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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.

Cytotoxic T Lymphocyte Antigen 4 (CTLA4) expressed on the surface of T cells is an important immunotherapeutic target in many cancers, including melanoma. However, we previously found highly upregulated expression of CTLA4 in BRAF/NRAS-mutant human melanoma cell lines and tissues. Although CTLA4 is almost entirely expressed intracellularly in melanoma cells, its intracellular functions have remained elusive. Ectopic expression of Ctla4 (Ctla4-ee) in mouse melanoma cell lines significantly promoted lung colonization in syngeneic mice as well as in immunocompromised mice, suggesting that Ctla4 had pro-tumorigenic effects independent of its extracellular immune checkpoint function. Proteomics analysis identified apoptosis to be a major affected pathway and we found that Ctla4 ectopic-expressing mouse melanoma cells (B2905-Ctla4-ee) were significantly resistant to doxorubicin-induced apoptosis as compared to empty vector (EV) controls. In a reciprocal experiment, knocking out CTLA4 in the Hs936T human melanoma cells, that exhibit high endogenous CTLA4 expression, showed significantly increased apoptosis than the parental cells. Moreover, significant upregulation of anti-apoptotic proteins (Bnip3, Birc6, Pak1, Mcl-1, Survivin, Rac1/Cdc42, Bcl-2, and Bnip3l) and downregulation of pro-apoptotic proteins (p53 and Bad) were observed in CTLA4-ee cells as compared to EV controls. CTLA4-ee cells also showed significantly higher invasion in Matrigel-coated Transwell assay. Interestingly, we also found that CTLA4 translocated to mitochondria, where it downregulated oxidative phosphorylation and increased glycolysis. These findings lead us to hypothesize that CTLA4 plays a significant role in regulating apoptosis and mitochondrial metabolic functions, leading to the promotion of melanoma progression and metastasis. Citation Format: Hasan Raza Kazmi, Xuan Mo, Bo Zhou, Sarah Preston Alp, Scott Gross, Hassaan Wajeeh, Carmen Merali, John Elrod, Jonathan Soboloff, Salim Merali, M. Raza Zaidi. Intracellular Ctla4 overexpression promotes melanoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2536.

The mitochondria-to-nucleus retrograde signaling (MtRS) pathway aids in cellular adaptation to stress. We earlier reported that the Ca(2+)- 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.

Cytotoxic T Lymphocyte Antigen 4 (CTLA4) is an important immune checkpoint protein and has been utilized as an immunotherapeutic target against melanoma. Our previous studies have shown that CTLA4 is expressed at low levels in primary human melanocytes but is upregulated in mutant-BRAF/NRAS melanoma cells and tissues. Despite predominantly intracellular localization of CTLA4, its intracellular function remains highly contradictory and unsubstantiated. We found that ectopic expression of Ctla4 in mouse melanoma cell lines substantially promoted lung colonization in allograft model systems in syngeneic mice. Moreover, similar results were observed in immunocompromised recipient mice, suggesting an intracellular cell-autonomous tumorigenic role of Ctla4 in melanoma. These finding led us to investigated intracellular functions of CTLA4 and its potential roles in melanomagenesis. Through the Ingenuity Pathway Analysis of our proteomics data, we found apoptosis as a major affected pathway in Ctla4-expressing mouse melanoma cells. We treated the cells (B2905-Ctla4-ee and EV control) with doxorubicin to induce apoptosis, followed by assessment by Annexin V assay. We found that Ctla4 expressing mouse melanoma cells were significantly resistant to doxorubicin-induced apoptosis. In reciprocal experiment, we generated (by CRISPR-Cas9) CTLA4-knockout A2058 human melanoma cells that exhibit high endogenous CTLA4 expression and we found that knockout cells showed significantly increased apoptosis than the parental cells. Moreover, we also observed significant upregulation of anti-apoptotic proteins (Bnip3, Birc6, Pak1, Mcl-1, Survivin, Rac1/Cdc42, Bcl-2, and Bnip3l) and downregulation of pro-apoptotic proteins (p53 and Bad) in CTLA4-expressing human melanoma cell lines. CTLA4-expressing cells also showed significantly higher invasion in Matrigel-coated transwell assay. These findings lead us to conclude that CTLA4 plays a significant role in regulating apoptosis and promoting melanoma progression in BRAF/NRAS mutant cells. Citation Format: Hasan Raza Kazmi, Xuan Mo, Bo Zhou, Sara Preston-Alp, Scott Gross, Hassaan Wajeeh, Carmen Merali, Jonathan Soboloff, Salim Merali, M. Raza Zaidi. A novel anti apoptotic function of CTLA4 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5652.

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. 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. 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

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

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.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.Significantly higher expression of survivin mRNA was observed in GBC (2.9-fold) and cholelithiasis (1.85-fold) as compared with control (P0.0001). In GBC, increased survivin expression (mRNA and protein) was significantly associated with higher tumor stage (stage III versus stage II) (P0.0001) and poor tumor differentiation (poor and moderate versus well) (P0.0001). 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).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.

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.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. p0.05 was considered statistically significant.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, p0.0001) 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 quantification1.5 as compared to those having relative quantification1.5. Similarly, significant differences in patient survival for maspin mRNA expression was observed for stage II (p=0.025) and III (p=0.011) cancer.Higher expression of maspin mRNA in gallbladder cancer has prognostic significance for stage II and III cancer, which needs to be investigated further.

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.

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 χ2 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.

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

Gallbladder Cancer (GBC) is the most common malignancy of the biliary tract with very high incidence in Northern India, especially around Gangetic belt. Late diagnosis and poor prognosis are the important features of this disease. For improving treatment strategies and outcomes, novel non invasive prognostic biomarkers are needed. Variations in peripheral blood mitochondrial DNA (mtDNA) copy number in various cancers are widely studied but information in relation to gallbladder malignancy is unknown. In this pilot study, we investigated leucocytes mtDNA copy number variation in relation to risk and prognosis of GBC. A total of 300 histologically confirmed or fine needle aspiration cytology proven GBC patients were recruited along with equal number of age and sex matched controls. The mtDNA copy number was measured by real-time polymerase chain reaction through absolute quantification using SYBR green chemistry. We also explore prognostic utility of mtDNA content in relation to overall survival of GBC patients. Statistical analysis was done by Student’s test and survival data was compared using Kaplan Meier method. Two tailed p value less than 0.05 was considered to be significant. We observed high mtDNA content in cases as compared with controls (mean±standard deviation, 1.21±0.88 vs. 1.02±0.76). Higher mtDNA copy number was significantly associated with increased gallbladder cancer risk (p=0.005). In this study, we compared overall survival (OS) for gallbladder cancer patients in relation to mtDNA copy number median value (median, 1.12). Kaplan Meir survival curve predicts decrease in OS (p=0.03, Hazard ratio=2.54; 95% CI = 1.79-5.86) for GBC patients in relation to high mtDNA content (>median) as compared with low mtDNA copy number ( This study supports an association between increased mtDNA copy number with susceptibility to gallbladder cancer in this endemic belt. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in relation to gallbladder cancer. Citation Format: Abhijit Chandra, Hasan R. Kazmi, Leena K. Satyam, Saket Kumar. Association of mitochondrial DNA copy number variation in peripheral blood leucocytes with risk and prognosis of gallbladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4729. doi:10.1158/1538-7445.AM2017-4729

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.

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

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. 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). 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

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 and protein 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.

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.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.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.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.