Resident fibroblasts that contact tumor epithelial cells (TEC) can become irreversibly activated as cancer-associated-fibroblasts (CAF) that stimulate oncogenic signaling in TEC. In this study, we ...evaluated the cross-talk between CAF and TEC isolated from tumors generated in a mouse model of KRAS/mut p53-induced pancreatic cancer (KPC mice). Transcriptomic profiling conducted after treatment with the anticancer compound Minnelide revealed deregulation of the TGFβ signaling pathway in CAF, resulting in an apparent reversal of their activated state to a quiescent, nonproliferative state. TEC exposed to media conditioned by drug-treated CAFs exhibited a decrease in oncogenic signaling, as manifested by downregulation of the transcription factor Sp1. This inhibition was rescued by treating TEC with TGFβ. Given promising early clinical studies with Minnelide, our findings suggest that approaches to inactivate CAF and prevent tumor-stroma cross-talk may offer a viable strategy to treat pancreatic cancer.
In an established mouse model of pancreatic cancer, administration of the promising experimental drug Minnelide was found to actively deplete reactive stromal fibroblasts and to trigger tumor regression, with implications for stromal-based strategies to attack this disease.
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Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) signaling have been shown to be dysregulated in multiple cancer types. Glucose regulatory protein 78 (GRP78), the master ...regulator of the UPR, plays a role in proliferation, invasion, and metastasis in cancer. Cancer stem cells (CSCs) make up a crucial component of the tumor heterogeneity in pancreatic cancer, as well as other cancers. "Stemness" in pancreatic cancer defines a population of cells within the tumor that have increased therapeutic resistance as well as survival advantage. In the current study, we investigated how GRP78 was responsible for maintaining "stemness" in pancreatic cancer thereby contributing to its aggressive biology. We determined that GRP78 downregulation decreased clonogenicity and self-renewal properties in pancreatic cancer cell lines in vitro. In vivo studies resulted in delayed tumor initiation frequency, as well as smaller tumor volume in the shGRP78 groups. Additionally, downregulation of GRP78 resulted in dysregulated fatty acid metabolism in pancreatic tumors as well as the cells. Further, our results showed that shGRP78 dysregulates multiple transcriptomic and proteomic pathways that involve DNA damage, oxidative stress, and cell death, that were reversed upon treatment with a ROS inhibitor, N-acetylcysteine. This study thus demonstrates for the first time that the heightened UPR in pancreatic cancer may be responsible for maintenance of the "stemness" properties in these cells that are attributed to aggressive properties like chemoresistance and metastasis.
We studied the effects of gut microbiome depletion by oral antibiotics on tumor growth in subcutaneous and liver metastases models of pancreatic cancer, colon cancer, and melanoma. Gut microbiome ...depletion significantly reduced tumor burden in all the models tested. However, depletion of gut microbiome did not reduce tumor growth in Rag1-knockout mice, which lack mature T and B cells. Flow cytometry analyses demonstrated that gut microbiome depletion led to significant increase in interferon gamma–producing T cells with corresponding decrease in interleukin 17A and interleukin 10–producing T cells. Our results suggest that gut microbiome modulation could emerge as a novel immunotherapeutic strategy.
Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with an immunosuppressive microenvironment that is resistant to most therapies. IL17 is involved in pancreatic tumorigenesis, but ...its role in invasive PDAC is undetermined. We hypothesized that IL17 triggers and sustains PDAC immunosuppression. We inhibited IL17/IL17RA signaling using pharmacological and genetic strategies alongside mass cytometry and multiplex immunofluorescence techniques. We uncovered that IL17 recruits neutrophils, triggers neutrophil extracellular traps (NETs), and excludes cytotoxic CD8 T cells from tumors. Additionally, IL17 blockade increases immune checkpoint blockade (PD-1, CTLA4) sensitivity. Inhibition of neutrophils or Padi4-dependent NETosis phenocopies IL17 neutralization. NMR spectroscopy revealed changes in tumor lactate as a potential early biomarker for IL17/PD-1 combination efficacy. Higher expression of IL17 and PADI4 in human PDAC corresponds with poorer prognosis, and the serum of patients with PDAC has higher potential for NETosis. Clinical studies with IL17 and checkpoint blockade represent a novel combinatorial therapy with potential efficacy for this lethal disease.
Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic background of the tumor and better ...understanding of the tumor microenvironment, immune checkpoint inhibitor therapy (targeting CTLA4, PD1, PDL1) has not been very successful against PDAC. The robust desmoplastic stroma, along with an extensive extracellular matrix (ECM) that is rich in hyaluronan, plays an integral role in this immune evasion. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on the one hand and influence the hyaluronan synthesis in the ECM on the other. The rate-limiting enzyme of the pathway, glutamine-fructose amidotransferase 1 (GFAT1), uses glutamine and fructose 6-phosphate to eventually synthesize uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). In the current manuscript, we targeted this glutamine-utilizing enzyme by a small molecule glutamine analog (6-diazo-5-oxo-l-norleucine DON). Our results showed that DON decreased the self-renewal potential and metastatic ability of tumor cells. Further, treatment with DON decreased hyaluronan and collagen in the tumor microenvironment, leading to an extensive remodeling of the ECM and an increased infiltration of CD8+ T cells. Additionally, treatment with DON sensitized pancreatic tumors to anti-PD1 therapy, resulting in tumor regression and prolonged survival.
The lack of tools for early detection of pancreatic ductal adenocarcinoma (PDAC) is directly correlated with the abysmal survival rates in patients. In addition to several potential detection tools ...under active investigation, we tested the gut microbiome and its metabolic complement as one of the earliest detection tools that could be useful in patients at high risk for PDAC. We used a combination of 16s rRNA pyrosequencing and whole-genome sequencing of gut fecal microbiota in a genetically engineered PDAC murine model (KRASG12DTP53R172HPdxCre or KPC). Metabolic reconstruction of microbiome was done using the HUMAnN2 pipeline. Serum polyamine levels were measured from murine and patient samples using chromogenic assay. Our results showed a Proteobacterial and Firmicutes dominance in gut microbiota in early stages of PDAC development. Upon in silico reconstruction of active metabolic pathways within the altered microbial flora, polyamine and nucleotide biosynthetic pathways were significantly elevated. These metabolic products are known to be actively assimilated by the host and eventually utilized by rapidly dividing cells for proliferation validating their importance in the context of tumorigenesis. In KPC mice, as well as PDAC patients, we show significantly elevated serum polyamine concentrations. Therefore, at the early stages of tumorigenesis, there is a strong correlation between microbial changes and release of metabolites that foster host tumorigenesis, thereby fulfilling the 'vicious cycle hypothesis' of the role of microbiome in health and disease states. Our results provide a potential, precise, noninvasive tool for early detection of PDAC, which may result in improved outcomes.
Pancreatic cancer stromal microenvironment is considered to be the major reason for failure of conventional and targeted therapy for this disease. The desmoplastic stroma, comprising mainly collagen ...and glycosaminoglycans like hyaluronan (HA), is responsible for compression of vasculature in the tumor resulting in impaired drug delivery and poor prognosis. Minnelide, a water-soluble prodrug of triptolide currently in phase I clinical trial, has been very effective in multiple animal models of pancreatic cancer. However, whether Minnelide will have efficacious delivery into the tumor despite the desmoplastic stroma has not been evaluated before.
Patient tumor-derived xenografts (PDX) and spontaneous pancreatic cancer mice were treated with 0.42 and 0.21 mg/kg body weight for 30 days. Stromal components were determined by IHC and ELISA-based assays. Vascular functionality and drug delivery to the tumor were assessed following treatment with Minnelide.
Our current study shows that treatment with Minnelide resulted in reduction of ECM components like HA and collagen in the pancreatic cancer stroma of both the spontaneous KPC mice as well as in patient tumor xenografts. Furthermore, treatment with Minnelide improved functional vasculature in the tumors resulting in four times more functional vessels in the treated animals compared with untreated animals. Consistent with this observation, Minnelide also resulted in increased drug delivery into the tumor compared with untreated animals. Along with this, Minnelide also decreased viability of the stromal cells along with the tumor cells in pancreatic adenocarcinoma.
In conclusion, these results are extremely promising as they indicate that Minnelide, along with having anticancer effects is also able to deplete stroma in pancreatic tumors, which makes it an effective therapy for pancreatic cancer.
Resistance to therapy is one of the major factors that contribute to dismal survival statistics in pancreatic cancer. While there are many tumor intrinsic and tumor microenvironment driven factors ...that contribute to therapy resistance, whether pre-existing metabolic diseases like type 2 diabetes (T2D) contribute to this has remained understudied. It is well accepted that hyperglycemia associated with type 2 diabetes changes the gut microbiome. Further, hyperglycemia also enriches for a "stem-like" population within the tumor. In the current study, we observed that in a T2D mouse model, the microbiome changed significantly as the hyperglycemia developed in these animals. Our results further showed that, tumors implanted in the T2D mice responded poorly to gemcitabine/paclitaxel (Gem/Pac) standard of care compared to those in the control group. A metabolomic reconstruction of the WGS of the gut microbiota further revealed that an enrichment of bacterial population involved in drug metabolism in the T2D group. Additionally, we also observed an increase in the CD133+ tumor cells population in the T2D model. These observations indicated that in an animal model for T2D, microbial dysbiosis is associated with increased resistance to chemotherapeutic compounds.
Pancreatic cancer is one of the most lethal human malignancies with an all-stage 5-year survival frequency of <5%, which highlights the urgent need for more effective therapeutic strategies. We have ...previously shown that triptolide, a diterpenoid, is effective against pancreatic cancer cells in vitro as well as in vivo. However, triptolide is poorly soluble in water, limiting its clinical use. We therefore synthesized a water-soluble analog of triptolide, named Minnelide. The efficacy of Minnelide was tested both in vitro and in multiple independent yet complementary in vivo models of pancreatic cancer: an orthotopic model of pancreatic cancer using human pancreatic cancer cell lines in athymic nude mice, a xenograft model where human pancreatic tumors were transplanted into severe combined immunodeficient mice, and a spontaneous pancreatic cancer mouse model (KRas(G12D); Trp53(R172H); Pdx-1Cre). In these multiple complementary models of pancreatic cancer, Minnelide was highly effective in reducing pancreatic tumor growth and spread, and improving survival. Together, our results suggest that Minnelide shows promise as a potent chemotherapeutic agent against pancreatic cancer, and support the evaluation of Minnelide in clinical trials against this deadly disease.
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