Exosomal S100A4 derived from highly metastatic hepatocellular carcinoma cells promotes metastasis by activating STAT3.

Intercellular cross-talk plays important roles in cancer progression and metastasis. Yet how these cancer cells interact with each other is still largely unknown. Exosomes released by tumor cells have been proved to be effective cell-to-cell signal mediators. We explored the functional roles of exosomes in metastasis and the potential prognostic values for hepatocellular carcinoma (HCC). Exosomes were extracted from HCC cells of different metastatic potentials. The metastatic effects of exosomes derived from highly metastatic HCC cells (HMH) were evaluated both in vitro and in vivo. Exosomal proteins were identified with iTRAQ mass spectrum and verified in cell lines, xenograft tumor samples, and functional analyses. Exosomes released by HMH significantly enhanced the in vitro invasion and in vivo metastasis of low metastatic HCC cells (LMH). S100 calcium-binding protein A4 (S100A4) was identified as a functional factor in exosomes derived from HMH. S100A4rich exosomes significantly promoted tumor metastasis both in vitro and in vivo compared with S100A4low exosomes or controls. Moreover, exosomal S100A4 could induce expression of osteopontin (OPN), along with other tumor metastasis/stemness-related genes. Exosomal S100A4 activated OPN transcription via STAT3 phosphorylation. HCC patients with high exosomal S100A4 in plasma also had a poorer prognosis. In conclusion, exosomes from HMH could promote the metastatic potential of LMH, and exosomal S100A4 is a key enhancer for HCC metastasis, activating STAT3 phosphorylation and up-regulating OPN expression. This suggested exosomal S100A4 to be a novel prognostic marker and therapeutic target for HCC metastasis.

Insulin-like growth factor 1-induced enolase 2 deacetylation by HDAC3 promotes metastasis of pancreatic cancer.

Enolase 2 (ENO2) is a key glycolytic enzyme in the metabolic process of glycolysis, but its potential function in pancreatic ductal adenocarcinoma (PDAC) is unclear. In this study, we observed a significant overexpression of ENO2 in PDAC tissues, and its expression was correlated with metastasis and poor prognosis in PDAC patients. K394 was identified as a major acetylation site in ENO2 that regulates its enzymatic activity, cell metabolism and PDAC progression. Knockdown of ENO2 suppressed tumor growth and liver metastasis in PDAC. Re-expression of wild-type (WT) ENO2, but not the K394 acetylation mimetic mutant, could reverse the decreased tumor malignancy. We further characterized histone deacetylase 3 (HDAC3) and P300/CBP-associated factor (PCAF) as the potential deacetylase and acetyltransferase for ENO2, respectively. HDAC3-mediated deacetylation was shown to lead to ENO2 activation and enhancement of glycolysis. Importantly, insulin-like growth factor-1 (IGF-1) was found to decrease K394 acetylation and stimulate ENO2 activity in a dose- and time-dependent manner. The PI3K/AKT/mTOR pathway facilitated the phosphorylation of HDAC3 on S424, which promoted K394 deacetylation and activation of ENO2. Linsitinib, an oral small-molecule inhibitor of IGF-1R, could inhibit IGF-1-induced ENO2 deacetylation by HDAC3 and the PI3K/AKT/mTOR pathway. Furthermore, linsitinib showed a different effect on the growth and metastasis of PDAC depending on the overexpression of WT versus K394-mutant ENO2. Our results reveal a novel mechanism by which acetylation negatively regulates ENO2 activity in the metastasis of PDAC by modulating glycolysis. Blockade of IGF-1-induced ENO2 deacetylation represents a promising strategy to prevent the development of PDAC.

miR-345 inhibits migration and stem-like cell phenotype in gastric cancer via inactivation of Rac1 by targeting EPS8.

Tumor metastasis is the main cause of treatment failure and death in patients with late stage of gastric cancer (GC). Studies showed that microRNAs (miRNAs) are important regulators in the process of tumor metastasis. In this study, we used miRNA array analysis to search for metastasis-associated miRNAs in primary and matched metastasis tissues of patients with GC and found that miR-345-5p (miR-345) was significantly higher in primary sites. Decreased expression of miR-345 was observed in GC tissues and cell lines, which was correlated with aggressive stage and grade. Patients with a higher level of miR-345 had a better prognosis. miR-345 could inhibit the migration and spheroid formation abilities in GC cell lines in transwell assay and spheroid formation assay. RNA sequencing and bioinformatics analysis revealed that miR-345 downregulated the epidermal growth factor receptor pathway substrate 8 (EPS8) and its downstream Rac1 signaling. Mechanistically, we confirmed that miR-345 could target EPS8 by directly binding to its 3' untranslated region by luciferase reporter assay. Further rescue assay showed that the ability of miR-345 in inhibiting the migration, stem-like cell phenotype, and epithelial-mesenchymal transition (EMT) in GC was partly dependent on targeting EPS8. In conclusion, miR-345 plays an inhibitory role in GC metastasis through inhibiting cell migration, EMT, and cancer stem cell phenotype via inactivation of Rac1 signaling by targeting EPS8, which provides the potential therapeutic and predictive value of miR-345 in GC.

Therapeutic Strategies Targeting Cancer Stem Cells and Their Microenvironment.

Cancer stem cells (CSCs) have been demonstrated in a variety of tumors and are thought to act as a clonogenic core for the genesis of new tumor growth. This small subpopulation of cancer cells has been proposed to help drive tumorigenesis, metastasis, recurrence and conventional therapy resistance. CSCs show self-renewal and flexible clonogenic properties and help define specific tumor microenvironments (TME). The interaction between CSCs and TME is thought to function as a dynamic support system that fosters the generation and maintenance of CSCs. Investigation of the interaction between CSCs and the TME is shedding light on the biologic mechanisms underlying the process of tumor malignancy, metastasis, and therapy resistance. We summarize recent advances in CSC biology and their environment, and discuss the challenges and future strategies for targeting this biology as a new therapeutic approach.

Mutated EPHA2 is a target for combating lymphatic metastasis in intrahepatic cholangiocarcinoma.

Exploring the genetic aberrations favoring metastasis is important for understanding and developing novel strategies to combat cancer metastasis. It remains lack of effective treatment for the dismal prognosis of intrahepatic cholangiocarcinoma (ICC). Here, we aimed to study genetic alternations during lymph node metastasis of ICC and investigate potential mechanisms and clinical strategy focused on mutations. We performed whole-exome sequencing and transcriptome sequencing on samples from 30 ICC patients, including lymph node metastases from five of the patients. We identified the alterations of genetic pattern related to lymph node metastases of ICC. EPHA2, a member of the tyrosine kinase family, was found to be frequently mutated in ICC. Correlation analysis indicated that EPHA2 mutations were closely associated with lymph node metastasis of ICC. In vitro and in vivo experiments revealed that EPHA2 mutations could lead to ligand independent phosphorylation of Ser897, and promote lymphatic metastasis of ICC, in which NOTCH1 signaling pathway played an important role. In both in vitro assays and patient-derived xenografts, an inhibitor of Ser897 phosphorylation effectively suppressed the metastasis of ICC with mutated EPHA2. Our findings demonstrated that EPHA2 mutants may be an attractive therapeutic target for lymphatic metastasis of ICC.

Osteopontin alters DNA methylation through up-regulating DNMT1 and sensitizes CD133+/CD44+ cancer stem cells to 5 azacytidine in hepatocellular carcinoma.

BACKGROUND: In hepatocellular carcinoma (HCC), CD133+/CD44+ cells are one subgroup with high stemness and responsible for metastatic relapse and resistance to treatment. Our previous studies have demonstrated that osteopontin (OPN) plays critical roles in HCC metastasis. We further investigated the molecular mechanism underlying the role of OPN in regulating the stemness of HCC epigenetically and explored possible targeting strategy. METHODS: CD133+/CD44+ subgroup sorting from HCC cell lines and HCC tissues was used to investigate the effects of OPN knockdown on stemness. iTRAQ and MedIP-sequencing were applied to detect the protein profile and epigenetic modification of CD133+/CD44+ subgroup with or without OPN knockdown. The antitumor effects of 5 Azacytidine were examined in cultured HCC cells and patient derived xenograft (PDX) models. RESULTS: OPN was accumulated in CD133+/CD44+ subgroup of HCC cells. Knocking down OPN significantly inhibited the sphere formation and stemness-related genes expression, and delayed tumor initiation of CD133+/CD44+ subgroup of HCC cells. Employing MedIP-sequencing, dot blot and iTRAQ analyses of CD133+/CD44+ SCR and CD133+/CD44+ shOPN cells, we found that OPN knockdown leaded to reduction in DNA methylation with particular enrichment in CGI. Meanwhile, DNA (cytosine-5)-methyltransferase 1 (DNMT1), the main methylation maintainer, was downregulated via proteomics analysis, which mediated OPN altering DNA methylation. Furthermore, DNMT1 upregulation could partially rescue the properties of CD133+/CD44+ shOPN cells. Both in vitro and in vivo assays showed that CD133+/CD44+ cells with high OPN levels were more sensitive to DNA methylation inhibitor, 5 Azacytidine (5 Aza). The above findings were validated in HCC primary cells, a more clinically relevant model. CONCLUSIONS: OPN induces methylome reprogramming to enhance the stemness of CD133+/CD44+ subgroup and provides the therapeutic benefits to DNMT1 targeting treatment in HCC.

A Nomogram for Distinction and Potential Prediction of Liver Metastasis in Breast Cancer Patients.

Liver metastasis from breast cancer has poor prognosis. We aimed at developing a reliable tool for making a distinction and prediction for liver metastasis in breast cancer patients, thus helping clinical diagnosis and treatment. In this study, totally 6238 patients from SEER database with known distant metastasis status and clinicopathologic variables were enrolled and divided randomly into training and validating groups. Logistic regression was used to screen variables and a nomogram was constructed. After multivariate logistic regression, sex, histology type, N stage, grade, age, ER, PR, HER2 status as significant variables for constructing the nomogram. The nomogram for distinguishing and predicting liver metastasis in breast cancer passed the calibration and validation steps and the areas under the receiver operating characteristic curve of the training set and the validation set were 0.6602 and 0.6511 respectively. Our nomogram is a reliable and robust tool for the distinction and prediction of liver metastasis in breast cancer patients, thus helping better choose medical examinations and optimize therapeutic regimen under the cooperation among medical oncologists and surgeons.

Gender-related prognostic value and genomic pattern of intra-tumor heterogeneity in colorectal cancer.

Intra-tumor heterogeneity (ITH) is crucial in tumorigenesis and resistance to target therapy. Here, we used mutant-allele tumor heterogeneity (MATH) to measure ITH based on next-generation sequencing data and high MATH was proven as an independent risk prognostic factor in male CRC patients in both a training set of 284 colorectal cancer (CRC) patients with from The Cancer Genome Atlas (TCGA) and a validating set of 187 CRC patients from International Cancer Genome Consortium (ICGC). Further, the genomic pattern according to MATH demonstrated that mutation rates of TP53, IRF5 and KRAS were independently associated with MATH, and the latter two were only significant in male patients. As MATH increased, the fraction of somatic copy number alteration (SCNA) elevated. Moreover, more SCNA events was independently associated with MATH in male than in female. WNT pathway, TGF-ß pathway and DNA repair deficiency was enriched in high MATH group and the latter two showed up only in male patients. In summary, we reveal the gender-related prognostic value of MATH and relevant genomic pattern in CRC. Potential mechanisms are provided and it remains to be proven whether they are drivers of subclone formation and ITH. Taking MATH into consideration in clinical trial might contribute to better therapeutic strategies in CRC with researches added on in the future.

The influence of marital status on the stage at diagnosis, treatment, and survival of adult patients with gastric cancer: a population-based study.

BACKGROUND & AIMS: Marital status was reported as a prognostic factor in many cancers. However, its role in gastric cancer (GC) hasn't been thoroughly explored. In this study, we aimed to investigate the effect of marital status on survival, stage, treatment, and survival in subgroups. METHODS: We used the Surveillance, Epidemiology and End Results (SEER) database and identified 16910 GC patients. These patients were categorized into married (58.44%) and unmarred (41.56%) groups. Pearson chi-square, Wilcoxon-Mann-Whitney, Log-rank, multivariate Cox regression, univariate and multivariate binomial or multinomial logistic regression analysis were used in our analysis. Subgroup analyses of married versus unmarried patients were summarized in a forest plot. RESULTS: Married patients had better 5-year overall survival (OS) (32.09% VS 24.61%, P<0.001) and 5-year cancer-caused special survival (CSS) (37.74% VS 32.79%, P<0.001) than unmarried ones. Then we studied several underlying mechanisms. Firstly, married patients weren't in earlier stage at diagnosis (P=0.159). Secondly, married patients were more likely to receive surgery (P < 0.001) or radiotherapy (P < 0.001) compared with the unmarried. Thirdly, in subgroup analyses, married patients still had survival advantage in subgroups with stage II-IV and no radiotherapy. CONCLUSIONS: These results showed that marital status was an independently prognostic factor for both OS and CSS in GC patients. Undertreatment and lack of social support in unmarried patients were potential explanations. With the knowledge of heterogeneous effects of marriage in subgroups, we can target unmarried patients with better social support, especially who are diagnosed at late stage and undergo no treatment.