Opioid Use in Murine Model Results in Severe Gastric Pathology that May Be Attenuated by Proton Pump Inhibition.

Opioids are the gold standard for chronic and acute pain management; however, their consequence on gastric function is relatively understudied. Opioid users have a higher incidence of gastric dysfunction, worse quality of life, increased hospitalizations, and increased use of antiemetic and pain modulator medications. The current study shows that morphine treatment in the murine model results in greater disruption of gastric epithelial cell morphology, increased gastric cell apoptosis, elevated inflammatory cytokines, and matrix metallopeptidase-9 secretion. Morphine treatment also increases gastric acid secretion and causes delays in gastric emptying. Moreover, morphine treatment causes an increase in systemic IL-6 level, which plays an important role in morphine-induced delayed gastric emptying and gastric damage. IL-6 knockout mice show a significant level of reduction in morphine-induced gastric delaying, acid retention, and gastric damage. Thus, morphine-mediated gastric damage is a consequence of the accumulation of acid in the stomach due to increased gastric acid secretion and delayed gastric emptying. Treatment with a proton pump inhibitor resulted in a significant reduction in morphine-induced gastric inflammation, gastric delaying, and improved morphine tolerance. Hence, these studies attribute morphine-mediated induction in gastric acidity and inflammatory cytokines as drivers for morphine-associated gastric pathology and show the therapeutic use of proton pump inhibitors as an inexpensive approach for clinical management of morphine-associated pathophysiology and analgesic tolerance.

Microbial dysbiosis and polyamine metabolism as predictive markers for early detection of pancreatic cancer.

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.

Type 2 diabetes induced microbiome dysbiosis is associated with therapy resistance in pancreatic adenocarcinoma.

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.

Association of MMP7 -181A→G Promoter Polymorphism with Gastric Cancer Risk: INFLUENCE OF NICOTINE IN DIFFERENTIAL ALLELE-SPECIFIC TRANSCRIPTION VIA INCREASED PHOSPHORYLATION OF cAMP-RESPONSE ELEMENT-BINDING PROTEIN (CREB).

Elevated expression of matrix metalloproteinase7 (MMP7) has been demonstrated to play a pivotal role in cancer invasion. The -181A→G (rs11568818) polymorphism in the MMP7 promoter modulates gene expression and possibly affects cancer progression. Here, we evaluated the impact of -181A→G polymorphism on MMP7 promoter activity and its association with gastric cancer risk in eastern Indian case-control cohorts (n = 520). The GG genotype as compared with the AA genotype was predisposed (p = 0.02; odds ratio = 1.9, 95% confidence interval = 1.1-3.3) to gastric cancer risk. Stratification analysis showed that tobacco addiction enhanced gastric cancer risk in GG subjects when compared with AA subjects (p = 0.03, odds ratio = 2.46, and 95% confidence interval = 1.07-5.68). Meta-analysis revealed that tobacco enhanced the risk for cancer more markedly in AG and GG carriers. Activity and expression of MMP7 were significantly higher in GG than in AA carriers. In support, MMP7 promoter-reporter assays showed greater transcriptional activity toward A to G transition under basal/nicotine-induced/cAMP-response element-binding protein (CREB) overexpressed conditions in gastric adenocarcinoma cells. Moreover, nicotine (a major component of tobacco) treatment significantly up-regulated MMP7 expression due to enhanced CREB phosphorylation followed by its nuclear translocation in gastric adenocarcinoma cells. Furthermore, chromatin immunoprecipitation experiments revealed higher binding of phosphorylated CREB with the -181G than the -181A allele. Altogether, specific binding of phosphorylated CREB to the G allele-carrying promoter enhances MMP7 gene expression that is further augmented by nicotine due to increased CREB phosphorylation and thereby increases the risk for gastric cancer.

Prescription opioids induced microbial dysbiosis worsens severity of chronic pancreatitis and drives pain hypersensitivity.

Opioids, such as morphine and oxycodone, are widely used for pain management associated with chronic pancreatitis (CP); however, their impact on the progression and pain sensitivity of CP has never been evaluated. This report investigates the impact of opioid use on the severity of CP, pain sensitivity, and the gut microbiome. C57BL/6 mice were divided into control, CP, CP with morphine/oxycodone, and either morphine or oxycodone alone groups. CP was induced by administration of caerulein (50ug/kg/h, i.p. hourly x7, twice a week for 10 weeks). The mouse-to-pancreas weight ratio, histology, and Sirius red staining were performed to measure CP severity. Tail flick and paw pressure assays were used to measure thermal and mechanical pain. DNA was extracted from the fecal samples and subjected to whole-genome shotgun sequencing. Germ-free mice were used to validate the role of gut microbiome in sensitizing acute pancreatic inflammation. Opioid treatment exacerbates CP by increasing pancreatic necrosis, fibrosis, and immune-cell infiltration. Opioid-treated CP mice exhibited enhanced pain hypersensitivity and showed distinct clustering of the gut microbiome compared to untreated CP mice, with severely compromised gut barrier integrity. Fecal microbiota transplantation (FMT) from opioid-treated CP mice into germ-free mice resulted in pancreatic inflammation in response to a suboptimal caerulein dose. Together, these analyses revealed that opioids worsen the severity of CP and induce significant alterations in pain sensitivity and the gut microbiome in a caerulein CP mouse model. Microbial dysbiosis plays an important role in sensitizing the host to pancreatic inflammation.

Morphine use induces gastric microbial dysbiosis driving gastric inflammation through TLR2 signalling which is attenuated by proton pump inhibition.

BACKGROUND AND PURPOSE: Opioids are the standard drug for pain management; however, their effects on gastric dysfunction are relatively understudied. Opioid users have a higher incidence of gastric pathology leading to increased hospitalization. Herein, we investigated the consequences of morphine use on gastric pathology and the underlying mechanisms. We further investigated the therapeutic benefit of proton pump inhibition to overcome morphine-mediated gastric inflammation. EXPERIMENTAL APPROACH: Mice were implanted with 25 mg slow-release morphine and placebo pellets. Gastric microbiome analyses were performed. Gastric damage was assayed. Gastric pH was measured. Germ-free and TLR2KO mice were used to investigate the mechanisms. Gastroprotective studies were performed with the proton pump inhibitor (PPI) omeprazole. KEY RESULTS: Chronic morphine treatment alters gastric microbial composition and induces preferential expansion of pathogenic bacterial communities such as Streptococcus and Pseudomonas. Morphine causes disruption of the gastric mucosal layer, increases apoptosis, and elevates inflammatory cytokines. Moreover, morphine-mediated gastric pathology was significantly attenuated in germ-free mice, and reconstitution of morphine gastric microbiome in germ-free mice resulted gastric inflammation. In addition, morphine-mediated gastric inflammation was attenuated in TLR2KO mice. Morphine causes a decrease in gastric pH, which contributes to gastric dysbiosis leads to gastric inflammation. Omeprazole treatment inhibits gastric acidity, rescuing morphine-induced gastric dysbiosis and preventing inflammation. CONCLUSION AND IMPLICATIONS: This study attributes morphine-induced gastric acidity as a driver of gastric dysbiosis and pathology and proposes the therapeutic use of PPI as an inexpensive approach for the clinical management of morphine-associated pathophysiology.

Matrix metalloproteinase3 gene promoter polymorphisms and their haplotypes are associated with gastric cancer risk in eastern Indian population.

Single nucleotide polymorphisms (SNPs) of matrix metalloproteinase3 (MMP3) promoter in the development and progression of gastric cancer of whole stomach has never been investigated in any population. We conducted a hospital-based case-control study to explore the MMP3 SNPs and their haplotypes with the risk of gastric cancer for the first time in eastern Indian population. A total of 218 gastric cancer patients and 175 healthy controls were genotyped for MMP3-1612 5A/6A (rs3025058) by PCR-RFLP and rechecked 10% by DNA sequencing. MMP3-707 A/G (rs522616) and MMP3-375 C/G (rs617819) were genotyped by DNA sequencing among 209 patients and 154 controls. MMP3-1612 5A6A genotype (P = 0.026, odds ratio (OR) = 1.756, confidence interval (CI) = 1.070-2.883), combined 5A5A and 5A6A genotype (P = 0.015, OR = 1.791, CI = 1.122-2.858) and 5A allele (P = 0.002, OR = 1.75, CI = 1.21-2.53) and; MMP3-707 GG genotype (P = < 0.0001; OR = 9.612; 95% CI = 3.403-27.147), combined GG and AG genotype (P = 0.001, OR = 2.201, CI = 1.385-3.498) and G allele (P = <0.0001, OR = 2.189, CI = 1.582-3.033) conferred significant risk for gastric cancer development. Also, tobacco addicted individuals with combined 5A5A and 5A6A genotype (P = 0.005, OR = 2.952, CI = 1.377-6.327) at -1612 position of MMP3 promoter displayed a higher risk to gastric cancer development. The genotypic combinations of all three MMP3 promoter polymorphisms and their haplotypes with increasing risk allele in a dose-dependent manner showed a potential risk for developing gastric cancer. The analyses suggested that the MMP3-707 G/G and MMP3-1612 5A/6A polymorphisms are potential independent predictors of gastric cancer risk development.

Obesity enriches for tumor protective microbial metabolites and treatment refractory cells to confer therapy resistance in PDAC.

Obesity causes chronic inflammation and changes in gut microbiome. However, how this contributes to poor survival and therapy resistance in patients with pancreatic cancer remain undetermined. Our current study shows that high fat diet-fed obese pancreatic tumor bearing mice do not respond to standard of care therapy with gemcitabine and paclitaxel when compared to corresponding control diet-fed mice. C57BL6 mice were put on control and high fat diet for 1 month following with pancreatic tumors were implanted in both groups. Microbiome of lean (control) and obese (high fat diet fed) mice was analyzed. Fecal matter transplant from control mice to obese mice sensitized tumors to chemotherapy and demonstrated extensive cell death. Analysis of gut microbiome showed an enrichment of queuosine (Q) producing bacteria in obese mice and an enrichment of S-adenosyl methionine (SAM) producing bacteria in control diet-fed mice. Further, supplementation of obese animals with SAM sensitized pancreatic tumors to chemotherapy. Treatment of pancreatic cancer cells with Q increased PRDX1 involved in oxidative stress protection. In parallel, tumors in obese mice showed increase in CD133+ treatment refractory tumor populations compared to control animals. These observations indicated that microbial metabolite Q accumulation in high fat diet-fed mice protected tumors from chemotherapy induced oxidative stress by upregulating PRDX1. This protection could be reversed by treatment with SAM. We conclude that relative concentration of SAM and queuosine in fecal samples of pancreatic cancer patients can be developed as a potential biomarker and therapeutic target in chemotherapy refractory pancreatic cancer.

Hypoxia-Driven Oncometabolite L-2HG Maintains Stemness-Differentiation Balance and Facilitates Immune Evasion in Pancreatic Cancer.

In pancreatic cancer, the robust fibroinflammatory stroma contributes to immune suppression and renders tumors hypoxic, altering intratumoral metabolic pathways and leading to poor survival. One metabolic enzyme activated during hypoxia is lactate dehydrogenase A (LDHA). As a result of its promiscuous activity under hypoxia, LDHA produces L-2 hydroxyglutarate (L-2HG), an epigenetic modifier, that regulates the tumor transcriptome. However, the role of L-2HG in remodeling the pancreatic tumor microenvironment is not known. Here we used mass spectrometry to detect L-2HG in serum samples from patients with pancreatic cancer, comprising tumor cells as well as stromal cells. Both hypoxic pancreatic tumors as well as serum from patients with pancreatic cancer accumulated L-2HG as a result of promiscuous activity of LDHA. This abnormally accumulated L-2HG led to H3 hypermethylation and altered gene expression, which regulated a critical balance between stemness and differentiation in pancreatic tumors. Secreted L-2HG inhibited T-cell proliferation and migration, suppressing antitumor immunity. In a syngeneic orthotopic model of pancreatic cancer, inhibition of LDH with GSK2837808A decreased L-2HG, induced tumor regression, and sensitized tumors to anti-PD1 therapy. In conclusion, hypoxia-mediated promiscuous activity of LDH produces L-2HG in pancreatic tumor cells, regulating the stemness-differentiation balance and contributing to immune evasion. Targeting LDH can be developed as a potential therapy to sensitize pancreatic tumors to checkpoint inhibitor therapy. SIGNIFICANCE: This study shows that promiscuous LDH activity produces L-2HG in pancreatic tumor and stromal cells, modulating tumor stemness and immune cell function and infiltration in the tumor microenvironment.

Therapy Resistance, Cancer Stem Cells and ECM in Cancer: The Matrix Reloaded.

The extracellular matrix (ECM) has remained an enigmatic component of the tumor microenvironment. It drives metastasis via its interaction with the integrin signaling pathway, contributes to tumor progression and confers therapy resistance by providing a physical barrier around the tumor. The complexity of the ECM lies in its heterogeneous composition and complex glycosylation that can provide a support matrix as well as trigger oncogenic signaling pathways by interacting with the tumor cells. In this review, we attempt to dissect the role of the ECM in enriching for the treatment refractory cancer stem cell population and how it may be involved in regulating their metabolic needs. Additionally, we discuss how the ECM is instrumental in remodeling the tumor immune microenvironment and the potential ways to target this component in order to develop a viable therapy.

Stroma secreted IL6 selects for "stem-like" population and alters pancreatic tumor microenvironment by reprogramming metabolic pathways.

Pancreatic adenocarcinoma is a devastating disease with an abysmal survival rate of 9%. A robust fibro-inflammatory and desmoplastic stroma, characteristic of pancreatic cancer, contribute to the challenges in developing viable therapeutic strategies in this disease. Apart from constricting blood vessels and preventing efficient drug delivery to the tumor, the stroma also contributes to the aggressive biology of cancer along with its immune-evasive microenvironment. In this study, we show that in pancreatic tumors, the developing stroma increases tumor initiation frequency in pancreatic cancer cells in vivo by enriching for CD133 + aggressive "stem-like" cells. Additionally, the stromal fibroblasts secrete IL6 as the major cytokine, increases glycolytic flux in the pancreatic tumor cells, and increases lactate efflux in the microenvironment via activation of the STAT signaling pathway. We also show that the secreted lactate favors activation of M2 macrophages in the tumor microenvironment, which excludes CD8 + T cells in the tumor. Our data additionally confirms that the treatment of pancreatic tumors with anti-IL6 antibody results in tumor regression as well as decreased CD133 + population within the tumor. Furthermore, inhibiting the lactate efflux in the microenvironment reduces M2 macrophages, and makes pancreatic tumors more responsive to anti-PD1 therapy. This suggests that stromal IL6 driven metabolic reprogramming plays a significant role in the development of an immune-evasive microenvironment. In conclusion, our study shows that targeting the metabolic pathways affected by stromal IL6 can make pancreatic tumors amenable to checkpoint inhibitor therapy.

Targeting tumor-intrinsic hexosamine biosynthesis sensitizes pancreatic cancer to anti-PD1 therapy.

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.

Labeling Cancer Stem Cells with 13C6 Glucose and 13C5 Glutamine for Metabolic Flux Analysis.

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are a population of cells present within tumor that have increased self-renewal, chemoresistance, and aggressiveness, thereby contributing to tumor relapse. Literature shows that CSCs or TICs typically originate within the hypoxic niches of the tumor, making hypoxia one of the driving factors for generation of this population. Hypoxic stress promotes adaptation to low oxygen tension in the tissues by altering metabolic properties of the CSCs. This leads to a number of altered enzymatic activities in the CSC population that further contribute to the survival of the CSCs leading to resistance to standard therapy. Hence, understanding this altered metabolic pathways as well as targeting key nodes in these may pave the way for cancer management.Glucose and glutamine are the major substrates utilized by cancer cells and feed into multiple biosynthetic pathways. Hence, labeling and tracking these compounds may reveal some novel metabolic pathways exploited by cancer stem cells to acquire survival advantage. In these current book chapters, we elaborately summarized the basic steps required for isolation, characterization, and metabolic labeling (13C6 glucose and 13C5 glutamine) of CSC for flux analysis.

Long non-coding RNA GAS5 acts as proliferation "brakes" in CD133+ cells responsible for tumor recurrence.

Presence of quiescent, therapy evasive population often described as cancer stem cells (CSC) or tumor initiating cells (TIC) is often attributed to extreme metastasis and tumor recurrence. This population is typically enriched in a tumor as a result of microenvironment or chemotherapy induced stress. The TIC population adapts to this stress by turning on cell cycle arrest programs that is a "fail-safe" mechanism to prevent expansion of malignant cells to prevent further injury. Upon removal of the "stress" conditions, these cells restart their cell cycle and regain their proliferative nature thereby resulting in tumor relapse. Growth Arrest Specific 5 (GAS5) is a long-non-coding RNA that plays a vital role in this process. In pancreatic cancer, CD133+ population is a typical representation of the TIC population that is responsible for tumor relapse. In this study, we show for the first time that emergence of CD133+ population coincides with upregulation of GAS5, that reprograms the cell cycle to slow proliferation by inhibiting GR mediated cell cycle control. The CD133+ population further routed metabolites like glucose to shunt pathways like pentose phosphate pathway, that were predominantly biosynthetic in spite of being quiescent in nature but did not use it immediately for nucleic acid synthesis. Upon inhibiting GAS5, these cells were released from their growth arrest and restarted the nucleic acid synthesis and proliferation. Our study thus showed that GAS5 acts as a molecular switch for regulating quiescence and growth arrest in CD133+ population, that is responsible for aggressive biology of pancreatic tumors.

A Common Tag Nucleotide Variant in MMP7 Promoter Increases Risk for Hypertension via Enhanced Interactions With CREB (Cyclic AMP Response Element-Binding Protein) Transcription Factor.

MMP (matrix metalloproteinase)-7-a potent extracellular matrix degrading enzyme-is emerging as a new regulator of cardiovascular diseases. However, potential contributions of MMP7 genetic variations to hypertension remain unknown. In this study, we probed for the association of a tag single-nucleotide polymorphism in the MMP7 promoter (-181A/G; rs11568818) with hypertension in an urban South Indian population (n=1501). The heterozygous AG genotype significantly increased risk for hypertension as compared with the wild-type AA genotype (odds ratio, 1.60 [95% CI, 1.25-2.06]; P=2.4×10-4); AG genotype carriers also displayed significantly higher diastolic blood pressure and mean arterial pressure than wild-type AA individuals. The study was replicated in a North Indian population (n=949) (odds ratio, 1.52 [95% CI, 1.11-2.09]; P=0.01). Transient transfection experiments using MMP7 promoter-luciferase reporter constructs revealed that the variant -181G allele conferred greater promoter activity than the -181A allele. Computational prediction and structure-based conformational and molecular dynamics simulation studies suggested higher binding affinity for the CREB (cyclic AMP response element-binding protein) to the -181G promoter. In corroboration, overexpression/downregulation of CREB and chromatin immunoprecipitation experiments provided convincing evidence for stronger binding of CREB with the -181G promoter. The -181G promoter also displayed enhanced responses to hypoxia and epinephrine treatment. The higher promoter activity of -181G allele translated to increased MMP7 protein level, and MMP7-181AG heterozygous individuals displayed elevated plasma MMP7 levels, which positively correlated with blood pressure. In conclusion, the MMP7 A-181G promoter polymorphism increased MMP7 expression under pathophysiological conditions (hypoxic stress and catecholamine excess) via increased interactions with CREB and enhanced the risk for hypertension in its carriers.

O-GlcNAc modification of Sox2 regulates self-renewal in pancreatic cancer by promoting its stability.

Pancreatic adenocarcinoma (PDAC) claims more than 90% of the patients diagnosed with the disease owing to its aggressive biology that is manifested by high rate of tumor recurrence. Aberrant upregulation in the transcriptional activity of proteins involved in self-renewal like Sox2, Oct4 and Nanog is instrumental in these recurrence phenomena. In cancer, Sox2 is aberrantly "turned-on" leading to activation of downstream genes those results in relapse of the tumor. Molecular mechanisms that regulate the activity of Sox2 in PDAC are not known. In the current study, we have studied the how glycosylation of Sox2 by O-GlcNAc transferase (OGT) can affect its transcriptional activity and thus regulate self-renewal in cancer. Methods: RNA-Seq analysis of CRISPR-OGTi PDAC cells indicated a deregulation of differentiation and self-renewal pathways in PDAC. Pancreatic tumor burden following inhibition of OGT in vivo was done by using small molecule inhibitor, OSMI, on subcutaneous implantation of PDAC cells. Sox2 activity assay was performed by Dual Luciferase Reporter Assay kit. Results: Our study shows for the first time that in PDAC, glycosylation of Sox2 by OGT stabilizes it in the nucleus. Site directed mutagenesis of this site (S246A) prevents this modification. We further show that inhibition of OGT delayed initiation of pancreatic tumors by inhibition of Sox2. We also show that targeting OGT in vivo with a small molecule-inhibitor OSMI, results in decreased tumor burden in PDAC. Conclusion: Understanding this mechanism of SOX2 regulation by its glycosylation is expected to pave the way for development of novel therapy that has the potential to eradicate the cells responsible for tumor-recurrence.

Metastasis and chemoresistance in CD133 expressing pancreatic cancer cells are dependent on their lipid raft integrity.

Metabolic rewiring is an integral part of tumor growth. Among metabolic pathways, the Mevalonic-Acid-Pathway (MVAP) plays a key role in maintaining membrane architecture through cholesterol synthesis, thereby affecting invasiveness. In the current study, we show for the first time that CD133Hi pancreatic tumor initiating cells (TIC) have increased expression of MVAP enzymes, cholesterol-content and Caveolin expression. Further, we show that CD133 in these cells is localized in the lipid-rafts (characterized by Cav-1-cholesterol association). Disruption of lipid-rafts by either depleting Cav-1 or by inhibiting MVAP by lovastatin decreased metastatic-potential and chemoresistance in CD133Hi cells while not affecting the CD133lo cells. Additionally, disruption of lipid-raft results in deregulation of FAK-signaling, decreasing invasiveness in pancreatic-TICs. Furthermore, this also inhibits ABC-transporter activity resulting in sensitizing TICs to standard chemotherapeutic agents. Repurposing existing drugs for new clinical applications is one of the safest and least resource intensive approaches to improve therapeutic options. In this context, our study is extremely timely as it shows that targeting lipid-rafts with statins can sensitize the normally resistant pancreatic TICHi-cells to standard chemotherapy and decrease metastasis, thereby defining a novel strategy for targeting the TICHi-PDAC.

Inactivation of Cancer-Associated-Fibroblasts Disrupts Oncogenic Signaling in Pancreatic Cancer Cells and Promotes Its Regression.

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 TGFß 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 TGFß. 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.Significance: 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. Cancer Res; 78(5); 1321-33. ©2018 AACR.

Attenuation of Helicobacter pylori-induced gastric inflammation by prior cag- strain (AM1) infection in C57BL/6 mice.

BACKGROUND: Helicobacter pylori, colonize in stomach of ~50% of the world population. cag pathogenicity Island of H. pylori is one of the important virulent factors that attributed to gastric inflammation. Coinfection with H. pylori strain with different genetic makeup alters the degree of pathogenicity and susceptibility towards antibiotics. The present study investigates host immunomodulatory effects of H. pylori infection by both cag+ strain (SS1) and cag- strain (AM1). C57BL/6 mice were infected with AM1 or SS1 strain as well as AM1 followed by SS1 (AM1/SS1) and vice versa. RESULTS: Mice infected with AM1/SS1 strain exhibited less gastric inflammation and reduced proMMP9 and proMMP3 activities in gastric tissues as compared to SS1/SS1 and SS1/AM1 infected groups. The expression of both MMP9 and MMP3 followed similar trend like activity in infected tissues. Both Th1 and Th17 responses were induced by SS1 strain more profoundly than AM1 strain infection which induced solely Th1 response in spleen and gastric tissues. Moreover, IFN-γ, TNF-α, IL-1ß and IL-12 were significantly downregulated in mice spleen and gastric tissues infected by AM1/SS1 compared to SS1/SS1 but not with SS1/AM1 coinfection. Surprisingly, IL-17 level was dampened significantly in AM1/SS1 compared to SS1/AM1 coinfected groups. Furthermore, number of Foxp3+ T-regulatory (Treg) cells and immunosuppressive cytokines like IL-10 and TGF-ß were reduced in AM1/SS1 compared to SS1/SS1 and SS1/AM1 coinfected mice gastric tissues. CONCLUSIONS: These data suggested that prior H. pylori cag- strain infection attenuated the severity of gastric pathology induced by subsequent cag+ strain in C57BL/6 mice. Prior AM1 infection induced Th1 cytokine IFN-γ, which reduced the Th17 response induced by subsequent SS1 infection. The reduced gastritis in AM1/SS1-infected mice might also be due to enrichment of AM1- primed Treg cells in the gastric compartment which inhibit Th1 and Th17 responses to subsequent SS1 infection. In summary, prior infection by non-virulent H. pylori strain (AM1) causes reduction of subsequent virulent strain (SS1) infection by regulation of inflammatory cytokines and MMPs expression.

An Overview of Matrix Metalloproteinase 9 Polymorphism and Gastric Cancer Risk.

Matrix metalloproteinase (MMP) 9, a key member of multifunctional family of zinc dependent endopeptidases has been found to be upregulated during inflammation and in some cancers. MMPs cleave extracellular matrix (ECM) proteins and play critical roles in cellular apoptosis, angiogenesis, tumor growth and metastasis. Several genetic polymorphisms have been identified that show allele specific effects on MMP9 regulation and are associated with gastric cancer, the fourth most common malignancy in the world. Besides Helicobacter pylori infection, genetic predisposition is another documented risk factor for gastric carcinoma. The single nucleotide polymorphism (SNP) at position -1562C/T of MMP9 results in the modulation for binding of transcription factors to the MMP9 gene promoter and thereby causes differences in protein expression and enzymatic activity. MMP9 transcriptional regulation during gastric cancer development remains poorly known although several studies have demonstrated associations between MMP9 -1562 C/T polymorphism with different diseases. Knowledge on mechanisms of MMP9 upregulation during gastric cancer may provide new paradigm in diagnostics and therapeutics.