Clonal relationship and alcohol consumption-associated mutational signature in synchronous hypopharyngeal tumours and oesophageal squamous cell carcinoma.

BACKGROUND: The patients with dual oesophageal squamous cell carcinoma (ESCC) and hypopharyngeal cancer (HPC) have poor prognosis; their underlying genetic pathogenesis is unclear. We hypothesise that development of synchronous ESCC/HPC depends on multicentricity or independent origin, rather than multifocality due to local or lateral spreading. METHOD: Multiple region whole-exome sequencing (M-WES) and clonality analysis were used to assess clonal relationship and spatial inter- or intra-tumour heterogeneity (ITH) in 62 tumour regions from eight dual ESCC/HPC and ten ESCC patients. RESULTS: All synchronous ESCC/HPC patients had COSMIC 16 mutation signatures, compared to only 40% ESCC in the current study (p = 0.013) and public data set (n = 165, p = 0.003). This alcohol consumption-related mutation signature 16, commonly involved in multiple alcohol-related cancers, was significantly associated with drinking and alcohol metabolism-related ADH1B rs1229984. The mutational landscape and copy number profiles were completely distinct between the two primary tumours; clonality analysis further suggested the two primary tumours shared no or only one clone accompanying independent subclone evolution. M-WES strategy demonstrated higher sensitivity and accuracy for detection of mutational prevalence and the late branch mutations among different regions in the ESCC tumours, compared to traditional sequencing analysis based on single biopsy strategy. Patients with high ITH assessed by cancer cell fraction analysis after M-WES were significantly associated with both relapse and survival. CONCLUSIONS: Our hypothesis-generating M-WES ITH assessment data have implications for prognostication. Collectively, our findings support multicentric independent clonal evolution, the field cancerisation theory, and suggest novel insights implicating an aetiologic role of alcohol metabolism in dual ESCC/HPC carcinogenesis.

BRCA2 loss-of-function germline mutations are associated with esophageal squamous cell carcinoma risk in Chinese.

Esophageal squamous cell carcinoma (ESCC) occurs with highest frequency in China with over 90% mortality, highlighting the need for early detection and improved treatment strategies. We aimed to identify ESCC cancer predisposition gene(s). Our study included 4,517 individuals. The discovery phase using whole-exome sequencing (WES) included 186 familial ESCC patients from high-risk China. Targeted gene sequencing validation of 598 genes included 3,289 Henan and 1,228 moderate-risk Hong Kong Chinese. A WES approach identified BRCA2 loss-of-function (LOF) mutations in 3.23% (6/186) familial ESCC patients compared to 0.21% (9/4300) in the ExAC East Asians (odds ratio [OR] = 15.89, p = 2.48 × 10-10 ). BRCA2 LOF mutation frequency in the combined Henan cohort has significantly higher prevalence (OR = 10.55, p = 0.0035). Results were independently validated in an ESCC Hong Kong cohort (OR = 10.64, p = 0.022). One Hong Kong pedigree was identified to carry a BRCA2 LOF mutation. BRCA2 inactivation in ESCC was via germline LOF mutations and wild-type somatic allelic loss via loss of heterozygosity. Gene-based association analysis, including LOF mutations and rare deleterious missense variants defined with combined annotation dependent depletion score ≥30, confirmed the genetic predisposition role of BRCA2 (OR = 9.50, p = 3.44 × 10-5 ), and provided new evidence for potential association of ESCC risk with DNA repair genes (POLQ and MSH2), inflammation (TTC39B) and angiogenesis (KDR). Our findings are the first to provide compelling evidence of the role of BRCA2 in ESCC genetic susceptibility in Chinese, suggesting defective homologous recombination is an underlying cause in ESCC pathogenesis, which is amenable to therapeutic options based on synthetic lethality approaches such as targeting BRCA2 with PARP1 inhibitors in ESCC.

Whole-exome sequencing reveals critical genes underlying metastasis in oesophageal squamous cell carcinoma.

Oesophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers, owing to a high frequency of metastasis. However, little is known about the genomic landscape of metastatic ESCC. To identify the genetic alterations that underlie ESCC metastasis, whole-exome sequencing was performed for 41 primary tumours and 15 lymph nodes (LNs) with metastatic ESCCs. Eleven cases included matched primary tumours, synchronous LN metastases, and non-neoplastic mucosa. Approximately 50-76% of the mutations identified in primary tumours appeared in the synchronous LN metastases. Metastatic ESCCs harbour frequent mutations of TP53, KMT2D, ZNF750, and IRF5. Importantly, ZNF750 was recurrently mutated in metastatic ESCC. Combined analysis from current and previous genomic ESCC studies indicated more frequent ZNF750 mutation in diagnosed cases with LN metastasis than in those without metastasis (14% versus 3.4%, n = 629, P = 1.78 × 10-5 ). The Cancer Genome Atlas data further showed that ZNF750 genetic alterations were associated with early disease relapse. Previous ESCC studies have demonstrated that ZNF750 knockdown strongly promotes proliferation, migration, and invasion. Collectively, these results suggest a role for ZNF750 as a metastasis suppressor. TP53 is highly mutated in ESCC, and missense mutations are associated with poor overall survival, independently of pathological stage, suggesting that these missense mutations have important functional impacts on tumour progression, and are thus likely to be gain-of-function (GOF) mutations. Additionally, mutations of epigenetic regulators, including KMT2D, TET2, and KAT2A, and chromosomal 6p22 and 11q23 deletions of histone variants, which are important for nucleosome assembly, were detected in 80% of LN metastases. Our study highlights the important role of critical genetic events including ZNF750 mutations, TP53 putative GOF mutations and nucleosome disorganization caused by genetic lesions seen with ESCC metastasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Nuclear Localization of DNAJB6 Is Associated With Survival of Patients With Esophageal Cancer and Reduces AKT Signaling and Proliferation of Cancer Cells.

BACKGROUND & AIMS: The DnaJ (Hsp40) homolog, subfamily B, member 6 (DNAJB6) is part of a family of proteins that regulates chaperone activities. One of its isoforms, DNAJB6a, contains a nuclear localization signal and regulates ß-catenin signaling during breast cancer development. We investigated the role of DNAJB6 in the pathogenesis of esophageal squamous cell carcinoma (ESCC). METHODS: We performed immunohistochemical analyses of primary ESCC samples and lymph node metastases from a cohort of 160 patients who underwent esophagectomy with no preoperative chemoradiotherapy at Hong Kong Queen Mary Hospital. Data were collected on patient outcomes over a median time of 12.1 ± 2.9 months. Retrospective survival association analyses were performed. Wild-type and mutant forms of DNAJB6a were overexpressed in cancer cell lines (KYSE510, KYSE 30TSI, KYSE140, and KYSE70TS), which were analyzed in proliferation and immunoblot assays, or injected subcutaneously into nude mice. Levels of DNAJB6 were knocked down in ESCC cell lines (KYSE450 and T.Tn), immortalized normal esophageal epithelial cell lines (NE3 and NE083), and other cells with short hairpin RNAs, or by genome engineering. Bimolecular fluorescence complementation was used to study interactions between proteins in living cells. RESULTS: In primary ESCC samples, patients whose tumors had high nuclear levels of DNAJB6 had longer overall survival times (19.2 ± 1.8 months; 95% confidence interval [CI], 15.6-22.8 mo) than patients whose tumors had low nuclear levels of DNAJB6 (12.6 ± 1.4 mo; 95% CI, 9.8-15.4 mo; P = .004, log-rank test). Based on Cox regression analysis, patients whose tumors had high nuclear levels of DNAJB6 had a lower risk of death than patients with low levels (hazard ratio, 0.562; 95% CI, 0.379-0.834; P = .004). Based on log-rank analysis and Cox regression analysis, the combination of the nuclear level of DNAJB6 and the presence of lymph node metastases at diagnosis could be used to stratify patients into groups with good or bad outcomes (P < .0005 for both analyses). There was a negative association between the nuclear level of DNAJB6 and the presence of lymph node metastases (P = .022; Pearson χ(2) test). Cancer cell lines that overexpressed DNAJB6a formed tumors more slowly in nude mice than control cells or cells that expressed a mutant form of DNAJB6a that did not localize to the nucleus. DNAJB6 knockdown in cancer cell lines promoted their growth as xenograft tumors in mice. A motif of histidine, proline, and aspartic acid in the J domain of DNAJB6a was required for its tumor-suppressive effects and signaling via AKT1. Loss of DNAJB6a resulted in up-regulation of AKT signaling in cancer cell lines and immortalized esophageal epithelial cells. Expression of a constitutively active form of AKT1 restored proliferation to tumor cells that overexpressed DNAJB6a, and DNAJB6a formed a complex with AKT1 in living cells. The expression of DNAJB6a reduced the sensitivity of ESCC to AKT inhibitors; the expression level of DNAJB6a affected AKT signaling in multiple cancer cell lines. CONCLUSIONS: Nuclear localization of DNAJB6 is associated with longer survival times of patients with ESCC. DNAJB6a reduces AKT signaling, and DNAJB6 expression in cancer cells reduces their proliferation and growth of xenograft tumors in mice. DNAJB6a might be developed as a biomarker for progression of ESCC.

Integrated network findings reveal ubiquitin-specific protease 44 overexpression suppresses tumorigenicity of liver cancer.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and third leading cause of cancer-related deaths worldwide. HCC is a multistep disease marked by various signaling alterations. A better understanding of the new molecular drivers of HCC could therefore provide an opportunity to develop effective diagnostic and therapeutic targets. Ubiquitin-specific protease 44 (USP44), a member of the cysteine protease family, has been reported to play a role in many cancer types. However, its contribution to HCC development remains unknown. In the present study, we observed suppression of USP44 expression in HCC tissue. Clinicopathologic analysis further showed that low USP44 expression correlated with poorer survival and a later tumor stage in HCC, suggesting that USP44 could be a predictor of poor prognosis in patients with HCC. Gain-of-function analysis in vitro demonstrated the importance of USP44 in HCC cell growth and G0/G1 cell cycle arrest. To investigate the downstream targets of USP44 and the molecular mechanisms underlying its regulation of cell proliferation in HCC, we conducted a comparative transcriptomic analysis and identified a cluster of proliferation-related genes, including CCND2, CCNG2, and SMC3. Ingenuity Pathway Analysis further delineated the gene networks controlled by USP44 through the regulation of membrane proteins and receptors, enzymes, transcriptional factors, and cyclins involved in the control of cell proliferation, metastasis, and apoptosis in HCC. To summarize, our results highlight, for the first time, the tumor-suppression role of USP44 in HCC and suggest a new prognostic biomarker in this disease.

Transcriptomic analysis reveals the oncogenic role of S6K1 in hepatocellular carcinoma.

The p70 ribosomal protein S6 kinase 1 (S6K1), a serine/threonine kinase, is commonly overexpressed in a variety of cancers. However, its expression level and functional roles in hepatocellular carcinoma (HCC), which ranks as the third leading cause of cancer-related death worldwide, is still largely unknown. In the current report, we show the in vivo and in vitro overexpression of S6K1 in HCC. In the functional analysis, we demonstrate that S6K1 is required for the proliferation and colony formation abilities in HCC. By using comparative transcriptomic analysis followed by gene ontology enrichment analysis and Ingenuity Pathway Analysis, we find that the depletion of S6K1 can elevate the expression of a cluster of apoptotic genes, tumor suppressor genes and immune responsive genes. Moreover, the knockdown of S6K1 is predicted to reduce the tumorigenicity of HCC through the regulation of hubs of genes including STAT1, HDAC4, CEBPA and ONECUT1. In conclusion, we demonstrate the oncogenic role of S6K1 in HCC, suggesting the possible use of S6K1 as a therapeutic target for HCC treatment.

Identification of miR-29c and its Target FBXO31 as a Key Regulatory Mechanism in Esophageal Cancer Chemoresistance: Functional Validation and Clinical Significance.

Rationale: Dysregulated microRNA (miRNA) expressions in cancer can contribute to chemoresistance. This study aims to identify miRNAs that are associated with fluorouracil (5-FU) chemoresistance in esophageal squamous cell carcinoma (ESCC). The potential of miR-29c as a novel diagnostic, prognostic and treatment-predictive marker in ESCC, and its mechanisms and therapeutic implication in overcoming 5-FU chemoresistance were explored. Methods: The miRNA profiles of an ESCC cell model with acquired chemoresistance to 5-FU were analyzed using a Taqman miRNA microarray to identify novel miRNAs associated with 5-FU chemoresistance. Quantitative real-time PCR was used to determine miR-29c expression in tissue and serum samples of patients. Bioinformatics, gain- and loss-of-function experiments, and luciferase reporter assay were performed to validate F-box only protein 31 (FBXO31) as a direct target of miR-29c, and to identify potential transcription factor binding events that control miR-29c expression. The potential of systemic miR-29c oligonucleotide-based therapy in overcoming 5-FU chemoresistance was evaluated in tumor xenograft model. Results: MiR-29c, under the regulatory control of STAT5A, was frequently downregulated in tumor and serum samples of patients with ESCC, and the expression level was correlated with overall survival. Functional studies showed that miR-29c could override 5-FU chemoresistance in vitro and in vivo by directly interacting with the 3'UTR of FBXO31, leading to repression of FBXO31 expression and downstream activation of p38 MAPK. Systemically administered miR-29c dramatically improved response of 5-FU chemoresistant ESCC xenografts in vivo. Conclusions: MiR-29c modulates chemoresistance by interacting with FBXO31, and is a promising non-invasive biomarker and therapeutic target in ESCC.

Elevated levels of serum nidogen-2 in esophageal squamous cell carcinoma.

BACKGROUND: Nidogen-2 (NID2), a secretory basement membrane protein, has been implicated as a potential biomarker in ovarian cancer and hepatocellular carcinoma. OBJECTIVE: In this study, we aimed to investigate the utility of detecting serum NID2 levels for identification of esophageal squamous cell carcinoma (ESCC) patients and prediction of poor survival outcome. METHODS: Using an in-house NID2 enzyme-linked immunosorbent assay (ELISA), serum samples from 101 ESCC patients and 50 healthy controls were screened for their NID2 levels. RESULTS: The serum NID2 levels in ESCC patients (median 24.4 µg/L) are significantly higher (p= 4.3e-09) than that of the healthy controls (median 15.85 µg/L). The receiver operating characteristic (ROC) curve demonstrated an area under the curve of 0.756. At the threshold of 17.95 µg/L, the sensitivity and specificity achieved are 0.76 and 0.63, respectively. Kaplan-Meier survival analysis revealed that patients with high serum NID2 levels (⩾ 32.6 µg/L) have significantly higher risk of death (HR = 1.984, 95% CI: 1.175-3.349; log-rank p-value = 0.012) compared to those with low serum NID2 levels (< 20.0 µg/L). CONCLUSIONS: In conclusion, we show that detecting the elevation of serum NID2 levels has potential diagnostic and prognostic value for ESCC patients.

Role of AMPK signaling in mediating the anticancer effects of silibinin in esophageal squamous cell carcinoma.

OBJECTIVE: Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK) may suppress cancer growth. Identification of novel AMPK activators is therefore crucial to exploit AMPK as a potential target for cancer prevention and treatment. RESEARCH DESIGN AND METHODS: We determined the expression status and role of AMPK in esophageal squamous cell carcinoma (ESCC) and investigated whether silibinin, a nontoxic natural product, could activate AMPK to inhibit ESCC development. RESULTS: Our results from 49 pairs of human ESCC and normal tissues showed that AMPK was constitutively inactive in the majority (69.4%) of ESCC. We found that silibinin induced apoptosis, and inhibited ESCC cell proliferation in vitro and tumorigenicity in vivo without any adverse effects. Silibinin also markedly suppressed the invasive potential of ESCC cells in vitro and their ability to form lung metastasis in nude mice. The anticancer effects of silibinin were abrogated by the presence of compound C or shRNA against AMPK. More importantly, silibinin enhanced the sensitivity of ESCC cells and tumors to the chemotherapeutic drugs, 5-fluorouracil and cisplatin. CONCLUSIONS: This preclinical study supports that AMPK is a valid therapeutic target and suggests that silibinin may be a potentially useful therapeutic agent and chemosensitizer for esophageal cancer.

Protein phosphatase magnesium-dependent 1δ is a novel tumor marker and target in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a lethal liver malignancy worldwide. In this study, we reported that protein phosphatase magnesium-dependent 1δ (PPM1D) was highly expressed in the majority of HCC cases (approximately 59%) and significantly associated with high serum α-fetoprotein (AFP) level (P = 0.044). Kaplan- Meier and Cox regression data indicated that PPM1D overexpression was an independent predictor of HCCspecific overall survival (HR, 2.799; 95% CI, 1.346-5.818, P = 0.006). Overexpressing PPM1D promoted cell viability and invasion, whereas RNA interference-mediated knockdown of PPM1D inhibited proliferation, invasion, and migration of cultured HCC cells. In addition, PPM1D suppression by small interfering RNA decreased the tumorigenicity of HCC cells in vivo. Overall, results suggest that PPM1D is a potential prognostic marker and therapeutic target for HCC.

Competitive Binding Between Id1 and E2F1 to Cdc20 Regulates E2F1 Degradation and Thymidylate Synthase Expression to Promote Esophageal Cancer Chemoresistance.

PURPOSE: Chemoresistance is a major obstacle in cancer therapy. We found that fluorouracil (5-FU)-resistant esophageal squamous cell carcinoma cell lines, established through exposure to increasing concentrations of 5-FU, showed upregulation of Id1, IGF2, and E2F1. We hypothesized that these genes may play an important role in cancer chemoresistance. EXPERIMENTAL DESIGN: In vitro and in vivo functional assays were performed to study the effects of Id1-E2F1-IGF2 signaling in chemoresistance. Quantitative real-time PCR, Western blotting, immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assays were used to investigate the molecular mechanisms by which Id1 regulates E2F1 and by which E2F1 regulates IGF2. Clinical specimens, tumor tissue microarray, and Gene Expression Omnibus datasets were used to analyze the correlations between gene expressions and the relationships between expression profiles and patient survival outcomes. RESULTS: Id1 conferred 5-FU chemoresistance through E2F1-dependent induction of thymidylate synthase expression in esophageal cancer cells and tumor xenografts. Mechanistically, Id1 protects E2F1 protein from degradation and increases its expression by binding competitively to Cdc20, whereas E2F1 mediates Id1-induced upregulation of IGF2 by binding directly to the IGF2 promoter and activating its transcription. The expression level of E2F1 was positively correlated with that of Id1 and IGF2 in human cancers. More importantly, concurrent high expression of Id1 and IGF2 was associated with unfavorable patient survival in multiple cancer types. CONCLUSIONS: Our findings define an intricate E2F1-dependent mechanism by which Id1 increases thymidylate synthase and IGF2 expressions to promote cancer chemoresistance. The Id1-E2F1-IGF2 regulatory axis has important implications for cancer prognosis and treatment.

Regulation of T helper 17 by bacteria: an approach for the treatment of hepatocellular carcinoma.

T helper 17 (T(H)17) is a novel subset of T helper cells that has recently been identified in the hepatocellular carcinoma (HCC) tumor environment. Its presence seems to be linked with HCC progression, possibly via facilitating angiogenesis. The origin of tumor-associated T(H)17 may be related to the gut, in which the differentiation of T cells, especially T(H)17 cells, is affected by microbiota. As T(H)17 may appear to be a new therapeutic target against tumor-promoting inflammation, strategies such as using probiotics to polarize the response away from T(H)17 may be beneficial to slow down tumor progression. This paper will attempt to discuss the potential linkage between HCC progression, T(H)17, and gut microbiota and the possible therapeutic implications of probiotics to modulate T(H)17-mediated response for tumor growth.