Matrix Metalloproteinase-1 (MMP-1) Promoter Polymorphisms are Well Linked with Lower Stomach Tumor Formation in Eastern Indian Population

Expression of matrix metalloproteinase-1 (MMP-1), an interstitial collagenase, plays a major role in cellular invasion during development of gastric cancer, a leading cause of death worldwide. A single-nucleotide polymorphism (SNP) 21607 1G/2G site of the MMP-1 gene promoter has been reported to alter transcription level. While the importance’s of other SNPs in the MMP-1 promoter have not yet been studied in gastric cancer, our aim was to investigate MMP-1 gene promoter polymorphisms and gastric cancer susceptibility in eastern Indian population. A total of 145 gastric cancer patients and 145 healthy controls were genotyped for MMP-1 21607 1G/2G (rs1799750) by PCR-restriction fragment length polymorphism (RFLP), while MMP-1 2519 A/G (rs1144393), MMP-1 2422 T/A (rs475007), MMP-1 2340 T/C (rs514921) and MMP-1 2320 T/C (rs494379) were genotyped by DNA sequencing. A positive association was found with MMP-1 2422 T/A SNP that showed significant risk for regional lymph node metastasis (P = 0.021, Odd’s ratio (OR) = 3.044, Confidence intervals (CI) = 1.187– 7.807). In addition, we found a significant association with lower stomach tumor formation among gastric cancer patients for three adjacent polymorphisms near the transcriptional start sites of [MMP-1 2422 T/A (P = 0.043, OR = 2.182, CI = 1.03– 4.643), MMP-1 2340 T/C (P = 0.075, OR = 1.97, CI = 0.94–4.158) and MMP-1 2320 T/C (P = 0.034, OR = 2.224, CI = 1.064– 40731)]. MMP-1 level in patients’ serum was correlated with MMP-1 promoter haplotypes conferring these three SNPs to evaluate the functional importance of these polymorphisms in lower stomach tumor formation and significant correlation was observed. Furthermore, MMP-1 2519 A/G polymorphism displayed poor cellular differentiation (P = 0.024, OR = 3.8, CI = 1.69–8.56) attributing a higher risk of cancer progression.In conclusion, MMP-1 proximal promoter SNPs are associated with the risk of lower stomach tumor formation and node metastasis in eastern Indian population

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

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-1β 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-β 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 expressio

Genetic and epigenetic signature of matrix metalloproteinase and tissue inhibitor of metalloproteinases and their association with risk of gastric carcinoma

Cancer (medical term: malignant neoplasm) is a disease of uncontrolled cellular growth, invasion that sometimes spread to other locations in the body via lymph or blood. Six potential alterations occur in cell physiology during cancer progression: (a) self support in growth signals (b) insensitivity to growth-inhibitory signals (c) escape from apoptosis (d) infinite replication (e) sustained angiogenesis and (f) tissue invasion and metastasis (Hanahan and Weinberg 2011, 2000). Tumor cells follow a multistep process namely: tumor cell detachment, local invasion, cell motility, angiogenesis, vessel invasion, survival in circulation, adhesion to endothelial cells, extravasation and regrowth in different organs during metastasis (Hanahan and Weinberg 2000). Stomach cancer or gastric cancer is developed from the lining of the stomach and found most often in people of older age. The disease affects men twice as often as women. Although the overall incidence rate of gastric cancer is declining over the last several decades however, gastric cancer remains the forth most common type of cancer and is the second leading cause of cancerrelated death worldwide (Jemal et al. 2011; Bertuccio et al. 2009). The steady decline in the incidence and mortality of stomach cancer in most developed countries is due to changes in nutrition pattern, improvement in food storage, advanced treatment and control of H. pylori infection (Jarosz et al. 2011; Amiri, Janssen, and Kunst 2011; Zhu and Sonnenberg 2012; Katanoda and Yako-Suketomo 2009; Bertuccio et al. 2009). Early gastric cancer symptoms may be associated with indigestion or a burning sensation (heartburn), which most of the cases remain neglected in developing countries. However, about 2% referred for endoscopy due to indigestion are diagnosed as gastric cancer. Unfortunately, in most of the cases, gastric cancer diagnose at late stage when the disease reaches in an advanced stage (Maconi, Manes, and Porro 2008). Advanced or metastatic gastric cancer treatment achieved little progress with survival of less than 1 year (Wilke and Van Cutsem 2003)

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. (Am J Pathol 2022, 192: 1136-1150; https://doi.org/10.1016/j.ajpath.2022.04.005

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

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 PurposeOpioids 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 ApproachMice 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 ResultsChronic 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 ImplicationsThis 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

Abstract 5284: Hypoxia promote self-renewal in pancreatic cancer by modulating L-2-Hydroxyglutarate level

Abstract Pancreatic cancer cells undergo extensive metabolic reprogramming to fulfil the demands of metabolic nutrients to fuel their rapid proliferation. This results in abnormal accumulation of various metabolites which causes both metabolic and nonmetabolic dysregulation and potential transformation to malignancy and therefore termed as “oncometabolites”. Hypoxia is a major hallmark of pancreatic tumors which associated with increase in self-renewal. The present study investigates the role of Hypoxia mediated altered oncometabolite in regulating self-renewal in pancreatic cancer. GC-mass spectrometric analysis of pancreatic cancer cells under hypoxia showed significant accumulation of 2-hydroxyglutarate (2-HG) both in cells and culture supernatant. Further analysis of hypoxic cells showed selective accumulation L- enantiomer form. Self-renewal requires critical balance between stemness and differentiation. Present study showed for the first time that L-2-HG regulates self-renewal by increasing expression of genes associated with stemness (Sox-2, CD133) and by decreasing expression of differentiation genes (PdX-1, HB9, NKX6.1). Further analysis showed that L-2-HG mediated epigenetic changes regulates these self-renewal gens in pancreatic cancer. Our results indicated that hypoxia mediated metabolic changes results in accumulation of L-2-HG which upregulate self-renewal by shifting critical balance of gene expression towards stemness in pancreatic cancer which contribute to chemoresistance and metastasis in Pancreatic cancer. Citation Format: Vineet K. Gupta, Nikita Sharma, Kousik Kesh, Roey Hadad, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. Hypoxia promote self-renewal in pancreatic cancer by modulating L-2-Hydroxyglutarate level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5284

Abstract A67: Microbial polyamines and early detection of pancreatic cancer

Abstract The lack of tools for early detection of pancreatic ductal adenocarcinoma (PDAC) is directly correlated to the abysmal survival rate in patients. In addition to several potential detection tools under active investigation, we present 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. A combination of 16s pyrosequencing and whole-genome sequencing of gut microbiota in a spontaneous genetically engineered PDAC murine model (KRASG12DTP53R172HPdxCre or KPC) showed a progressive 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 found to be 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 concentration. Therefore, at the early stages of tumorigenesis, the gut microbial composition changes in a way to promote inflammation and release metabolites that foster host tumorigenesis, thereby fulfilling the “vicious cycle hypothesis” of the role of the microbiome in health and disease states. Our results provide a potential, precise, noninvasive tool for early detection of PDAC, which will result in improved outcomes. Citation Format: Roberto Mendez, Kousik Kesh, Nivedita Arora, Leá Di Martino, Florencia McAllister, Nipun Merchant, Sulagna Banerjee, Santanu Banerjee. Microbial polyamines and early detection of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A67

Abstract 4353: Stroma drive metabolic reprogramming and induces stemness properties in pancreatic cancer cells

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most devastating human malignancies with poor survival rate owing to chemoresistance and relapse. Small population of Tumor Initiating Cells (TIC) are considered major players driving these traits. Recent literature has demonstrated the importance of microenvironment “niche” in enrichment of these TIC populations. The desmoplastic stroma in pancreatic cancer comprising of stromal cells and immune cells are instrumental in pathogenesis and progression of PDAC. Among many cytokines, IL-6 a pro-inflammatory cytokine plays a major role in it. In this study, we show that stromal cell secreted IL6 altered metabolic reprogramming and increased cancer stemness in PDAC cells. PDAC cells cultured with pancreatic stellate cells condition media (PSC-CM) or recombinant IL6 and stem ness markers expression were investigated. Cytometry Bead Array (CBA) were used for cytokine profiling. Metabolic alteration along with TIC gene expression were measured using flow cytometry and Seahorse. PDAC cells when cultured with PSC-CM exhibited increase glucose uptake along with an enrichment of CD133+ population. An analysis of the conditioned media using CBA reveled that PSC-CM is enriched with cytokine IL-6 and TNF-α. Treatment of PDAC cells with recombinant IL-6 increased expression of self-renewal genes SOX2, OCT4, Nanog along with biochemical marker ALDH. In addition, TIC marker CD133 and invasion marker gene MMP7 expression also increased upon IL6 induction. Moreover, IL6 treatment increased glucose uptake and lactate secretion. Small molecule inhibitor Stattic (inhibitor of STAT3) inhibit glucose uptake and stemness gene expression.Our study shows that stromal IL6 altered metabolic reprogramming in cancer cells as well as increased cancer stemness and invasion. In future, the underlying mechanisms of IL-6 driven metabolic alteration and its impact on stemness and metastasis will be investigated. Citation Format: Kousik K. Kesh, Vineet K. Gupta, Nikita Sharma, Roey Hadad, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. Stroma drive metabolic reprogramming and induces stemness properties in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4353