). In the MET-independent NCI-H1299 orthotopic model, treatment with LY2801653 showed a significant inhibition of primary tumor growth but not metastasis. Conclusions: Collectively, these results support clinical evaluation of LY2801653 in NSCLCs and suggest that differences in the MET activation of tumors may be predictive of response. Clin Cancer Res; 19(20); 5699–710. ©2013 AACR .