Heterodimerization of cholecystokinin 1 and cholecystokinin 2 receptors in gallbladder cancer: a new mechanism for carcinogenesis.

PURPOSE: Cholecystokinin is present in abundance in gallbladder tissue and mediates function through two structurally related receptors, CCK1R and CCK2R. Heterodimerization of these receptors is known to impact cell growth in vitro. However, the significance of these heterodimers in gallbladder carcinogenesis is relatively unknown. METHODS: Therefore, we evaluated the expression and the dimerization status of CCK1 and CCK2 receptors in human gallbladder carcinoma cell line (GBC-SD) and resected gallbladder tissue from normal (n = 10), cholelithiasis (n = 25) and gallbladder cancer (n = 25) by immunofluorescence/immunohistochemistry and western blot. The dimerization status of CCK1R and CCK2R was evaluated by co-immunoprecipitation. To understand the effect of heterodimerization of these receptors on growth-related signaling pathways, the expression of p-AKT, rictor, raptor and p-ERK was evaluated by western blot. RESULTS: We demonstrated the expression and heterodimerization of CCK1 and CCK2 receptor in GBC-SD gall bladder carcinoma cell line. Knockdown of CCK1R and CCK2R in the cell line led to significant reduction in p-AKT (P = 0.005; P = 0.0001) and rictor (P < 0.001; P < 0.001) levels. In tissue samples, significantly higher expression of CCK1R and CCK2R was observed in gallbladder cancer when compared to other groups both by immunohistochemistry (P = 0.008 and P = 0.013) and western blot (P = 0.009 and P = 0.003). An increase in heterodimer formation of CCK1R with CCK2R was observed in gallbladder cancer when compared to normal and cholelithiasis tissues. No significant difference in the expression of p-AKT and p-ERK was observed between the three groups. CONCLUSION: Our results provide the first evidence of heterodimerization of CCK1R and CCK2R in gallbladder tissue, and its association with development of gallbladder cancer. This finding has potential clinical and therapeutic significance.

Interferon-gamma signaling promotes melanoma progression and metastasis.

Interferon-gamma (IFNG) has long been regarded as the flag-bearer for the anti-cancer immunosurveillance mechanisms. However, relatively recent studies have suggested a dual role of IFNG, albeit there is no direct experimental evidence for its potential pro-tumor functions. Here we provide in vivo evidence that treatment of mouse melanoma cell lines with Ifng enhances their tumorigenicity and metastasis in lung colonization allograft assays performed in immunocompetent syngeneic host mice, but not in immunocompromised host mice. We also show that this enhancement is dependent on downstream signaling via Stat1 but not Stat3, suggesting an oncogenic function of Stat1 in melanoma. The experimental results suggest that melanoma cell-specific Ifng signaling modulates the tumor microenvironment and its pro-tumorigenic effects are partially dependent on the γδ T cells, as Ifng-enhanced tumorigenesis was inhibited in the TCR-δ knockout mice. Overall, these results show that Ifng signaling may have tumor-promoting effects in melanoma by modulating the immune cell composition of the tumor microenvironment.

Interferon-gamma induces melanogenesis via post-translational regulation of tyrosinase.

Melanogenesis (melanin pigment production) in melanocytes is canonically stimulated by the alpha melanocyte stimulating hormone (αMSH), which activates the cyclic-AMP-mediated expression of the melanocyte inducing transcription factor (MITF) and its downstream melanogenic genes, including the principal rate-limiting melanogenic enzyme tyrosinase (TYR). Here, we report that interferon-gamma (IFNG; type II interferon), but not interferon-alpha (a type I interferon), induces a noncanonical melanogenic pathway in mouse and human melanocytic cells. Inhibition of IFNG pathway by the JAK1/2 inhibitor ruxolitinib or knocking out Stat1 gene abrogated the IFNG-induced melanogenesis. Interestingly, IFNG-induced melanogenesis was independent of MITF. IFNG markedly increased the TYR protein expression but did not affect the mRNA expression, suggesting a post-translational regulatory mechanism. In contrast, IFNG had no effect on the expression of other melanogenesis-related proteins, for example, tyrosinase-related protein 1 (TYRP1) and dopachrome tautomerase (DCT). Glycosidase digestion assays revealed that IFNG treatment increased the mature glycosylated form of TYR, but not its de novo synthesis. Moreover, cycloheximide chase assay showed that degradation of TYR was decreased in IFNG-treated cells. These results suggest that the IFNG-STAT1 pathway regulates melanogenesis via regulation of the post-translational processing and protein stability of TYR.

Cytochrome c oxidase dysfunction enhances phagocytic function and osteoclast formation in macrophages.

The mitochondria-to-nucleus retrograde signaling (MtRS) pathway aids in cellular adaptation to stress. We earlier reported that the Ca2+- and calcineurin-dependent MtRS induces macrophage differentiation to bone-resorbing osteoclasts. However, mechanisms through which macrophages sense and respond to cellular stress remain unclear. Here, we induced mitochondrial stress in macrophages by knockdown (KD) of subunits IVi1 or Vb of cytochrome c oxidase (CcO). Whereas both IVi1 and Vb KD impair CcO activity, IVi1 KD cells produced higher levels of cellular and mitochondrial reactive oxygen species with increased glycolysis. Additionally, IVi1 KD induced the activation of MtRS factors NF-κB, NFAT2, and C/EBPδ as well as inflammatory cytokines, NOS 2, increased phagocytic activity, and a greater osteoclast differentiation potential at suboptimal RANK-L concentrations. The osteoclastogenesis in IVi1 KD cells was reversed fully with an IL-6 inhibitor LMT-28, whereas there was minimal rescue of the enhanced phagocytosis in these cells. In agreement with our findings in cultured macrophages, primary bone marrow-derived macrophages from MPV17-/- mice, a model for mitochondrial dysfunction, also showed higher propensity for osteoclast formation. This is the first report showing that CcO dysfunction affects inflammatory pathways, phagocytic function, and osteoclastogenesis.-Angireddy, R., Kazmi, H. R., Srinivasan, S., Sun, L., Iqbal, J., Fuchs, S. Y., Guha, M., Kijima, T., Yuen, T., Zaidi, M., Avadhani, N. G. Cytochrome c oxidase dysfunction enhances phagocytic function and osteoclast formation in macrophages.

Significance of methylation status of MASPIN gene and its protein expression in prognosis of gallbladder cancer.

AIM: We investigated prognostic significance of methylation status of MASPIN gene and its protein expression in normal subjects, cholelithiasis and gallbladder cancer (GBC) patients. METHODS: MASPIN protein expression and its promoter methylation in gallbladder tissue of cholelithiasis (n = 36), GBC (n = 46) and controls (n = 25) were investigated. Clinicopathological parameters and prognosis of patients with GBC were correlated with protein expression of MASPIN. Survival analysis of GBC patients was performed using Kaplan-Meier method. RESULTS: Significant increased (P < 0.0001) protein expression of MASPIN was observed in GBC as compared to cholelithiasis, whereas negligible expression was found in normal tissues. Methylation-specific PCR revealed statistical significant (P = 0.005) difference in methylation status of MASPIN promoter between gallstone and GBC patients. Significant association was observed between methylation profile with protein expression of MASPIN (P = 0.004), stage (P = 0.011) and cellular differentiation (P = 0.012) for GBC. Also, significant (P = 0.004) difference in survival was observed for malignant patients having demethylated MASPIN promoter. Further significant negative correlation (Pearson's coefficient [r] = -0.617; P < 0.0001) was observed between MASPIN expression and survival of cancer patients after surgery. Overall survival was significantly shorter (P ≤ 0.0001; hazard ratio [HR] for death = 2.84; 95% confidence interval [CI], 0.09865-0.3683) in patients of GBC with MASPIN expression ≥169.56 pg/mg (median survival; 10 months) with compared to those with expression <169.56 pg/mg (median survival; 16 months). CONCLUSION: Overexpression of MASPIN protein may play an important role in malignant progression and is correlated with a poor prognosis. Also MASPIN DNA methylation can be used as a novel therapeutic target for treating GBC.

Epigenetic Mechanisms and Events in Gastric Cancer-Emerging Novel Biomarkers.

Gastric cancer is one of the most common malignancy worldwide. The various genetic and epigenetic events have been found to be associated with its carcinogenesis. The epigenetic is a heritable and transient/reversible change in the gene expression that is not accompanied by modification in the DNA sequence. This event is characterized by the alteration in the promoter CpG island of the gene or histone modification. These events are associated with silencing of critical tumor suppressor gene and activation of oncogenes leading to carcinogenesis. The DNA methylation is a chemical change in the DNA sequence that most commonly occurs at cytosine moiety of CpG dinucleotide and histone, primarily on N- terminal tail that ultimately effect the interaction of DNA with chromatin modifying protein.Hypermethylation of tumor suppressor genes and global hypomethylation of oncogenes are widely studied epigenetic modifications. There are large number of publish reports regarding epigenetic events involving gastric cancer. These changes are potentially useful in identifying markers for early diagnosis and management of this lethal malignancy. Also, role of specific miRNAs and long non coding RNAs in regulation of gene expression is gaining interest and is a matter of further investigation. In this review, we aimed to summarize major epigenetic events (DNA methylation) in gastric cancer along with alteration in miRNAs and long non coding RNAs which plays an important role in pathology of this poorly understood malignancy.

A let-7 microRNA binding site polymorphism in the KRAS 3'UTR is associated with increased risk and reduced survival for gallbladder cancer in North Indian population.

PURPOSE: Gallbladder cancer is a lethal malignancy of hepato-biliary system with high incidence in North India, especially along gangetic plain. The let-7 microRNAs play a key role in regulating KRAS expression and a polymorphism in 3' untranslated region (rs61764370, T/G) of KRAS leads to its higher expression. This polymorphism is known to be associated with increased risk and prognosis of various cancers but its association with gallbladder cancer has not been evaluated. To address this research question, we evaluated whether rs61764370 variant is associated with gallbladder cancer susceptibility and clinical outcomes. METHODS: In present case-control study, we enrolled 541 patients with gallbladder malignancy and 307 controls. Genomic DNA was obtained from peripheral blood and genotyping was performed using Taqman allelic discrimination assay. RESULTS: Heterozygous (TG) individuals are at a significant higher risk for GBC as compared with wild genotype (TT) (p = 0.007, odds ratio = 2.56, 95 % CI 1.27-5.18). At allelic level, allele G has significant higher risk for GBC as compared with T allele (p = 0.008, odds ratio = 2.5, 95 % CI 1.25-5.01). Survival analysis reveals decrease in overall survival for heterozygous genotype (p < 0.0001, hazard ratio = 3.42, 95 % CI 1.21-4.20). Also, significant decrease in overall survival was observed for patient carrying allele G (p < 0.0001, HR = 2.89, 95 % CI 1.21-4.20) as compared with allele C. CONCLUSIONS: We conclude that KRAS rs61764370 polymorphism is significantly associated with risk and prognosis of gallbladder malignancy in this endemic belt.

Polymorphism and Expression Profile of Cholecystokinin Type A Receptor in Relation to Gallstone Disease Susceptibility.

In the present study, we investigated expression pattern of Cholecystokinin type A receptor (CCKAR) in relation to its commonly studied polymorphism (rs1800857, T/C) in gallstone disease (GSD) patients and controls. A total of 502 subjects (272 GSD and 230 controls) were enrolled, and genotyping was performed by evaluating restriction fragments of PstI digested DNA. For analyzing expression pattern of CCKAR in relation to polymorphism, gallbladder tissue samples from 80 subjects (GSD-55; control-25) were studied. Expression of CCKAR mRNA was evaluated by reverse transcriptase-PCR and confirmed using real-time PCR. Protein expression was evaluated by enzyme-linked immunosorbent assay. We observed significantly (p < 0.0001) lower expression of CCKAR mRNA and protein in GSD tissues as compared with control. Significantly higher frequency of A1/A1 genotype (C/T transition) (p = 0.0005) was observed for GSD as compared with control. Expression of CCKAR protein was found to be significantly lower (p < 0.0001) in A1/A1 genotype as compared with other genotypes for GSD patients. Perhaps, this is the first report providing evidence of alteration in CCKAR expression in relation to its polymorphism elucidating the molecular pathway of the disease. Additional investigations with lager sample size are needed to confirm these findings.

Prognostic significance of survivin in resected gallbladder cancer.

BACKGROUND: Survivin, a novel inhibitor of apoptosis, plays a role in oncogenesis and has been correlated with poor prognosis. We investigated its expression in gallbladder tissues of control, cholelithiasis, and gallbladder cancer (GBC). Survivin expression was correlated with different clinicopathologic parameters including prognosis in patients with GBC. MATERIALS AND METHODS: Gallbladder tissue samples were collected from GBC (n = 39), cholelithiasis (n = 30), and control (n = 25). Expression of survivin messenger RNA (mRNA) was evaluated by real time polymerase chain reaction. Protein quantification was done by enzyme-linked immunosorbent assay. RESULTS: Significantly higher expression of survivin mRNA was observed in GBC (2.9-fold) and cholelithiasis (1.85-fold) as compared with control (P < 0.0001). In GBC, increased survivin expression (mRNA and protein) was significantly associated with higher tumor stage (stage III versus stage II) (P < 0.0001) and poor tumor differentiation (poor and moderate versus well) (P < 0.0001). No significant correlation was observed with any of the other clinicopathologic factors studied. Increased expression of survivin was associated with shorter survival (median survival 11.5 mo versus 18 mo). CONCLUSIONS: Differential expression of survivin in GBC suggests its possible role in gallbladder carcinogenesis. Its overexpression is associated with poor prognosis. Assessment of survivin might be used to stratify GBC patients for optimal treatment modalities, including targeted therapy.

Expression of serum survivin protein in diagnosis and prognosis of gallbladder cancer: a comparative study.

The role of survivin in gallbladder cancer (GBC) has not been evaluated. We investigated survivin protein expression in serum of patients with gallbladder diseases (cholelithiasis, n = 30; GBC, n = 39) and compared with healthy controls (n = 25). Clinicopathological parameters, diagnosis and prognosis of patients with GBC were correlated with the expression of serum survivin by enzyme-linked immunosorbent assay. Significantly higher (P < 0.0001) expression of survivin protein was observed in GBC as compared to cholelithiasis and control. Increased survivin expression was significantly associated with higher tumor stage (stage III vs. stage II; P < 0.0001) and cellular differentiation (poor and moderate vs. well differentiated; P < 0.0001) in GBC. No significant correlation was observed with any of the other clinico-pathological parameters studied. The cutoff value of survivin protein of 79 pg/ml with sensitivity of 81.16 % and specificity of 80 % differentiated the diseased group (cholelithiasis or GBC) from control group were as the cutoff value of 109 pg/ml differentiated GBC from cholelithiasis with a sensitivity of 82.05 % and specificity of 93.33 %. Though not significant, increased expression of survivin was associated with median overall survival (12 vs. 18 months; P = 0.05) in GBC patients. Our study suggests that survivin protein in serum could be both a useful diagnostic marker and an important prognostic factor for GBC.

Differential expression of cholecystokinin A receptor in gallbladder cancer in the young and elderly suggests two subsets of the same disease?

BACKGROUND: Cholecystokinin type A receptor (CCKAR) is known to be overexpressed in variety of human malignancies but information regarding its expression in gallbladder cancer (GBC) is limited. Attempts were now made to investigate expression pattern of CCKAR mRNA and protein in controls and GBC patients and correlate it with various clinicopathological parameters following surgical resection. MATERIALS AND METHODS: Gallbladder tissue samples from 64 subjects (GBC: 39; control: 25) were studied. Expression of CCKAR mRNA was evaluated by reverse transcriptase-polymerase chain reaction and confirmed using real-time polymerase chain reaction. Protein expression was studied by enzyme-linked immunosorbent assay. RESULTS: Significantly higher expression of CCKAR mRNA (P < 0.0001) and protein (P < 0.0001) was observed in GBC tissues. Overexpression was also observed for stage III and in moderately and poorly differentiated tumors. When the clinicopathological parameters were compared, we found age dependent decrease in CCKAR expression. Relatively higher expression of CCKAR was observed in younger patients (age < 45 years) having more aggressive disease when compared with elderly ones (age ≥ 45 years). CONCLUSIONS: Age related differential expression of CCKAR in GBC may suggest two possible variants of the disease in this endemic belt.

Expression of survivin mRNA in gallbladder cancer: a diagnostic and prognostic marker?

Survivin, an inhibitor of apoptosis, has been shown to be expressed in various malignancies. However, its role in gallbladder cancer (GBC) has not been evaluated yet. We investigated its expression in peripheral blood of patients with gallbladder diseases (gallstone disease (GSD), n = 30; GBC, n = 39) and compared with healthy controls (n = 25). Survivin expression was correlated with clinicopathological parameters, diagnosis, and prognosis of patients with GBC. Expression of survivin messenger RNA (mRNA) in blood was evaluated by real-time PCR. Significantly higher (P < 0.0001) expression of survivin mRNA was observed in GBC (2.2-fold) and GSD (1.52-fold) as compared to control. In GBC, increased survivin expression was significantly associated with higher tumor stage (stage III vs. stage II; P < 0.0001) and tumor differentiation (poor and moderate vs. well differentiated; P < 0.0001). No significant correlation was observed with any of the other clinicopathological parameters (age, gender, and presence or absence of gallstones) studied. Cutoff value of survivin mRNA relative quantification (RQ) was 1.08, with a sensitivity of 98.55 % and specificity of 100 % for the diseased group (GSD or GBC). RQ value of 1.71 differentiated GBC from GSD with a sensitivity of 89.74 % and specificity of 100 %. Increased expression of survivin was associated with a shorter median overall survival (12 vs. 18 months) in GBC patients. Differential expression of survivin in GBC suggests its possible role and association with poor prognosis. Expression of survivin in peripheral blood could be useful both in the diagnosis and prognosis of GBC.

TNF-α, IL-6, and IL-8 cytokines and their association with TNF-α-308 G/A polymorphism and postoperative sepsis.

INTRODUCTION: Early prediction of postoperative sepsis remains an enormous clinical challenge. Association of TNF-α-308 G/A polymorphism with sepsis remains controversial. We, therefore, investigated this polymorphism with serum levels of cytokines TNF-α, IL-6, and IL-8 in relation to development of sepsis following major gastrointestinal surgery. METHODS: Two hundred and thirty-nine patients undergoing major gastrointestinal surgery were enrolled. Polymorphism was studied through the analysis of restriction fragments of Nco1-digested DNA with the polymerase chain reaction. All patients were followed for 1 month following surgery for evidence of sepsis. Levels of serum cytokines TNF-α, IL-6, and IL-8 were measured preoperatively and postoperatively by enzyme-linked immunosorbent assay (ELISA). RESULTS: Forty-seven (19.66 %) patients developed postoperative sepsis. Patients with postoperative sepsis were significantly (p = 0.002) more likely to possess AA homozygous genotype with higher capacity to produce cytokines TNF-α (p < 0.0001), IL-6 (p < 0.0001), and IL-8 (p < 0.0001) as compared to other genotypes. When compared with patients carrying at least one G allele, the AA genotype was associated with a significantly higher probability (odds ratio (OR) = 4.17; p = 0.003; 95 % confidence interval (CI) = 1.5-11.48) of developing sepsis. Compared with the GG genotype, AA was associated with a significantly higher probability (OR = 5.18; p = 0.0008; 95 % CI = 1.82-14.76) of sepsis development. CONCLUSION: TNF-α-308 G/A polymorphism is significantly associated with the development of postoperative sepsis and with increased expression of cytokines TNF-α, IL-6, and IL-8.

Elevated expression of maspin mRNA as a predictor of survival in stage II and III gallbladder cancer cases.

BACKGROUND: Maspin expression is a potential prognostic factor for various malignancies but its relation with gallbladder cancer is unknown and needs to be investigated needs to be investigated. We therefore here focused on maspin mRNA expression in normal, gall stone disease and gallbladder cancer subjects, with particular attention to prognostic importance in individuals with malignancies. MATERIALS AND METHODS: This study was carried out at the Department of Surgical Gastroenterology, King George's Medical University, Lucknow, India. Gallbladder samples from normal (n=25), gall stone disease (n=25) and cancer patients (n=38) were analysed for maspin mRNA expression by semi-quantitative reverse transcriptase PCR and quantitative real time PCR. Statistical analysis was carried out using the Students t test or ANOVA. Survival analysis was conducted according to the Kaplan-Meier method and correlations were assessed using the Pearson correlation method. p<0.05 was considered statistically significant. RESULTS: Significant increase (p=0.028) in expression of maspin mRNA was observed in gallbladder cancer as compared to gall stone disease, whereas no expression was found in normal tissues. Significant correlation (Pearson's coefficient(r)=-0.798, p<0.0001) was observed between relative quantification of maspin mRNA and survival of cancer patients after surgery, with significantly shorter (p=0.002) survival in patients having relative quantification >1.5 as compared to those having relative quantification <1.5. Similarly, significant differences in patient survival for maspin mRNA expression was observed for stage II (p=0.025) and III (p=0.011) cancer. CONCLUSIONS: Higher expression of maspin mRNA in gallbladder cancer has prognostic significance for stage II and III cancer, which needs to be investigated further.

Prognostic significance of K-ras codon 12 mutation in patients with resected gallbladder cancer.

BACKGROUND: Point mutation of K-ras is associated with carcinogenesis and overall survival in various cancers. We investigated the mutational spectrum of K-ras codon 12 in resected normal and gallbladder cancer tissue samples in a Northern Indian population and correlated it with different clinicopathological parameters. PATIENTS AND METHODS: Gallbladder tissues from normal (n = 24) and cancer patients (n = 39) were analyzed for K-ras codon 12 mutation by restriction fragment length polymorphism. Statistical analysis was carried out using the χ(2) test or Fisher's exact test. Survival was estimated using the Kaplan-Meier method, and the difference between survival curves was analyzed by the log-rank test. RESULTS: The frequency of K-ras mutation was significantly higher (p = 0.001) in gallbladder cancer tissue samples (16/39) compared to normal samples (1/24). Patients with K-ras mutation had a significantly decreased overall survival (p = 0.003), particularly for stage II (p = 0.021) and III (p = 0.009) cancers. No significant correlation was observed with any of the other clinicopathological factors studied. CONCLUSIONS: Gallbladder cancer has a high frequency of K-ras codon 12 mutation with poorer outcomes in resected stage II and III disease. K-ras mutational analysis has important prognostic implications that need to be investigated further.