Metabolic Interactions Between Tumor and Stromal Cells in the Tumor Microenvironment.

In this chapter, we provide information about metabolic reprogramming in cancer cells, molecular interactions between tumor and stromal cells in the tumor microenvironment, focusing primarily on CAFs and tumor cell interaction. We have covered the role of cytokines, chemokines, and lactate in driving tumor-stroma interactions in the microenvironment. Here, we have discussed the pro-tumorigenic molecular interactions in between tumor cells and CAFs mediated via altered signaling pathways, cytokines, chemokines, and lactate in the tumor vicinity. A better understanding of the complex cancer cell-CAF interactions will help in designing successful therapeutic strategies targeting the stromal-rich tumors in the clinic.

Tumor stroma interaction is mediated by monocarboxylate metabolism.

Human breast tumors contain significant amounts of stromal cells. There exists strong evidence that these stromal cells support cancer development and progression by altering various pathways (e.g. downregulation of tumor suppressor genes or autocrine signaling loops). Here, we suggest that stromal carcinoma-associated fibroblasts (CAFs), shown to be generated from bone marrow-derived mesenchymal stem cells, may (i) recycle tumor-derived lactate for their own energetic requirements, thereby sparing glucose for neighboring glycolytic tumor cells, and (ii) subsequently secrete surplus energetically and biosynthetically valuable metabolites of lactate oxidation, such as pyruvate, to support tumor growth. Lactate, taken up by stromal CAFs, is converted to pyruvate, which is then utilized by CAFs for energy needs as well as excreted and shared with tumor cells. We have interrogated lactate oxidation in CAFs to determine what metabolites may be secreted, and how they may affect the metabolism and growth of MDA-MB-231 breast cancer cells. We found that CAFs secrete pyruvate as a metabolite of lactate oxidation. Further, we show that pyruvate is converted to lactate to promote glycolysis in MDA-MB-231 cells and helps to control elevated ROS levels in these tumor cells. Finally, we found that inhibiting or interfering with ROS management, using the naturally occurring flavonoid phloretin (found in apple tree leaves), adds to the cytotoxicity of the conventional chemotherapeutic agent doxorubicin. Our work demonstrates that a lactate-pyruvate, reciprocally-supportive metabolic relationship may be operative within the tumor microenvironment (TME) to support tumor growth, and may be a useful drug target.

Distribution of hepatitis B virus genotype and cancer predicting precore and basal core promoter mutations.

BACKGROUND: Chronic hepatitis B (CHB) infection which is associated with an increased risk of developing liver disease including cirrhosis and hepatocellular carcinoma. Viral factors that may increase the risk for HCC development include HBV DNA level, genotypes, and naturally occurring mutations such as hepatitis B virus precore (PC) (G1896A) and basal core promoter (BCP) A1762T/G1764A double mutations. HBV genotypes and subgenotypes can significantly influence HBeAg seroconversion rates, viremia levels, mutational patterns that could significantly influence the heterogeneity in clinical manifestations and even response to antiviral therapy. METHOD: 94 CHB infected individuals with detectable serum HBV DNA levels were studied. HBsAg, HBeAg, anti-HBc IgM antibody estimations were done by ELISA. HBV DNA estimation was done. The HBV genotypes were determined by TSP-PCR and 10 samples randomly selected for DNA sequencing. PC and BCP mutations were determined by DNA sequence analysis of core region. RESULT: Of 94 study participant samples with detectable serum HBV DNA levels, 75 were successfully genotyped and sequenced for BCP/PC region. 30/75 (40%) harbored PC and BCP mutations. The total Double mutations of BCP at A1762T/G1764A nucleotide positions, and PC mutation at G1896A nucleotide position were seen in 29.3% and 21.3%, respectively. All 75 isolates were subtype D using TSP-PCR. However, by sequencing 2/10 were subtype A, while 8 were subtype D. CONCLUSION: Our study reinforces that D is the predominant genotype in Indian population. It reveals that Indian CHB subjects have increased prevalence of BCP & PC mutations, which possibly may lead to development of HCC.

Management of complicated Bouveret's syndrome.

Bouveret's syndrome is an uncommon cause of gastric outlet obstruction caused by the impaction of large gallstones in the duodenal lumen. The gallstones pass into the duodenal lumen through a cholecystogastric or a cholecystoduodenal fistula. Endoscopic retrieval with or without lithotripsy is the first line of management, often with variable success. We present a case of a woman in her 70s who presented with signs of gastric outlet obstruction and was diagnosed with Bouveret's syndrome with a 5 cm diameter gallstone in the third part of her duodenum. Following several unsuccessful attempts of endoscopic extraction, she underwent successful jejunal enterotomy with fragmentation and extraction of the calculus using an Allis tissue holding forceps. Postoperative recovery was uneventful.

Auto-immune pancreatitis with unusual presentations - A case series.

Auto-immune pancreatitis (AIP) is a rare benign disease commonly presented with painless obstructive jaundice and biliary obstruction with rare complications like pseudocyst. We present a case series of two patients of AIP with unusual presentations; one case presented with periorbital swelling, jaundice, and pseudocyst, and the other case presented with abdominal pain and biliary obstruction without jaundice; both showed good response with steroids.

Enhanced degradation of dihydrofolate reductase through inhibition of NAD kinase by nicotinamide analogs.

Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin's lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment.

Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment.

Human mesenchymal stem cells (hMSCs) are bone marrow-derived stromal cells, which play a role in tumor progression. We have shown earlier that breast cancer cells secrete higher levels of interleukin-6 (IL-6) under hypoxia, leading to the recruitment of hMSCs towards hypoxic tumor cells. We found that (i) MDA-MB-231 cells secrete significantly higher levels of lactate (3-fold more) under hypoxia (1% O(2)) than under 20% O(2) and (ii) lactate recruits hMSCs towards tumor cells by activating signaling pathways to enhance migration. The mRNA and protein expression of functional MCT1 in hMSCs is increased in response to lactate exposure. Thus, we hypothesized that hMSCs and stromal carcinoma associated fibroblasts (CAFs) in the tumor microenvironment have the capacity to take up lactate expelled from tumor cells and use it as a source of energy. Our (13)C NMR spectroscopic measurements indicate that (13)C-lactate is converted to (13)C-alpha ketoglutarate in hMSCs and CAFs supporting this hypothesis. To our knowledge this is the first in vitro model system demonstrating that hMSCs and CAFs can utilize lactate produced by tumor cells.

Secretome of Stromal Cancer-Associated Fibroblasts (CAFs): Relevance in Cancer.

The cancer secretome reflects the assortment of proteins released by cancer cells. Investigating cell secretomes not only provides a deeper knowledge of the healthy and transformed state but also helps in the discovery of novel biomarkers. Secretomes of cancer cells have been studied in the past, however, the secretome contribution of stromal cells needs to be studied. Cancer-associated fibroblasts (CAFs) are one of the predominantly present cell populations in the tumor microenvironment (TME). CAFs play key role in functions associated with matrix deposition and remodeling, reciprocal exchange of nutrients, and molecular interactions and signaling with neighboring cells in the TME. Investigating CAFs secretomes or CAFs-secreted factors would help in identifying novel CAF-specific biomarkers, unique druggable targets, and an improved understanding for personalized cancer diagnosis and prognosis. In this review, we have tried to include all studies available in PubMed with the keywords "CAFs Secretome". We aim to provide a comprehensive summary of the studies investigating role of the CAF secretome on cancer development, progression, and therapeutic outcome. However, challenges associated with this process have also been addressed in the later sections. We have highlighted the functions and clinical relevance of secretome analysis in stromal CAF-rich cancer types. This review specifically discusses the secretome of stromal CAFs in cancers. A deeper understanding of the components of the CAF secretome and their interactions with cancer cells will help in the identification of personalized biomarkers and a more precise treatment plan.

Visualizing Activated Myofibroblasts Resulting from Differentiation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells that exhibit two main characteristics which define stem cells: self-renewal and differentiation. MSCs can migrate to sites of injury, inflammation, and tumor. Moreover, MSCs undergo myofibroblast-like differentiation, including increased production of α-SMA in response to transforming growth factor-ß (TGF-ß), a growth factor commonly secreted by tumor cells to evade immune surveillance. Based on our previous findings, hMSCs become activated and resemble carcinoma-associated myofibroblasts upon prolonged exposure to a conditioned medium from MDAMB231 human breast cancer cells. In this section, we show using immunofluorescence that keratinocyte-conditioned medium (KCM) induces differentiation of MSCs to resemble dermal myofibroblast-like cells with punctate vinculin staining and F-actin filaments.

Esophageal Lichen Planus Unusual Presentations: A Case Series.

Esophageal Lichen Planus (ELP)is a rare manifestation of Lichen Planus(LP). We present a case series of 2 patients with oral mucosal involvement who presented with dysphagia. On endoscopy, one patient revealed proximal esophageal stricture and the other patient had proximal esophageal ulcer. LP considered as a differential diagnosis in middle aged women with dysphagia.

Post-prandial paradoxical filling of gall bladder in patients with acute hepatitis: Myth or reality?

BACKGROUND: A minority of patients of acute hepatitis may exhibit edematous GB walls with no visible lumen despite fasting and may also exhibit paradoxical GB response in the post-prandial state. METHODS: Patients of acute hepatitis underwent routine upper abdominal sonography after overnight fasting. Patients who demonstrated contracted GB with edematous and coapted walls without any visible lumen despite overnight fasting were studied in the post-prandial phase after having breakfast. Serial ultrasonography was carried out at 10-15 min interval for an hour and changes in GB morphology was noted at each stage. RESULTS: A total of 77 patients of acute hepatitis underwent USG of hepatobiliary system between Sept 2008 and Aug 2009. Contracted gall bladder with edematous and coapted walls without any visible lumen despite overnight fasting was noted in 11 patients and were studied in the post-prandial phase. Post-prandial paradoxical filling of GB was observed in all such cases. Serial post-prandial ultrasonography demonstrated onset of filling of GB as early as 10-15 min post-prandial. Maximal GB distension was observed between 30 and 60 min. USG at 60 min post-prandial showed slight reduction in GB volume. As the GB distended in the post-prandial state, the lumen became increasingly visible with marginal reduction in wall thickening. CONCLUSION: We have observed transient paradoxical filling of GB on serial USG in early post-prandial state, in a subset of patients of acute viral hepatitis who had contracted GB with coapted walls in the initial USG after overnight fasting.

Transformation of benign Barrett's epithelium by repeated acid and bile exposure over 65 weeks: a novel in vitro model.

The mechanism by which gastroesophageal reflux promotes metaplasia→dysplasia→carcinoma is unknown. The aim of the study is to determine if repeated exposure to acid and bile confers a tumorigenic phenotype in a telomerase (hTERT)-immortalized benign Barrett's cell line (BAR-T). BAR-T cells were exposed to acid (pH 4) (A) and bile salt (200 µM glycochenodeoxycholic acid) (B) daily for 5 min up to 65+ weeks. The control cells were grown in parallel without any A or B treatment. Cell morphology, proliferation, transformation, and molecular changes in the gene expression for COX-2, TC22, p53 and p53 target genes were analyzed at 8-12 weeks intervals. At 46 weeks BAR-T cells exposed to (A+B) showed distinct phenotypic changes: forming clusters and acini, and at 65 weeks displayed foci in monolayer, and formed distinct colonies in soft agar. Untreated cells did not show any such changes. In A+B-treated BAR-T cells, COX-2 mRNA increased 10- to 20-fold, TC22 mRNA increased by 2- to 3-fold at 22-65 weeks, p53, MDM2, PERP, and p21mRNA increased 2.5-, 6.4-, 4-, and 2.6-fold respectively when compared to untreated cells at 34 weeks. However, at 58 weeks onward, there was a sharp decline of p53 and its target genes to the baseline level. At 65 weeks A+B-treated BAR-T cells formed tumor in nude mice whereas untreated cells did not. We demonstrate a novel in vitro model of transformation of a benign Barrett's cell line following repeated exposure to A+B over the course of 65 weeks.

Protein kinase C zeta regulates interleukin-8-mediated stromal-derived factor-1 expression and migration of human mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are bone marrow-derived cells with multipotent differentiation capability that are mobilized into the circulation in response to injury and localize to areas of tissue damage including solid tumors. They have the capacity to adopt a phenotype similar to carcinoma-associated fibroblasts (CAFs) and, like CAFs, promote tumor growth. The molecular communication between tumor cells and MSCs has not been well defined. However, MSCs have increased expression of the chemokine stromal-derived factor 1 (SDF-1) when exposed to conditioned medium from tumor cells. Additionally, SDF-1 has been shown to be important in the promotion of tumor growth by CAFs. These data suggest that the SDF-1 signaling axis is a key feature of the tumor microenvironment. In this report, we demonstrate that interleukin 8 (IL-8) induces an increase in SDF-1 expression by MSCs. The increase in SDF-1 expression in response to IL-8 is mediated by the activation of the protein kinase C (PKC) zeta isoform. In a functional assay, activation of PKC is required for in vitro MSC migration in response to tumor conditioned medium. These results indicate that IL-8-mediated SDF-1 production by MSCs requires PKC zeta activation. This signaling pathway provides insight into possible molecular targets for cancer therapy aimed at disrupting the interaction between components of the tumor microenvironment.

Interleukin 6 mediated recruitment of mesenchymal stem cells to the hypoxic tumor milieu.

Mesenchymal stem cells (MSCs) are a heterogeneous population of non-hematopoietic precursor cells predominantly found in the bone marrow. They have been recently reported to home towards the hypoxic tumor microenvironment in vivo. Interleukin-6 is a multifunctional cytokine normally involved in the regulation of the immune and inflammatory response. In addition to its normal function, IL-6 signaling has been implicated in tumorigenesis. Solid tumors develop hypoxia as a result of inadequate O(2) supply. Interestingly, tumor types with increased levels of hypoxia are known to have increased resistance to chemotherapy as well as increased metastatic potential. Here, we present evidence that under hypoxic conditions (1.5% O(2)) breast cancer cells secrete high levels of IL-6, which serve to activate and attract MSCs. We now report that secreted IL-6 acts in a paracrine fashion on MSCs stimulating the activation of both Stat3 and MAPK signaling pathways to enhance migratory potential and cell survival. Inhibition of IL-6 signaling utilizing neutralizing antibodies leads to attenuation of MSC migration. Specifically, increased migration is dependent on IL-6 signaling through the IL-6 receptor. Collectively, our data demonstrate that hypoxic tumor cells specifically recruit MSCs, which through activation of signaling and survival pathways facilitate tumor progression.

Primary conservative treatment results in mortality comparable to surgery in patients with infected pancreatic necrosis.

BACKGROUND & AIMS: The standard treatment for patients with infected pancreatic necrosis (IPN) is surgical necrosectomy. We compared the outcomes of surgical treatment versus primary conservative treatment (patients kept in intensive care unit and treated with antibiotics, organ support, intensive nutritional support, and, if required, percutaneous drainage) among patients with IPN. METHODS: We performed retrospective comparative (with prospectively acquired database) and prospective observational studies; data were collected from all consecutive patients with acute pancreatitis (n = 804), and those with IPN formed the study group. Patients with IPN were divided into 2 groups on the basis of diagnosis of IPN during 1997-2002 (group 1, n = 30) or 2003-2006 (group 2, n = 50). Eighteen patients in group 1 were treated by surgical necrosectomy, and 40 patients in group 2 were given primary conservative treatment; surgery was performed on patients if conservative treatment failed (n = 10). The primary outcome measure was mortality. RESULTS: The mortality was comparable in group 1 versus group 2 (43% vs 28%; P = .22). During a period of 10 years, the patients who received primary conservative treatment had significantly higher survival rates than those who received surgery (76.9% vs 46.4%; P = .005). In the prospective study during 2007-2008, the mortality from infected necrosis was 29.6% after primary conservative treatment, confirming the results of the comparative study. CONCLUSIONS: In treating patients with IPN, a primary conservative strategy resulted in mortality that was comparable with that after surgery, and 76% of the patients were able to avoid surgery; 54.5% of IPN patients were successfully managed with the primary conservative strategy.

Activation of signal transducers and activators of transcription 3 and focal adhesion kinase by stromal cell-derived factor 1 is required for migration of human mesenchymal stem cells in response to tumor cell-conditioned medium.

Mesenchymal stem cells (MSCs) migrate to tumors both in vitro and in vivo. Gene expression profiling analysis reveals that stromal cell-derived factor 1 (SDF-1) is significantly upregulated in MSCs exposed to tumor cell-conditioned medium, when compared with cells treated with control medium, suggesting that SDF-1 signaling is important in mediating MSC migration. This study investigates downstream signaling during MSC migration in response to tumor cell-conditioned medium and recombinant SDF-1 protein treatments. We observed that both recombinant SDF-1 and tumor cell-conditioned medium were able to activate downstream signaling via signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) as revealed by increased phosphorylation of STAT3 and ERK1/2 in human MSCs (hMSCs). Significant impairment of in vitro migration was observed in the presence of MAPK/ERK kinase (MEK) inhibitor PD98059, whereas two Janus kinase 2 (Jak2) inhibitors completely abolished migration induced by tumor cell-conditioned medium. Impaired MSC migration correlated with decreased levels of phosphorylated STAT3 and ERK1/2, suggesting that SDF-1 stimulation activates Jak2/STAT3 as well as MEK/ERK1/2 signaling, which in turn promotes migration of MSCs toward tumor cells. Furthermore, stimulation of hMSCs with recombinant SDF-1 and tumor cell-conditioned medium also significantly activated the focal adhesion kinases (FAKs) and paxillin, which correlated with reorganization of F-actin filaments in hMSCs. Decreased phosphorylation of FAK and paxillin as well as disruption of cytoskeleton organization was observed following Jak2 and MEK inhibitor treatment. Taken together, our results provide insight into the molecular pathways responsible for MSC migration toward the tumor microenvironment and may provide the molecular basis for modifying MSCs for therapeutic purposes.

Correction: Mishra, et al.; Lactate Dehydrogenases as Metabolic Links between Tumor and Stroma in the Tumor Microenvironment. Cancers 2019, 11, 750.

The authors would like to make a correction to their published paper [...].

Nitric Oxide and S-Nitrosylation in Cancers: Emphasis on Breast Cancer.

Nitric oxide (NO) is a ubiquitous, endogenously produced, water-soluble signaling molecule playing critical roles in physiological processes. Nitric oxide plays pleiotropic roles in cancer and, depending on its local concentration, may lead to either tumor progression or tumor suppression. Addition of NO group to a cysteine residue within a protein, termed as S-nitrosylation, plays diverse regulatory roles and affects processes such as metabolism, apoptosis, protein phosphorylation, and regulation of transcription factors. The process of S-nitrosylation has been associated with development of different cancers, including breast cancer. The present review discusses different mechanisms through which NO acts, with special emphasis on breast cancers, and provides detailed insights into reactive nitrogen species, posttranslational modifications of proteins mediated by NO, dual nature of NO in cancers, and the implications of S-nitrosylation in cancers. Our review will generate interest in exploring molecular regulation by NO in different cancers and will have significant therapeutic implications in the management and treatment of breast cancer.

Species-specific differences in translational regulation of dihydrofolate reductase.

We have observed that rodent cell lines (mouse, hamster) contain approximately 10 times the levels of dihydrofolate reductase as human cell lines, yet the sensitivity to methotrexate (ED(50)), the folate antagonist that targets this enzyme, is similar. Our previous studies showed that dihydrofolate reductase protein levels increased after methotrexate exposure, and we proposed that this increase was due to the relief of feedback inhibition of translation as a consequence of methotrexate binding to dihydrofolate reductase. In the current report, we show that unlike what was observed in human cells, dihydrofolate reductase (DHFR) levels do not increase in hamster cells after methotrexate exposure. We provide evidence to show that although there are differences in the putative mRNA structure between hamster and human mRNA in the dihydrofolate reductase binding region previously identified, "hamsterization" of this region in human dihydrofolate reductase mRNA did not change the level of the enzyme or its induction by methotrexate. Further experiments showed that human dihydrofolate reductase is a promiscuous enzyme and that it is the difference between the hamster and human dihydrofolate reductase protein, rather than the DHFR mRNA, that determines the response to methotrexate exposure. We also present evidence to suggest that the translational up-regulation of dihydrofolate reductase by methotrexate in tumor cells is an adaptive mechanism that decreases sensitivity to this drug.

Translational modulation of proteins expressed from bicistronic vectors.

Bicistronic vectors are useful tools for exogenous expression of two gene products from a single promoter element; however, reduced expression of protein from the second cistron compared with the first cistron is a common limitation to this approach. To overcome this limitation, we explored use of dihydrofolate reductase (DHFR) complementary DNA encoded in bicistronic vectors to induce a second protein of interest by methotrexate (MTX) treatment. Previous studies have demonstrated that levels of DHFR protein and DHFR fusion protein can be induced translationally following MTX treatment of cells. We demonstrated that in response to MTX treatment, DHFR partner protein in a bicistronic construct is induced for longer periods of time when compared with endogenous DHFR and DHFR fusion protein, in vitro and in vivo. Using rapamycin pretreatment followed by MTX treatment, we also devised a strategy to modulate levels of two proteins expressed from a bicistronic construct in a cap-independent manner. To our knowledge, this is the first report demonstrating that levels of proteins in DHFR-based bicistronic constructs can be induced and modulated using MTX and rapamycin treatment.