Anti-PD-L1 prolongs survival and triggers T cell but not humoral anti-tumor immune responses in a human MUC1-expressing preclinical ovarian cancer model.

Monoclonal antibodies that block inhibitory immune checkpoint molecules and enhance anti-tumor responses show clinical promise in advanced solid tumors. Most of the preliminary evidence on therapeutic efficacy of immune checkpoint blockers comes from studies in melanoma, lung and renal cancer. To test the in vivo potential of programmed death-ligand 1 (PD-L1) blockade in ovarian cancer, we recently generated a new transplantable tumor model using human mucin 1 (MUC1)-expressing 2F8 cells. The MUC1 transgenic (MUC1.Tg) mice develop large number of intraperitoneal (IP) tumors following IP injection of 8 × 10(5) syngeneic 2F8 cells. The tumors are aggressive and display little T cell infiltration. Anti-PD-L1 antibody was administered IP every 2 weeks (200 µg/dose) for a total of three doses. Treatment was started 21 days post-tumor challenge, a time point which corresponds to late tumor stage. The anti-PD-L1 treatment led to substantial T cell infiltration within the tumor and significantly increased survival (p = 0.001) compared to isotype control-treated mice. When the same therapy was administered to wild-type mice challenged with 2F8 tumors, no survival benefit was observed, despite the presence of high titer anti-MUC1 antibodies. However, earlier treatment (day 11) and higher frequency of IP injections restored the T cell responses and led to prolonged survival. Splenocyte profiling via Nanostring using probes for 511 immune genes revealed a treatment-induced immune gene signature consistent with increased T cell-mediated immunity. These findings strongly support further preclinical and clinical strategies exploring PD-L1 blockade in ovarian cancer.

Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner.

A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity.

MUC1 positive, Kras and Pten driven mouse gynecologic tumors replicate human tumors and vary in survival and nuclear grade based on anatomical location.

Activating mutations of Kras oncogene and deletions of Pten tumor suppressor gene play important roles in cancers of the female genital tract. We developed here new preclinical models for gynecologic cancers, using conditional (Cre-loxP) mice with floxed genetic alterations in Kras and Pten. The triple transgenic mice, briefly called MUC1KrasPten, express human MUC1 antigen as self and carry a silent oncogenic KrasG12D and Pten deletion mutation. Injection of Cre-encoding adenovirus (AdCre) in the ovarian bursa, oviduct or uterus activates the floxed mutations and initiates ovarian, oviductal, and endometrial cancer, respectively. Anatomical site-specific Cre-loxP recombination throughout the genital tract of MUC1KrasPten mice leads to MUC1 positive genital tract tumors, and the development of these tumors is influenced by the anatomical environment. Endometrioid histology was consistently displayed in all tumors of the murine genital tract (ovaries, oviducts, and uterus). Tumors showed increased expression of MUC1 glycoprotein and triggered de novo antibodies in tumor bearing hosts, mimicking the immunobiology seen in patients. In contrast to the ovarian and endometrial tumors, oviductal tumors showed higher nuclear grade. Survival for oviduct tumors was significantly lower than for endometrial tumors (p = 0.0015), yet similar to survival for ovarian cancer. Oviducts seem to favor the development of high grade tumors, providing preclinical evidence in support of the postulated role of fallopian tubes as the originating site for high grade human ovarian tumors.

Oxidative stress sensitizes bladder cancer cells to TRAIL mediated apoptosis by down-regulating anti-apoptotic proteins.

PURPOSE: TRAIL, an endogenous protein involved in immunosurveillance and a novel drug in clinical trials, is of particular interest as cancer therapy because it can induce apoptosis in cancer cells but not in normal cells. Since some cancers develop resistance to TRAIL, safe and effective methods of TRAIL sensitization are of clinical interest. We explored how chemotherapy and oxidative stress affect TRAIL sensitivity and expression of proteins in the apoptotic pathway. MATERIALS AND METHODS: Sensitivity to TRAIL was assessed in viability assays. Apoptosis was measured by caspase-3/7 activity and/or nuclear condensation using Hoechst staining. Western blotting was used to determine cleavage, phosphorylation or alterations in protein expression. RESULTS: TRAIL decreased the viability of 5637 but not of J82 or T24 bladder carcinoma cells (ATCC(R)). Chemotherapy with doxorubicin or cisplatin (Ben Venue Laboratories, Bedford, Ohio) decreased the expression of the anti-apoptotic protein cFLIP(S) and increased caspase-8 cleavage, reversing TRAIL resistance in T24 cells. Specific targeting of cFLIP(S) by siRNA was insufficient for sensitization to TRAIL in T24 cells. However, chemotherapy mediated TRAIL sensitization was mimicked by low concentrations of H(2)O(2), which resulted in the phosphorylation of translation EF2 and decreased the expression of several short half-life, anti-apoptotic proteins, including FLIP(S), XIAP and survivin. CONCLUSIONS: Inducing oxidative stress by low H(2)O(2) concentrations may reverse TRAIL resistance. This warrants the further exploration of H(2)O(2) as an adjuvant intravesical treatment to lower the apoptotic threshold of bladder cancer cells.