Androgen blockade primes NLRP3 in macrophages to induce tumor phagocytosis.

Immune-based therapies induce durable remissions in subsets of patients across multiple malignancies. However, there is limited efficacy of immunotherapy in metastatic castrate-resistant prostate cancer (mCRPC), manifested by an enrichment of immunosuppressive (M2) tumor- associated macrophages (TAM) in the tumor immune microenvironment (TME). Therefore, therapeutic strategies to overcome TAM-mediated immunosuppression are critically needed in mCRPC. Here we discovered that NLR family pyrin domain containing 3 (NLRP3), an innate immune sensing protein, is highly expressed in TAM from metastatic PC patients treated with standard-of-care androgen deprivation therapy (ADT). Importantly, ex vivo studies revealed that androgen receptor (AR) blockade in TAM upregulates NLRP3 expression, but not inflammasome activity, and concurrent AR blockade/NLRP3 agonist (NLRP3a) treatment promotes cancer cell phagocytosis by immunosuppressive M2 TAM. In contrast, NLRP3a monotherapy was sufficient to enhance phagocytosis of cancer cells in anti-tumor (M1) TAM, which exhibit high de novo NLRP3 expression. Critically, combinatorial treatment with ADT/NLRP3a in a murine model of advanced PC resulted in significant tumor control, with tumor clearance in 55% of mice via TAM phagocytosis. Collectively, our results demonstrate NLRP3 as an AR-regulated "macrophage phagocytic checkpoint", inducibly expressed in TAM by ADT and activated by NLRP3a treatment, the combination resulting in TAM-mediated phagocytosis and tumor control.

Mixed Lineage Kinase 3 phosphorylates prolyl-isomerase PIN1 and potentiates GLI1 signaling in pancreatic cancer development.

The transcription factor Glioma-Associated Oncogene Homolog 1 (GLI1) is activated by sonic hedgehog (SHH) cascade and is an established driver of pancreatic ductal adenocarcinoma (PDAC). However, therapies targeting upstream hedgehog signaling have shown little to no efficacy in clinical trials. Here, we identify Mixed Lineage Kinase 3 (MLK3) as a druggable regulator of oncogenic GLI1. Earlier, we reported that MLK3 phosphorylated a peptidyl-prolyl isomerase PIN1 on the S138 site, and the PIN1-pS138 translocated to the nucleus. In this report, we identify GLI1 as one of the targets of PIN1-pS138 and demonstrate that PIN1-pS138 is upregulated in human PDAC and strongly associates with the upregulation of GLI1 and MLK3 expression. Moreover, we also identified two new phosphorylation sites on GLI1, T394, and S1089, which are directly phosphorylated by MLK3 to promote GLI1 nuclear translocation, transcriptional activity, and cell proliferation. Additionally, pharmacological inhibition of MLK3 by CEP-1347 promoted apoptosis in PDAC cell lines, reduced tumor burden, extended survival, and reduced GLI1 expression in the Pdx1-Cre x LSL-KRASG12D x LSL-TP53R172H (KPC) mouse model of PDAC. These findings collectively suggest that MLK3 is an important regulator of oncogenic GLI1 and that therapies targeting MLK3 warrant consideration in the management of PDAC patients.

Role of stromal activin A in human pancreatic cancer and metastasis in mice.

Pancreatic ductal adenocarcinoma (PDAC) has extensive stromal involvement and remains one of the cancers with the highest mortality rates. Activin A has been implicated in colon cancer and its stroma but its role in the stroma of PDAC has not been elucidated. Activin A expression in cancer and stroma was assessed in human PDAC tissue microarrays (TMA). Activin A expression in human TMA is significantly higher in cancer samples, with expression in stroma correlated with shorter survival. Cultured pancreatic stellate cells (PSC) were found to secrete high levels of activin A resulting in PDAC cell migration that is abolished by anti-activin A neutralizing antibody. KPC mice treated with anti-activin A neutralizing antibody were evaluated for tumors, lesions and metastases quantified by immunohistochemistry. KPC mice with increased tumor burden express high plasma activin A. Treating KPC mice with an activin A neutralizing antibody does not reduce primary tumor size but decreases tumor metastases. From these data we conclude that PDAC patients with high activin A expression in stroma have a worse prognosis. PSCs secrete activin A, promoting increased PDAC migration. Inhibition of activin A in mice decreased metastases. Hence, stroma-rich PDAC patients might benefit from activin A inhibition.

p110γ deficiency protects against pancreatic carcinogenesis yet predisposes to diet-induced hepatotoxicity.

Pancreatic ductal adenocarcinoma (PDAC) is notorious for its poor survival and resistance to conventional therapies. PI3K signaling is implicated in both disease initiation and progression, and specific inhibitors of selected PI3K p110 isoforms for managing solid tumors are emerging. We demonstrate that increased activation of PI3K signals cooperates with oncogenic Kras to promote aggressive PDAC in vivo. The p110γ isoform is overexpressed in tumor tissue and promotes carcinogenesis via canonical AKT signaling. Its selective blockade sensitizes tumor cells to gemcitabine in vitro, and genetic ablation of p110γ protects against Kras-induced tumorigenesis. Diet/obesity was identified as a crucial means of p110 subunit up-regulation, and in the setting of a high-fat diet, p110γ ablation failed to protect against tumor development, showing increased activation of pAKT and hepatic damage. These observations suggest that a careful and judicious approach should be considered when targeting p110γ for therapy, particularly in obese patients.

TM4SF18 is aberrantly expressed in pancreatic cancer and regulates cell growth.

Current therapies for pancreatic ductal adenocarcinoma (PDAC) only modestly impact survival and can be highly toxic. A greater understanding of the molecules regulating this disease is critical for identifying new drug targets and developing more effective therapies. The L6 family of proteins are known to be positive regulators of tumor growth and metastasis among various cancers. However, little is known about the L6 family member TM4SF18. We investigated the expression and localization of the TM4SF18 protein in normal human pancreas and in PDAC tissue. Utilizing immunohistochemistry (IHC) and western blot analysis, our studies for the first time demonstrate that TM4SF18 is highly expressed in PDAC tumor epithelium. Furthermore, we identified TM4SF18 to be expressed in normal acinar tissue and weakly expressed in normal ducts. Although there is minimal expression in normal ducts, we observed increased TM4SF18 levels in preneoplastic ducts and tumor epithelium. To investigate a functional role of TM4SF18 in PDAC we developed stably-expressing inducible shRNA pancreatic cancer cell lines. Knockdown of the TM4SF18 protein led to a significant decrease in Capan-1 cell growth as measured by the MTT assay, demonstrating this molecule to be a novel regulator of PDAC. Uniquely there is no ortholog of the TM4SF18 gene in mouse or rat prompting us to seek other in vivo experimental models. Using IHC and western blot analysis, expression of TM4SF18 was confirmed in the porcine PDAC model, thus we establish an alternative model to investigate this gene. TM4SF18 represents a promising novel biomarker and therapeutic target for pancreatic cancer.

Interplay between interferon regulatory factor 1 and BRD4 in the regulation of PD-L1 in pancreatic stellate cells.

The fibrotic reaction is a characteristic feature of human pancreatic ductal adenocarcinoma (PDAC) tumors. It is associated with activation and proliferation of pancreatic stellate cells (PSCs), which are key regulators of fibrosis in vivo. While there is increasing interest in the regulation of PD-L1 expression in cancer and immune cells, the expression and regulation of PD-L1 in other stromal cells, such as PSCs, has not been fully evaluated. Here we show that PSCs in vitro express higher PD-L1 mRNA and protein levels compared to the levels present in PDAC cells. We show that inhibitors targeting bromodomain and extra-terminal (BET) proteins and BRD4 knockdown decrease interferon-γ (IFN-γ)-induced PD-L1 expression in PSCs. We also show that c-MYC, one of the well-established targets of BET inhibitors, does not mediate IFN-γ-regulated PD-L1 expression in PSCs. Instead we show that interferon regulatory factor 1 (IRF1) mediates IFN-γ-induced PD-L1 expression in PSCs. Finally, while we show that BET inhibitors do not regulate IFN-γ-induced IRF1 expression in PSCs, BET inhibitors decrease binding of IRF1 and BRD4 to the PD-L1 promoter. Together, these results demonstrate the interplay between IRF1 and BRD4 in the regulation of PD-L1 in PSCs.