IL-22 regulates MASTL expression in intestinal epithelial cells.

Microtubule-associated serine-threonine kinase-like (MASTL) has recently been identified as an oncogenic kinase given its overexpression in numerous cancers. Our group has shown that MASTL expression is upregulated in mouse models of sporadic colorectal cancer and colitis-associated cancer (CAC). CAC is one of the most severe complications of chronic inflammatory bowel disease (IBD), but a limited understanding of the mechanisms governing the switch from normal healing to neoplasia in IBD underscores the need for increased research in this area. However, MASTL levels in patients with IBD and its molecular regulation in IBD and CAC have not been studied. This study reveals that MASTL is upregulated by the cytokine interleukin (IL)-22, which promotes proliferation and has important functions in colitis recovery; however, IL-22 can also promote tumorigenesis when chronically elevated. Upon reviewing the publicly available data, we found significantly elevated MASTL and IL-22 levels in the biopsies from patients with late-stage ulcerative colitis compared with controls, and that MASTL upregulation was associated with high IL-22 expression. Our subsequent in vitro studies found that IL-22 increases MASTL expression in intestinal epithelial cell lines, which facilitates IL-22-mediated cell proliferation and downstream survival signaling. Inhibition of AKT activation abrogated IL-22-induced MASTL upregulation. We further found an increased association of carbonic anhydrase IX (CAIX) with MASTL in IL-22-treated cells, which stabilized MASTL expression. Inhibition of CAIX prevented IL-22-induced MASTL expression and cell survival. Overall, we show that IL-22/AKT signaling increases MASTL expression to promote cell survival and proliferation. Furthermore, CAIX associates with and stabilizes MASTL in response to IL-22 stimulation.NEW & NOTEWORTHY MASTL is upregulated in colorectal cancer; however, its role in colitis and colitis-associated cancer is poorly understood. This study is the first to draw a link between MASTL and IL-22, a proinflammatory/intestinal epithelial recovery-promoting cytokine that is also implicated in colon tumorigenesis. We propose that IL-22 increases MASTL protein stability by promoting its association with CAIX potentially via AKT signaling to promote cell survival and proliferation.

LINC02086 inhibits ferroptosis and promotes malignant phenotypes of pancreatic cancer via miR-342-3p/CA9 axis.

Long noncoding RNAs (lncRNAs) play important roles in modulating the tumorigenesis and progression of malignant tumors. LINC02086 is a newly identified oncogene associated with tumorigenesis, but its role in pancreatic cancer (PC) has not been fully elucidated. In this study we examined the expression levels of LINC02086, miR-342-3p, and CA9 in PC. The relationship of ferroptosis with these factors was analyzed by detecting the expression levels of Fe2+, reactive oxygen species (ROS), and ferroptosis marker proteins. The expression of these genes was altered to observe their effects on cell proliferation, migration, and invasion ability. Bioinformatics was used to predict target genes, and the binding relationship was verified luciferase reporter assay. Finally, the function of LINC02086 was evaluated in vivo. The findings suggest that LINC02086 is highly expressed in PC tissues and cell lines and is correlated with a poor prognosis. In vitro experiments demonstrated that LINC02086 knockdown promoted ferroptosis in PC cells to suppress their malignant phenotype. LINC02086 acts as a competitive endogenous RNA that adsorbed miR-342-3p. miR-342-3p hinders the malignant progression of PC by promoting ferroptosis. In addition, miR-342-3p targets CA9 and affects its function. Further mechanistic studies revealed that LINC02086 inhibits ferroptosis and promotes PC progression by acting as a sponge for miR-342-3p to upregulate CA9 expression. In vivo experiments further confirmed this mechanism. Taken together, LINC02086 upregulates CA9 expression by competitively binding with miR-342-3p, thereby inhibiting ferroptosis in PC cells and promoting their malignant phenotype. The results of our study provide new insights into how LINC02086 contributes to the progression of PC.

Vascular, adipose tissue, and/or calyceal invasion in clear cell tubulopapillary renal cell tumour: potentially problematic diagnostic scenarios.

AIMS: The 2022 WHO classification for kidney tumours recently downgraded clear cell tubulopapillary (also known as clear cell papillary) renal cell carcinoma (RCC) to a benign neoplasm (i.e. clear cell tubulopapillary renal cell tumour) based on the overwhelmingly banal nature of this neoplasm. However, it has been recognized that some clear cell tubulopapillary renal cell tumours demonstrate vascular, adipose or pelvicalyceal invasion, raising the possibility of more aggressive behaviour. The goal of this study was to determine if these 'high stage' features have an effect on tumour prognosis, warranting a carcinoma designation. METHODS AND RESULTS: After excluding cases with tissue artefact (i.e. prior core biopsy track changes) and other RCC subtypes with next-generation sequencing, nine clear cell tubulopapillary renal cell tumours with these so-called 'high stage' features, and otherwise classic morphologic and immunophenotypic findings, including low-grade cytology and 'cup-like' CA9 expression, were evaluated. Median tumour size was 2.2 cm with a range of 0.8 to 6.7 cm. Eight cases (89%) demonstrated perinephric or hilar adipose tissue invasion, although most of these cases showed a bulging (in contrast to an infiltrative) growth pattern. One case demonstrated renal vascular invasion in addition to hilar adipose tissue invasion, and one case demonstrated extension into the pelvicalyceal system. There were no recurrences or evidence of metastatic disease. CONCLUSION: These overall findings continue to support the benign designation for clear cell tubulopapillary renal cell tumours, despite morphologic features that might raise the possibility of a 'higher stage' neoplasm.

Vanillic acid restores homeostasis of intestinal epithelium in colitis through inhibiting CA9/STIM1-mediated ferroptosis.

The damage of integrated epithelial epithelium is a key pathogenic factor and closely associated with the recurrence of ulcerative colitis (UC). Here, we reported that vanillic acid (VA) exerted potent therapeutic effects on DSS-induced colitis by restoring intestinal epithelium homeostasis via the inhibition of ferroptosis. By the CETSA assay and DARTS assay, we identified carbonic anhydrase IX (CAIX, CA9) as the direct target of VA. The binding of VA to CA9 causes insulin-induced gene-2 (INSIG2) to interact with stromal interaction molecule 1 (STIM1), rather than SREBP cleavage-activating protein (SCAP), leading to the translocation of SCAP-SREBP1 from the endoplasmic reticulum (ER) to the Golgi apparatus for cleavage into mature SREBP1. The activation of SREBP1 induced by VA then significantly facilitated the transcription of stearoyl-CoA desaturase 1 (SCD1) to exert an inhibitory effect on ferroptosis. By inhibiting the excessive death of intestinal epithelial cells caused by ferroptosis, VA effectively preserved the integrity of intestinal barrier and prevented the progression of unresolved inflammation. In conclusion, our study demonstrated that VA could alleviate colitis by restoring intestinal epithelium homeostasis through CA9/STIM1-mediated inhibition of ferroptosis, providing a promising therapeutic candidate for UC.

Marine Prostanoids with Cytotoxic Activity from Octocoral Clavularia spp.

Octocoral of the genus Clavularia is a kind of marine invertebrate possessing abundant cytotoxic secondary metabolites, such as prostanoids and dolabellanes. In our continuous natural product study of C. spp., two previously undescribed prostanoids [clavulone I-15-one (1) and 12-O-deacetylclavulone I (2)] and eleven known analogs (3-13) were identified. The structures of these new compounds were elucidated based on analysis of their 1D and 2D NMR, HRESIMS, and IR data. Additionally, all tested prostanoids (1 and 3-13) showed potent cytotoxic activities against the human oral cancer cell line (Ca9-22). The major compound 3 showed cytotoxic activity against the Ca9-22 cells with the IC50 value of 2.11 ± 0.03 µg/mL, which echoes the cytotoxic effect of the coral extract. In addition, in silico tools were used to predict the possible effects of isolated compounds on human tumor cell lines and nitric oxide production, as well as the pharmacological potentials.

Downregulation of HHLA2 inhibits ovarian cancer progression via the NF-κB signaling pathway and suppresses the expression of CA9.

HHLA2 has been recently demonstrated to play multifaceted roles in several types of cancers. However, its underlying mechanism in the progression of human ovarian cancer (OC) remains largely unexplored. In the present study, we aimed to determine whether downregulation of HHLA2 inhibited malignant phenotypes of human OC cells and explore its specific mechanism. Our results revealed that downregulation of HHLA2 by transfection with a lentiviral vector significantly suppressed the viability, invasion, and migration of OC cells. Interaction study showed that downregulation of HHLA2 in OC cells reduced the expression of CA9 and increased the expressions of p-IKKß and p-RelA. Conversely, the viability, invasion, and migration of HHLA2-depleted OC cells were increased when CA9 was upregulated. In vivo, we found that downregulation of HHLA2 significantly inhibited tumor growth, which was reversed by CA9 overexpression. In addition, downregulation of HHLA2 inhibited the OC progression via activating the NF-κB signaling pathway and decreasing the expression of CA9. Collectively, our data suggested a link between HHLA2 and NF-κB axis in the pathogenesis of OC, and these findings might provide valuable insights into the development of novel potential therapeutic targets for OC.

Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.

The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.

Oxygen-independent disulfide bond formation in VEGF-A and CA9.

Low levels of oxygen (hypoxia) occurs in many (patho)physiological situations. Adaptation to hypoxia is in part mediated by proteins expressed in the extracellular space that mature in the endoplasmic reticulum (ER) prior to traversing the secretory pathway. The majority of such ER cargo proteins require disulfide bonds for structural stability. Disulfide bonds are formed co- and posttranslationally in a redox relay that requires a terminal electron acceptor such as oxygen. We have previously demonstrated that some ER cargo proteins such as low-density lipoprotein receptor (LDLR) and influenza hemagglutinin (Flu-HA) are unable to complete disulfide bond formation in the absence of oxygen, limiting their ability to pass ER quality control and their ultimate expression. Here, using radioactive pulse-chase immunoprecipitation analysis, we demonstrate that hypoxia-induced ER cargo proteins such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A) complete disulfide bond formation and mature with similar kinetics under hypoxia and normoxia. A global in silico analysis of ER cargo revealed that hypoxia-induced proteins on average contain fewer free cysteines and shorter-range disulfide bonds in comparison to other ER cargo proteins. These data demonstrate the existence of alternative electron acceptors to oxygen for disulfide bond formation in cellulo. However, the ability of different proteins to utilize an oxygen-independent pathway for disulfide bond formation varies widely, contributing to differential gene expression in hypoxia. The superior ability of hypoxia-induced proteins such as VEGF-A and CA9 to mature in hypoxia may be conferred by a simpler disulfide architecture.

Randomized multicenter phase II trial of prophylactic irradiation of para-aortic lymph nodes in advanced cervical cancer according to tumor hypoxia: Korean Radiation Oncology Group (KROG 07-01) study.

We conducted a prospective phase II study on whether extended-field irradiation (EFI) confers survival benefits depending on hypoxic markers in locally advanced uterine cervical cancer (LAUCC). RNA-seq was performed to identify immune and hypoxic gene signatures. A total of 288 patients were randomized to either EFI or pelvic radiotherapy (PRT). All patients completed chemoradiotherapy. Overall, significantly higher 5-year para-aortic recurrence free survival (PARFS) rate occurred in EFI (97.6%) than in PRT group (87.2%), with marginal tendency to improve disease-free survival (DFS; 78% vs 70%, P = .066). Subgroup analyses were performed based on carbonic anhydrase 9 (CA9)-only positive, CA9/hypoxia-inducible factor (HIF) double positive and CA9 negative. In the CA9-only positive, EFI successfully increased 5-year PARFS (100% vs 76.4%, P = .010), resulting in significantly improved long-term DFS (85.7% vs 54.7%, P = .023) compared to the PRT, while there was no such benefit of EFI in the CA9/HIFs double positive. RNA-seq analysis identified distinct immunehigh subgroup with negative correlation with hypoxia gene signatures (R = -.37, P < .01), which showed a higher 5-year DFS than the immunelow (P = .032). Hypoxia-related genes were upregulated in the CA9/HIFs double positive compared to CA9 negative (P < .05). Only 17.4% of patients in CA9-negative group showed immunelow signatures, while 40.0% of patients in the double-positive group exhibited immunelow signatures. In conclusion, EFI improved PARFS significantly in all patients, but therapeutic efficacy of EFI in terms of improved DFS was solely observed in CA9-only positive LAUCC, and not in CA9/HIFs double-positive subgroup. RNA-seq analysis suggested that hypoxia-induced immunosuppression may be related to treatment resistance in LAUCC.

OLA1 promotes colorectal cancer tumorigenesis by activation of HIF1α/CA9 axis.

BACKGROUND: Obg-like ATPase 1 (OLA1) is a highly conserved GTPase, which was over expressed in a variety of malignant tumors, but its role in colorectal cancer (CRC) was poorly studied. PATIENTS AND METHODS: Three public CRC gene databases were applied for OLA1 mRNA expression detection. The clinical data of 111 CRC patients were retrospectively collected from the Second Affiliated Hospital of Zhejiang University (SAHZU) for OLA1 protein expression and Kaplan-Meier Survival analysis. OLA1 stably knocked out CRC cell lines were conducted by CRISPR-Cas9 for experiments in vitro and in vivo. RESULTS: OLA1 was highly expressed in 84% CRC compared to matched surrounding tissues. Patients with OLA1 high expression had a significantly lower 5-year survival rate (47%) than those with OLA1 low expression (75%). OLA1 high expression was an independent factor of poor prognosis in CRC patients. OLA1-KO CRC cell lines showed lower ability of growth and tumorigenesis in vitro and in vivo. By mRNA sequence analysis, we found 113 differential express genes in OLA1-KO cell lines, of which 63 were hypoxic related. HIF1α was a key molecule in hypoxic regulation. Further molecular mechanisms showed HIF1α /CA9 mRNA and/or protein levels were heavily downregulated in OLA1-KO cell lines, which could explain the impaired tumorigenesis. According to previous studies, HIF1α was a downstream gene of GSK3ß, we verified GSK3ß was over-activated in OLA1-KO cell lines. CONCLUSION: OLA1 was a new gene that was associated with carcinogenesis and poor outcomes in CRC by activation of HIF1α/CA9 axis, which may be interpreted by GSK3ß.

MN/CA9 gene expression as a potential tumor marker for renal cell carcinoma.

MN/CA9 is a cell surface glycoprotein and a tumor-associated antigen. It plays a crucial role in the regulation of cell proliferation and oncogenesis. There is no ideal tumor marker currently available for renal cell carcinoma (RCC) with sufficient sensitivity and specificity. Therefore, we studied MN/CA9 gene expression in the tumor tissue, apparently normal kidney tissue, preoperative blood, and urine samples of patients with RCC. We included thirty cases of renal tumors (26 RCC and 4 benign tumors) in the study. We applied an RT-PCR assay for MN/CA9 gene expression to 26 RCC kidney tumor samples and four benign kidney tumor tissue samples. We also evaluated MN/CA9 gene expression in preoperative blood and urine samples of 15 of these cases. Additionally, thirty-five grossly normal renal tissue samples, including 21 from kidneys with RCC, were also evaluated for gene expression. The RT-PCR analysis revealed that twenty-one out of 26 RCC tissue samples showed MN/CA9 gene expression compared to three out of 35 non-malignant renal tissue samples (p < 0.05). Two out of four benign renal tissue samples also expressed this gene. We also observed MN/CA9 gene expression in nine out of 15 blood samples and four out of 15 urine samples. All patients with urinary MN/CA9 gene expression showed expression in blood and tumor tissue samples. We found a correlation in terms of MN/CA9 expression between blood and tumor tissue samples of RCC patients as those who exhibit MN/CA9 expression in blood were also positive at the tumor tissue levels. The difference in MN/CA9 gene expression in tumor tissue, blood, and urine samples in relation to the stage of the disease, nuclear grade, and histological cell-type was not statistically significant. However, all the three patients who had metastatic RCC had MN/CA9 gene expression in their blood. The existence of a tumor-associated antigen such as MN/CA9 may present a possible target for molecular diagnosis and management of RCC.

Epigenetic regulation of the DNMT1/MT1G/KLF4/CA9 axis synergises the anticancer effects of sorafenib in hepatocellular carcinoma.

Sorafenib, a multikinase inhibitor, has been widely used as a first-line anticancer drug for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance to sorafenib is frequently observed in clinical applications. Potential nonkinase targets of sorafenib have not been well documented and may provide insights into reversing drug resistance and enhancing drug efficacy. Herein, we report that sorafenib exerts its anticancer effects by activating metallothionein 1 G (MT1G) expression. MT1G is a novel marker in HCC that correlates well with patient survival. MT1G overexpression suppressed the cellular proliferation, migration, invasion, and tumour formation of HCC and sensitised cells to sorafenib treatment. However, the disruption of MT1G attenuated the anticancer effects of sorafenib. Mechanistically, sorafenib upregulated MT1G expression via hypomethylation of its promoter region by binding and inhibiting DNA methyltransferase 1 (DNMT1) and increasing its promoter accessibility in HCC cells. Activation of MT1G also inhibited CA9 transcription through the suppression of HIF1A as mediated by KLF4. Our collective data revealed that sorafenib exerts its anticancer effects through epigenetic regulation of the DNMT1/MT1G/KLF4/CA9 axis in HCC and the activation of MT1G might constitute a strategy for enhancing the effect of sorafenib to suppress HCC cells.

CA9 transcriptional expression determines prognosis and tumour grade in tongue squamous cell carcinoma patients.

CA9 is a member of the carbonic anhydrases' family, that is often expressed in cancer cells under hypoxic condition. However, the role of CA9 in the molecular mechanisms of tongue squamous cell carcinoma (TSCC) pathogenesis remains unclear. CA9 expression was analysed using the TCGA database, and its influence on survival was performed using Kaplan-Meier, LASSO and COX regression analyses. The correlation between CA9 and immune infiltration was investigated by CIBERSORT and ESTIMATE. Moreover, the relationship between CA9 expression and downstream molecular regulation pathways was analysed by GSEA, GO and WGCNA. CA9 expression correlated with clinical prognosis and tumour grade in TSCC. Moreover, CA9 expression potentially contributes to the regulation of cancer cell differentiation and mediates tumour-associated genes and signalling pathways, including apoptosis, hypoxia, G2M checkpoint, PI3K/AKR/mTOR signalling and TGF-beta signalling pathways. However, the follicular helper T cells, regulatory T cells, immune and stromal scores showed no significance between high and low CA9 expression groups. These findings suggested that CA9 plays a critical role of TSCC prognosis and tumour grade. CA9 expression significantly correlated with the regulation of cell differentiation, various oncogenes and cancer-associated pathways.

Sohlh2 alleviates malignancy of EOC cells under hypoxia via inhibiting the HIF1α/CA9 signaling pathway.

Epithelial ovarian cancer (EOC) is the most common and deadly ovarian cancer. Most of the patients have abdominal/pelvic invasion and metastasis at the time of diagnosis, but the underlying mechanism remains unclear. Insufficiency of blood perfusion and diffusion within most solid tumors can lead to a hypoxic tumor microenvironment and promotes tumor malignancy. In the present study, we detected the role of the spermatogenesis- and oogenesis-specific basic helix-loop-helix (bHLH) transcription factor 2 (sohlh2) on migration, invasion and epithelial-mesenchymal transition (EMT) of EOC cell lines under hypoxia in vitro. We also investigated the possible mechanism underlying it. The results showed that sohlh2 inhibited the migration, invasion and EMT of EOC cells and might function through suppression of the hypoxia-inducible factor 1α (HIF1α)/carbonic anhydrase 9 (CA9) signaling pathway. Our results may open a new avenue for the further development of diagnostic tools and novel therapeutics that will benefit EOC patients.

Carbonic anhydrase IX (CA9) expression in multiple renal epithelial tumour subtypes.

AIMS: Renal epithelial neoplasms (RENs) can be difficult to subclassify, owing to overlapping morphological features. Carbonic anhydrase 9 (CA9) is a common biomarker for clear cell renal cell carcinoma (CCRCC); however, the sensitivity and specificity across REN subtypes are less clear. The aim of this study was to investigate CA9 expression in RENs, especially those in the differential diagnosis with CCRCC and less common entities, to determine its reliability as a diagnostic biomarker. METHODS AND RESULTS: CA9 immunostaining was performed on 262 RENs, including 119 CCRCCs and 143 non-CCRCC. Immunostaining was evaluated as negative (0%), rare (1+, 1-10%), focal (2+, 11-50%), or diffuse (3+, >50%). CCRCCs were 3+ CA9-positive in 93% of cases; 4% were CA9-negative. Sixty-seven percent of papillary renal cell carcinomas (RCCs) were 1+/2+ CA9-positive, whereas 33% were CA9-negative. Chromophobe RCCs were nearly always CA9-negative (93%), with 7% showing rare cell reactivity. Clear cell tubulopapillary RCCs (CCTPRCCs) were consistently 3+ CA9-positive, but with a cup-like staining pattern. Fifty-three percent of Xp11.2 RCCs were CA9-negative; however, 6% were 3+ CA9-positive and 12% were 2+ CA9-positive. Two of eight fumarate hydratase-deficient RCCs were 3+ CA9-positive. A small subset of the remaining RCCs showed rare to focal CA9 expression. All oncocytomas and eosinophilic solid and cystic RCCs were CA9-negative. CONCLUSIONS: Overall, diffuse CA9 expression was identified in nearly all CCRCCs and in all CCTPRCCs (high sensitivity); however, CA9 was not entirely specific. At least focal CA9 expression can been seen in a subset of many RCCs, and such findings should be taken into consideration with other morphological, immunophenotypic and clinical findings.

CA9 Silencing Promotes Mitochondrial Biogenesis, Increases Putrescine Toxicity and Decreases Cell Motility to Suppress ccRCC Progression.

Carbonic anhydrase IX (CA9), a pH-regulating transmembrane protein, is highly expressed in solid tumors, and particularly in clear cell renal cell carcinoma (ccRCC). The catalytic mechanisms of CA9 are well defined, but its roles in mediating cell migration/invasion and survival in ccRCC remain to be determined. Here, we confirmed that the mRNA expression of CA9 in ccRCC was significantly higher than that in para-carcinoma tissues from analysis of the datasets in The Cancer Genome Atlas. CA9 knockdown upregulated oxidative phosphorylation-associated proteins and increased mitochondrial biogenesis, resulting in the reversal of the Warburg phenotype and the inhibition of cell growth. Our study revealed that CA9 knockdown upregulated mitochondrial arginase 2 (ARG2), leading to the accumulation of putrescine, which suppressed ccRCC proliferation. Surfaceomics analysis revealed that CA9 knockdown downregulated proteins associated with extracellular matrix (ECM)-receptor interaction and cell adhesion, resulting in decreased cell migration. CA9 silencing also downregulated amino acid transporters, leading to reduced cellular amino acids. Collectively, our data show that CA9 knockdown suppresses proliferation via metabolic reprogramming and reduced cell migration, reaffirming that CA9 is a potential therapeutic target for ccRCC treatment.

PADI4 stimulates esophageal squamous cell carcinoma tumor growth and up-regulates CA9 expression.

An increasing amount of evidence indicates that peptidylarginine deiminase isoform 4 (PADI4) plays an important role in tumorigenesis. However, the effects of PADI4 on tumor-bearing mice are unknown, and no studies have investigated this tumorigenic pathway in an animal model. In the present study, ECA109 cells originating from esophageal squamous cell carcinoma (ESCC) were transfected with PADI4-expressing lentivirus and were injected into BALB/c nude mice. Tumor size and weight were significantly increased in the mouse tumors established with PADI4-overexpressing ECA109 cells. PCR array analysis revealed increased CA9 expression in ECA109 cells transfected with a PADI4-expressing plasmid, while decreased CA9 expression levels were detected in cells transfected with anti-PADI4 siRNA. Furthermore, up-regulation of CA9 expression was detected in mouse tumors established with PADI4-overexpressing cells. Immunohistochemistry detected the increased expression and co-localization of PADI4 and CA9 in ESCC tissues compared with adjacent non-tumor tissues and normal tissue controls. These results were verified using Western blotting. Cell proliferation significantly increased or decreased in ECA109 and EC9706 (another ESCC-originating cell line) cells transfected with a PADI4-expressing plasmid or anti-PADI4 siRNA, respectively. The above findings suggest that increased PADI4 expression in ESCC stimulates tumor growth and up-regulates CA9 expression, which is known to promote metastatic properties in tumor cells.

Mucin 5B, carbonic anhydrase 9 and claudin 18 are potential theranostic markers of gallbladder carcinoma.

AIMS: Gallbladder cancer (GBC) is an aggressive tumour that is usually diagnosed at advanced stages and is characterised by a poor prognosis. Using public data of normal human tissues, we found that mRNA and protein levels of mucin 5B (MUC5B) and carbonic anhydrase 9 (CA9) were highly increased in gallbladder tissues. In addition, previous evidence has shown that claudin 18 (CLDN18) protein expression is higher in GBC. The aim of this study was to perform an analysis of these cell surface proteins during the histological progression of GBC in order to identify their theranostic potential. METHODS AND RESULTS: MUC5B expression, CA9 expression and CLDN18 expression were examined by immunohistochemistry in a series of 179 chronic cholecystitis (including 16 metaplastic tissues), 15 dysplasia and 217 GBC samples by the use of tissue microarray analysis. A composite staining score was calculated from staining intensity and percentage of positive cells. Immunohistochemical analysis showed high expression of MUC5B and CA9 among normal epithelium, metaplastic tissues, and dysplastic tissues. However, expression of both proteins was observed in roughly 50% of GBC samples. In contrast, CLDN18 was absent in normal epithelium, but its expression was higher in metaplastic cells. Among GBC cases, approximately half showed high CLDN18 expression. No associations were found between MUC5B, CA9 and CLDN18 expression and any clinicopathological features. CONCLUSIONS: CLDN18 is a new metaplasia marker in gallbladder tissues, and is conserved in approximately half of GBC cases. MUC5B and CA9 are highly conserved during GBC histological progression. The three markers are potential theranostic markers, in particular CA9 and CLDN18, for which there are already targeted therapies available.

High Beclin-1 and ARID1A expression corelates with poor survival and high recurrence in intrahepatic cholangiocarcinoma: a histopathological retrospective study.

BACKGROUND: Although surgical resection provides a cure for patients with intrahepatic cholangiocarcinoma (ICC), the risk of mortality and recurrence remains high. Several biomarkers are reported to be associated with the prognosis of ICC, including Beclin-1, ARID1A, carbonic anhydrase IX (CA9) and isocitrate dehydrogenase 1 (IDH1), but results are inconsistent. Therefore, a histopathological retrospective study was performed to simultaneously investigate the relationship of these four potential biomarkers with clinicopathological parameters and their prognostic values in patients with ICC. METHODS: A total of 113 patients with ICC were enrolled from Cancer Hospital of Chinese Academy of Medical Sciences between January 1999 and June 2015. The expression of Beclin-1, ARID1A, IDH1 and CA9 were determined by immunohistochemical staining. The prognostic values of the four biomarkers were analyzed by Cox regression and the Kaplan-Meier method. RESULTS: Beclin-1, ARID1A, CA9 and IDH1 were highly expressed in ICC tumor tissues. Higher mortality was positively associated with Beclin-1 expression (HR = 2.39, 95% CI = 1.09-5.24) and higher recurrence was positively associated with ARID1A expression (HR = 1.71, 95% CI = 1.06-2.78). Neither CA9 nor IDH1 expression was significantly associated with mortality or disease recurrence. Kaplan-Meier survival curves showed that ICC patients with higher Beclin-1 and ARID1A expression had a lower survival rate and a worse recurrence rate than patients with low Beclin-1 and ARID1A expression (p < 0.05). CONCLUSIONS: High Beclin-1 and ARIDIA expression are strongly associated with poor prognosis in ICC patients, and thus Beclin-1 and ARID1A should be simultaneously considered as potential prognostic biomarkers for ICC patients.

ERO1α is a novel endogenous marker of hypoxia in human cancer cell lines.

BACKGROUND: Hypoxia is an important factor that contributes to tumour aggressiveness and correlates with poor prognosis and resistance to conventional therapy. Therefore, identifying hypoxic environments within tumours is extremely useful for understanding cancer biology and developing novel therapeutic strategies. Several studies have suggested that carbonic anhydrase 9 (CA9) is a reliable biomarker of hypoxia and a potential therapeutic target, while pimonidazole has been identified as an exogenous hypoxia marker. However, other studies have suggested that CA9 expression is not directly induced by hypoxia and it is not expressed in all types of tumours. Thus, in this study, we focused on endoplasmic reticulum disulphide oxidase 1α (ERO1α), a protein that localises in the endoplasmic reticulum and is involved in the formation of disulphide bonds in proteins, to determine whether it could serve as a potential tumour-hypoxia biomarker. METHODS: Using quantitative real-time polymerase chain reaction, we analysed the mRNA expression of ERO1α and CA9 in different normal and cancer cell lines. We also determined the protein expression levels of ERO1α and CA9 in these cell lines by western blotting. We then investigated the hypoxia-inducible ERO1α and CA9 expression and localisation in HCT116 and HeLa cells, which express low (CA9-low) and high (CA9-high) levels of CA9, respectively. A comparative analysis was performed using pimonidazole, an exogenous hypoxic marker, as a positive control. The expression and localisation of ERO1α and CA9 in tumour spheres during hypoxia were analysed by a tumour sphere formation assay. Finally, we used a mouse model to investigate the localisation of ERO1α and CA9 in tumour xenografts using several cell lines. RESULTS: We found that ERO1α expression increased under chronic hypoxia. Our results show that ERO1α was hypoxia-induced in all the tested cancer cell lines. Furthermore, in the comparative analysis using CA9 and pimonidazole, ERO1α had a similar localisation to pimonidazole in both CA9-low and CA9-high cell lines. CONCLUSION: ERO1α can serve as a novel endogenous chronic hypoxia marker that is more reliable than CA9 and can be used as a diagnostic biomarker and therapeutic target for cancer.