The purpose of this study is to investigate the efficacy and the mechanism of Hsp90 inhibition of Withaferin A (WA), a steroidal lactone occurring in Withania somnifera, in pancreatic cancer in vitro and in vivo. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro (with IC50s of 1.24, 2.93 and 2.78 μM) in pancreatic cancer cell lines Panc-1, MiaPaca2 and BxPc3, respectively. Annexin V staining showed that WA induced significant apoptosis in Panc-1 cells in a dose dependent manner. Western blotting demonstrated that WA inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins (Akt, Cdk4 and glucocorticoid receptor), which was reversed by the proteasomal inhibitor, MG132. WA-Biotin pull-down assay of Hsp90 using Panc-1 cancer cell lysates and purified Hsp90 showed that WA-biotin binds to C-terminus of Hsp90, which was competitively blocked by unlabeled WA. Co-immunoprecipitation exhibited that WA (10 μM) disrupted Hsp90-Cdc37 complexes from 1–24 hour post treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6 mg/kg) inhibited tumor growth in pancreatic Panc-1 xenografts by 30% and 58%, respectively. These data demonstrate that Withaferin A binds Hsp90, inhibits Hsp90 chaperone activity through an ATP independent mechanism, results in Hsp90 client protein degradation, and exhibits in vivo anticancer activity against pancreatic cancer.

Objectives:Evidence suggests a possible role of Fusobacterium nucleatum in colorectal carcinogenesis, especially in right-sided proximal colorectum. Considering a change in bowel contents and microbiome from proximal to distal colorectal segments, we hypothesized that the proportion of colorectal carcinoma enriched with F. nucleatum might gradually increase along the bowel subsites from rectum to cecum.Methods:A retrospective, cross-sectional analysis was conducted on 1,102 colon and rectal carcinomas in molecular pathological epidemiology databases of the Nurses’ Health Study and the Health Professionals Follow-up Study. We measured the amount of F. nucleatum DNA in colorectal tumor tissue using a quantitative PCR assay and equally dichotomized F. nucleatum-positive cases (high vs. low). We used multivariable logistic regression analysis to examine the relationship of a bowel subsite variable (rectum, rectosigmoid junction, sigmoid colon, descending colon, splenic flexure, transverse colon, hepatic flexure, ascending colon, and cecum) with the amount of F. nucleatum.Results:The proportion of F. nucleatum-high colorectal cancers gradually increased from rectal cancers (2.5% 4/157) to cecal cancers (11% 19/178), with a statistically significant linear trend along all subsites (P<0.0001) and little evidence of non-linearity. The proportion of F. nucleatum-low cancers was higher in rectal, ascending colon, and cecal cancers than in cancers of middle segments.Conclusions:The proportion of F. nucleatum-high colorectal cancers gradually increases from rectum to cecum. Our data support the colorectal continuum model that reflects pathogenic influences of the gut microbiota on neoplastic and immune cells and challenges the prevailing two-colon (proximal vs. distal) dichotomy paradigm.

Importance Although patients with resected pancreatic adenocarcinoma are at high risk for disease recurrence, few markers are available to inform patient outcomes. Objective To evaluate alterations of the four main driver genes for pancreatic adenocarcinoma and patient outcomes after cancer resection. Design, Setting, and Participants We analyzed protein expression and DNA alterations for KRAS, CDKN2A, SMAD4, and TP53 by immunohistochemistry and next-generation sequencing in formalin-fixed, paraffin-embedded tumors from 356 patients with resected pancreatic adenocarcinoma evaluated at three U.S. centers. Associations of driver gene alterations with disease-free survival (DFS) and overall survival (OS) were evaluated using Cox proportional hazards regression with estimation of hazard ratios (HR) and 95% confidence intervals (CI) and adjustment for age, sex, tumor characteristics, institution, and peri-operative treatment. Main Outcomes DFS and OS among patients with resected pancreatic adenocarcinoma Results Patients with KRAS mutant tumors had worse DFS and OS compared to patients with KRAS wild-type tumors, with median OS of 20.3 versus 38.6 months and 5-year OS of 13.0% versus 30.2%, respectively. Particularly poor outcomes were identified in patients with KRAS G12D-mutant tumors, who had median OS of 15.3 months. Patients whose tumors lacked CDKN2A expression had worse DFS and OS compared to patients whose tumors retained CDKN2A, with median OS of 19.7 versus 24.6 months and 5-year OS of 11.9% versus 19.5%, respectively. SMAD4 status was not associated with DFS or OS, while TP53 status was associated only with DFS (P=0.04). Patients had worse DFS and OS with greater number of altered driver genes. Compared to patients with 0-2 altered genes, those with 4 altered genes had HR for DFS of 1.79 (1.24-2.59; P<0.01) and OS of 1.38 (0.98-1.94; P=0.06). Five-year OS was 18.4% for patients with 0-2 gene alterations, 14.1% for 3 alterations and 8.2% for 4 alterations. Alterations in the four driver genes were not significantly associated with local recurrence as the first site of disease recurrence. Conclusions and Relevance Patient outcomes are associated with alterations of the four main driver genes in resected pancreatic adenocarcinoma.

Nearly 10% of PDAC patients harbor germline variants, although the majority lack somatic second hits, the therapeutic significance of which warrants further study.