Pioglitazone inhibits growth of carcinoid cells and promotes TRAIL-induced apoptosis by induction of p21(waf1/cip1)

Background/Aims: We investigated the effect of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist pioglitazone on growth and TRAIL-induced apoptosis in carcinoid cells. Methods: Carcinoid cells were incubated without and with pioglitazone. Effects on growth were examined by cell count and cell cycle analysis. p21(waf1/cip1) expression was determined by Western blotting. Cytotoxicity assay was performed by FACS analysis. Results: Pioglitazone suppressed the growth and induced apoptosis of carcinoid cells. Additionally, pioglitazone significantly enhanced carcinoid cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). The enhancement of TRAIL-induced apoptosis was associated with an upregulation of cyclin-dependent kinase inhibitor p21(waf1/cip1) in pioglitazone-treated carcinoid cells. Importantly, overexpression of p21(waf1/cip1) in carcinoid cells by adenoviral gene transfer of p21 sensitized them to TRAIL-induced apoptosis. Conclusions: These results suggest that pioglitazone inhibits cell growth and sensitizes cells to TRAIL-induced apoptosis by induction of p21(waf1/cip1). Therefore, pioglitazone can be an effective therapeutic adjuvant for the treatment of carcinoid tumors. Copyright (C) 2001 S. Karger AG, Basel

The role of p53 in anti-tumor immunity and response to immunotherapy

p53 is a transcription factor that regulates the expression of genes involved in tumor suppression. p53 mutations mediate tumorigenesis and occur in approximately 50% of human cancers. p53 regulates hundreds of target genes that induce various cell fates including apoptosis, cell cycle arrest, and DNA damage repair. p53 also plays an important role in anti-tumor immunity by regulating TRAIL, DR5, TLRs, Fas, PKR, ULBP1/2, and CCL2; T-cell inhibitory ligand PD-L1; pro-inflammatory cytokines; immune cell activation state; and antigen presentation. Genetic alteration of p53 can contribute to immune evasion by influencing immune cell recruitment to the tumor, cytokine secretion in the TME, and inflammatory signaling pathways. In some contexts, p53 mutations increase neoantigen load which improves response to immune checkpoint inhibition. Therapeutic restoration of mutated p53 can restore anti-cancer immune cell infiltration and ameliorate pro-tumor signaling to induce tumor regression. Indeed, there is clinical evidence to suggest that restoring p53 can induce an anti-cancer immune response in immunologically cold tumors. Clinical trials investigating the combination of p53-restoring compounds or p53-based vaccines with immunotherapy have demonstrated anti-tumor immune activation and tumor regression with heterogeneity across cancer type. In this Review, we discuss the impact of wild-type and mutant p53 on the anti-tumor immune response, outline clinical progress as far as activating p53 to induce an immune response across a variety of cancer types, and highlight open questions limiting effective clinical translation

Disease Control With FOLFIRI Plus Ziv-aflibercept (zFOLFIRI) Beyond FOLFIRI Plus Bevacizumab: Case Series in Metastatic Colorectal Cancer (mCRC)

Background: The prognosis of patients with metastatic colorectal cancer (mCRC) is poor, especially after failure of initial systemic therapy. The VELOUR study showed modestly prolonged overall survival (OS) with ziv-aflibercept plus 5-fluorouracil, leucovorin, and irinotecan (zFOLFIRI) vs. placebo+FOLFIRI after progression on 5-fluoruracil, leucovorin, and oxaliplatin (FOLFOX) ± bevacizumab. The utility of zFOLFIRI after bevacizumab+FOLFIRI is unknown and not recommended in NCCN guidelines. We explored whether zFOLFIRI may be active beyond progression on bevacizumab+FOLFIRI.Methods: We undertook a retrospective analysis of patients treated in routine clinical practice. A chart review was conducted for a cohort (N = 19) of advanced cancer patients (18 mCRC) who received zFOLFIRI from 2014 to 2018 at Fox Chase Cancer Center (FCCC). Analysis included time on zFOLFIRI, PFS, OS, CEA trends and adverse events. A second mCRC cohort (N = 26) from the Flatiron Health EHR-derived database treated with zFOLFIRI after prior bevacizumab+FOLFOX and bevacizumab+FOLFIRI was analyzed for time-on-treatment and overall survival.Results: Median age of mCRC cohort at zFOLFIRI treatment was 54 (FCCC; N = 18) and 62 (Flatiron Health-cohort; N = 26). Of 18 FCCC mCRC patients, 1 patient had prior bevacizumab+FOLFOX and ramucirumab+irinotecan prior to zFOLFIRI for 8.5 months. Of 17 FCCC mCRC patients with prior bevacizumab+FOLFIRI who received zFOLFIRI, 13 had mutant-KRAS, 3 WT-KRAS, and one BRAF-V600E. The patient with BRAF-V600E mutation achieved stable disease on zFOLFIRI after multiple BRAF-targeted therapies. One patient (WT-KRAS mCRC) remained on zFOLFIRI for 14 months. Of 14 patients with mutated-KRAS, 8 remained on zFOLFIRI for >5 months including 3 for >15 months. The rate-of-change in CEA measures on zFOLFIRI was significantly different (p = 0.004) between rapid progressors and those with PFS>4 months. For mCRC patients treated with zFOLFIRI in the 3rd line or greater (N = 18), median PFS was 7.1 months (214 days) and median OS was 13.8 months (416 days). Median time-on-treatment with zFOLFIRI in the Flatiron Health cohort was 4.4 months, median OS was 7.8 months, and longest time-on-treatment with zFOLFIRI was 266 days.Conclusions: In these small real-world cohorts, clinical meaningful stable disease and overall survival on zFOLFIRI beyond progression on bevacizumab+FOLFIRI was observed in patients with mCRC. Further exploration of this approach is warranted

Murine Pancreatic Adenocarcinoma Dampens SHIP-1 Expression and Alters MDSC Homeostasis and Function

Pancreatic cancer is one of the most aggressive cancers, with tumor-induced myeloid-derived suppressor cells (MDSC) contributing to its pathogenesis and ineffective therapies. In response to cytokine/chemokine receptor activation, src homology 2 domain-containing inositol 5'-phosphatase-1 (SHIP-1) influences phosphatidylinositol-3-kinase (PI3K) signaling events, which regulate immunohomeostasis. We hypothesize that factors from murine pancreatic cancer cells cause the down-regulation of SHIP-1 expression, which may potentially contribute to MDSC expansion, and the suppression of CD8(+) T cell immune responses. Therefore, we sought to determine the role of SHIP-1 in solid tumor progression, such as murine pancreatic cancer.Immunocompetent C57BL/6 mice were inoculated with either murine Panc02 cells (tumor-bearing [TB] mice) or Phosphate Buffer Saline (PBS) (control mice). Cytometric Bead Array (CBA) analysis of supernatants of cultured Panc02 detected pro-inflammatory cytokines such as IL-6, IL-10 and MCP-1. TB mice showed a significant increase in serum levels of pro-inflammatory factors IL-6 and MCP-1 measured by CBA. qRT-PCR and Western blot analyses revealed the in vivo down-regulation of SHIP-1 expression in splenocytes from TB mice. Western blot analyses also detected reduced SHIP-1 activity, increased AKT-1 and BAD hyper-phosphorylation and up-regulation of BCL-2 expression in splenocytes from TB mice. In vitro, qRT-PCR and Western blot analyses detected reduced SHIP-1 mRNA and protein expression in control splenocytes co-cultured with Panc02 cells. Flow cytometry results showed significant expansion of MDSC in peripheral blood and splenocytes from TB mice. AutoMACS sorted TB MDSC exhibited hyper-phosphorylation of AKT-1 and over-expression of BCL-2 detected by western blot analysis. TB MDSC significantly suppressed antigen-specific CD8(+) T cell immune responses in vitro.SHIP-1 may regulate immune development that impacts MDSC expansion and function, contributing to pancreatic tumor progression. Thus, SHIP-1 can be a potential therapeutic target to help restore immunohomeostasis and improve therapeutic responses in patients with pancreatic cancer

Acoustic separation of circulating tumor cells

Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (~100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state.National Institutes of Health (U.S.) (Grant 1 R01 GM112048-01A1)National Institutes of Health (U.S.) (Grant 1R33EB019785-01)National Science Foundation (U.S.)Penn State Center for Nanoscale Science (Materials Research Science and Engineering Center Grant DMR-0820404)National Institutes of Health (U.S.) (Grant U01HL114476

Death receptor 5 signaling promotes hepatocyte lipoapoptosis.

Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR(-/-)) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor

ER stress regulates myeloid-derived suppressor cell fate through TRAIL-R–mediated apoptosis

Myeloid-derived suppressor cells (MDSCs) dampen the immune response thorough inhibition of T cell activation and proliferation and often are expanded in pathological conditions. Here, we studied the fate of MDSCs in cancer. Unexpectedly, MDSCs had lower viability and a shorter half-life in tumor-bearing mice compared with neutrophils and monocytes. The reduction of MDSC viability was due to increased apoptosis, which was mediated by increased expression of TNF-related apoptosis–induced ligand receptors (TRAIL-Rs) in these cells. Targeting TRAIL-Rs in naive mice did not affect myeloid cell populations, but it dramatically reduced the presence of MDSCs and improved immune responses in tumor-bearing mice. Treatment of myeloid cells with proinflammatory cytokines did not affect TRAIL-R expression; however, induction of ER stress in myeloid cells recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts. The ER stress response was detected in MDSCs isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes, and blockade of ER stress abrogated tumor-associated changes in TRAIL-Rs. Together, these data indicate that MDSC pathophysiology is linked to ER stress, which shortens the lifespan of these cells in the periphery and promotes expansion in BM. Furthermore, TRAIL-Rs can be considered as potential targets for selectively inhibiting MDSCs

Molecular characterization and clinical outcomes of pancreatic neuroendocrine tumors (pNENs) harboring PAK4-NAMPT alterations

Background: The mTOR inhibitor, Everolimus (EVE), is FDA-approved for the treatment of advanced PNENs on the basis of delay of progression. The RADIANT-3 trial showed an increase in PFS from 4.6 to 11 months compared to placebo with an ORR of only 5%. Prior studies suggest that targeting the aberrant expression of mTOR regulators p21 activated kinase 4 (PAK4) and nicotinamide adenine dinucleotide biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT) in PNENs sensitizes these tumors to EVE. To further qualify these observations, we queried a large real-world dataset of PNENs, characterizing the molecular and immune landscapes, as well as the clinical outcomes associated with aberrant PAK4 and NAMPT expression. Methods: 294 cases of PNENs were analyzed using Next Generation Sequencing (NextSeq) and Whole Exome and Whole Transcriptome Sequencing (NovaSeq) at Caris Life Sciences (Phoenix, AZ). For our analyses, we stratified our study cohort into four groups based on the median expression of PAK4 and NAMPT: PAK4-low/NAMPT-low, PAK4-low/ NAMPT-high, PAK4-high/NAMPT-low and PAK4-high/NAMPT-high. Significance was determined using chi-square, Fisher-Exact or Mann-Whitney U, and p-values were adjusted for multiple comparisons (q , 0.05). Results: High prevalence of mutations in PTEN (10.71% vs 1.18%; p \u3c 0.05, q \u3e 0.05), a tumor suppressor of the mTOR pathway and high expression of genes activated in response to mTOR activation such as SLC2A1 (3.07-fold), PFKP (3.32-fold), SCD (2.70-fold), MVK (2.92-fold) and G6PD (2.58-fold) were observed in PAK4-high/NAMPT-high compared to the PAK4-low/NAMPTlow tumors (all q , 0.05). A congruent enrichment of PI3K/AKT/mTOR and glycolysis pathways by single-sample gene set enrichment analysis was observed in these tumors (all q , 0.05). When querying the immune landscape, we observed enrichment in inflammatory response, IL6/JAK/STAT3, IL2/STAT5 signaling pathways and immune checkpoint genes such as PDCD1 (5.14-fold), CD274 (2.84-fold), PDCD1LG2 (2.44-fold), CD80 (3.00-fold), CD86 (2.82-fold), IDO1 (1.92-fold), LAG3 (3.09-fold), HAVCR2 (2.66-fold) and CTLA4 (4.49-fold) in the PAK4-high/NAMPT-high tumors (all q,0.05). Immune cell infiltration estimates revealed an increase in Neutrophils, NK cells and Tregs in the PAK4-high/NAMPT-high tumors (p \u3c 0.05, q \u3e 0.05). Conclusions: Our study demonstrates that PAK4-high/NAMPT-high PNENs are associated with distinct molecular and immune profiles. While the dual blockade of PAK4 and NAMPT has been reported to enhance the efficacy of EVE in PNENs, whether such a blockade would enhance the efficacy of immunotherapeutics warrants further investigation