Complete response to crizotinib in a metastatic adenocarcinoma of unknown primary harboring MET amplification and NTRK1 co-occurring mutation.

Carcinomas of unknown primary (CUPs) have poor prognosis due to the paucity of data on their clinical characteristics and laboratory features, and empirical chemotherapy still remains the critical management for this kind of disease. This study aimed to present the knowledge of treating an elderly man with metastatic adenocarcinoma of unknown primary and also with a history of long-term hypertension and renal cysts. He was identified to harbor mesenchymal-epithelial transition factor (MET) gene amplification and neurotrophic tyrosine receptor kinase 1 (NTRK1) gene co-occurring mutation by targeted next-generation sequencing analysis upon the progression of empirical chemotherapy. He was then treated with a standard dose of crizotinib (250 mg, twice daily), which exhibited a satisfactory complete response (CR) of the targeted lesions after 1 month of treatment. When the number of renal cysts increased and renal inadequacy occurred after treatment for 2 months, crizotinib was reduced to half-dose (250 mg, once daily), and still conferred maintenance of CR for another 6.5 months and good quality life of the patient. These results suggested that treatments based on driver genes rather than primary tumor types could be a promising manipulation for achieving better treatment outcome, and a half-dose of crizotinib might be both effective and tolerable for MET-overexpressed CUPs with underlying renal diseases.

Connexin 32 downregulation is critical for chemoresistance in oxaliplatin-resistant HCC cells associated with EMT.

Background: Oxaliplatin (OXA)-based chemotherapy is critical in the management of advanced hepatocellular carcinoma (HCC); however, acquired drug resistance has largely restricted its clinical efficacy. This study aims to explore the key mechanisms and regulatory factors determining chemosensitivity in HCC. Methods: We developed OXA-resistant (OR) HCC cells and used multiple methods, including real-time RT-PCR, Western blot, immunofluorescence, transwell invasion assay, wound-healing assay, MTT assay, gene transfection, and immunohistochemistry to achieve our goals. Results: We found that OR HCC cells showed a typical epithelial-mesenchymal transition (EMT) phenotype. Meanwhile, the expression of Cx32, a major member of the liver connexin (Cx) family, was lowly expressed in OR HCC cells. Downregulation of Cx32 in parental HCC cells led to EMT induction and thereby reduced OXA cytotoxicity, while Cx32 upregulation in OR HCC cells could reverse the EMT phenotype and partially restore chemosensitivity to OXA. Finally, in human HCC tissue samples, Cx32 was positively correlated with the expression of the EMT marker E-cadherin and negatively correlated with the expression of Vimentin. Conclusion: Our findings demonstrated that downregulation of Cx32 may be an important determinant for HCC cells to acquire EMT-related acquired drug resistance to OXA, and targeting Cx32 could be a novel strategy to overcome OXA resistance in HCC.

Downregulated connexin32 promotes EMT through the Wnt/ß-catenin pathway by targeting Snail expression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the common malignances in the world and is associated with high mortality and poor prognosis, partly due to early invasion and metastasis. Cx32 has been indicated to be involved in the progression of many cancers including HCC, but its relationship with tumor invasion and metastasis is still controversial. In the present study, the downregulated Cx32 in HCC tissue was found negatively correlated with histological grade and lymph node metastasis. Cx32 regulated HCC migration and invasion in vitro and inhibited tumor metastasis in xenograft models in vivo. We subsequently identified that Cx32 mediated epithelial-mesenchymal transition (EMT) by regulating Snail expression, and the enhanced Snail was due to activation of Wnt/ß-catenin signaling in response to Cx32 inhibition. Finally, decreased expression of Cx32 showed strong correlation with loss/reduction of E-cadherin, higher expression of Snail, and nuclear accumulation of ß-catenin in HCC tissues. Taken together, our results suggest that Cx32 inhibits HCC invasion and metastasis through Snail-mediated EMT, Cx32 and this signaling pathway molecules may offer potential targets for HCC cancer therapy.

Role of IL-33 expression in oncogenesis and development of human hepatocellular carcinoma.

Interleukin-33 (IL-33), a newly-discovered cytokine belonging to the IL-1 family, serves an important role in inflammation. However, it is not clear whether IL-33 is of clinical significance in hepatocarcinogenesis. The present study was designed to investigate the role of IL-33 during oncogenesis and development of hepatocellular carcinoma (HCC). IL-33 protein expression was detected in 76 HCC (including 36 para-carcinoma), 33 cirrhosis, 30 hepatitis, and 20 normal liver tissues using immunohistochemistry. IL-33 mRNA expression in carcinoma and para-carcinoma tissues was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The possible correlation between IL-33 and clinicopathological parameters of HCC was also analyzed. Significant differences in IL-33 expression were not observed among normal, hepatic, and cirrhotic tissues (P>0.05), whereas the level of protein positive rate was markedly reduced in HCC tissues (P<0.01). Positive staining of IL-33 in non-cancerous liver (NCL) tissues (i.e. normal, hepatitis, and liver cirrhosis) was located predominantly in the nucleus and occasionally in the cytoplasm of hepatocytes; however, the expression in HCC tissues was mostly restricted to the cytoplasm. A significant alteration in protein localization was observed in HCC tissues as compared with NCL tissues (P<0.01). In comparison with HCC tissues, cytoplasmic staining of IL-33 was increased in para-carcinoma tissues. RT-PCR assay further confirmed relatively high mRNA expression levels of IL-33 in para-carcinoma tissues. IL-33 expression was significantly negatively associated with tumor histological grade (r=-0.279, P=0.015), but not with year, gender, tumor size, clinical stage, HCC with hepatitis and cirrhosis background, lymph node metastasis or intrahepatic vascular embolism (P>0.05). Therefore, the aberrant expression of IL-33 is associated with oncogenesis and progression of HCC and the cytoplasmic accumulation of the protein may serve a role in hepatocarcinogenesis.

Impaired gap junctions in human hepatocellular carcinoma limit intrinsic oxaliplatin chemosensitivity: A key role of connexin 26.

Hepatocellular carcinoma (HCC) is generally believed to have low sensitivity to chemotherapeutic agents including oxaliplatin (OXA). Studies have demonstrated that gap junctions (GJs) composed of connexin (Cx) proteins have the potential to modulate drug chemosensitivity in multiple tumor cells. In the present study, we investigated the characteristics of Cx and GJs in HCC at both histologic and cytologic levels, and the effects of GJ and its effective components on OXA cytotoxicity in HCC cells in vitro. Immunohistochemistry was performed in 76 HCCs and 20 normal liver tissues to detect and locate the expression of Cx26, Cx32 and Cx43. At cytologic levels, the expression and localization of Cxs were evaluated by RT-PCR, western blot and immunofluorescence assay, respectively. The GJ function between adjacent cells was detected using dye transfer assay. The role of GJs in the modulation of OXA toxicity in HCC cells was explored using pharmacologic and molecular biologic methods. We found that Cx expression in HCC tissues was significantly lower than in normal liver tissues, and the 'internalization' from cell membrane to cytoplasm was remarkable. In vitro experiments revealed the presence of functional GJs in the SMMC-7721 HCC cells due to a small amount of Cx protein along the plasma membrane at cell-cell contacts. Regulation of this part of GJs positively influenced OXA cytotoxicity. Using RNA interference, only specific inhibition of Cx26 but not Cx32 or Cx43 reduced OXA cytotoxicity. Conversely, Cx26 overexpression by transfection of Cx26 plasmid DNA enhanced OXA cytotoxicity. This study demonstrated that during hepatocarcinogenesis, the reduced expression and internalization of Cx proteins impaired the GJ function, which further attenuated OXA cytotoxicity. Impaired GJ function may contribute to low intrinsic chemosensitivity of HCC cells to OXA, mediated by Cx26.

[Expression of gap junction protein connexin 26 in human hepatocellular carcinoma and its significance].

OBJECTIVE: To investigate the expression of gap junction protein connexin 26(Cx26) in hepatocellular carcinoma(HCC) and its significance. METHODS: The expression of Cx26 in liver tissue was examined by immunohistochemistry staining in 159 paraffin-embeded liver sections, including 20 samples of normal liver tissue, 30 samples of chronic hepatitis, 33 samples of liver cirrhosis, and 76 samples of HCC. Normal hepatic cell line LO2 and HCC cell line SMMC-7721 were used in vitro to verify the characteristics of gap junction and Cx26 expression pattern. The expression and localization of Cx26 were measured by Western blotting and immunofluorescence assay, respectively. The function of gap junction between adjacent cells was detected by dye transfer assay. RESULTS: Compared to normal liver samples, the positive rate of Cx26 was markedly decreased in hepatitis, cirrhosis and HCC tissues(all P<0.05). The intensity of Cx26 staining was significantly increased in HCC tissues compared with those in non-carcinomatous liver(NCL) tissues(all P<0.05). In NCL tissues, there was a mild to moderated staining of Cx26 which located mainly on the membranes of hepatocytes at intercellular contacts. The positive staining of Cx26 in HCC tissues was observed mainly in cytoplasm. Positive Cx26 expression was positively associated with tumor size(P=0.036), but not with age, gender, histologic grade, clinical stage, underlying hepatitis and cirhosis, lymph node metastasis and intrahepatic vascular embolism(all P>0.05). Compared with LO2 cells, an aberrant expression and distribution of Cx26 in SMMC-7721 cells was confirmed, which may lead to a decreased function of gap junctions. CONCLUSIONS: The aberrant expression and distribution of Cx26 protein may be associated with hepatocarcinogenesis, and the residual gap junction in HCC may provide a new target for treatment of HCC.

[Expression of Tcf-4, MMP7 and Survivin in Colorectal Cancer and Its Clinical Significance].

OBJECTIVE: To explore the clinical and pathological significance and correlations among the xpressions of Tcf-4, MMP7 and survivin in colorectal cancer. METHODS: The expressions of Tcf-4, MMP7 and survivin mRNA in tumor tissues and adjacent normal mucosa from 50 colorectal cancer patients were detected by reverse transcription PCR (RT-PCR). The expressed proteins of Tcf-4, MMP7 and survivin were measured using mmunohistochemistry staining technique (Elivision) in 100 colorectal cancer samples and 60 normal mucosa tissue samples. We analyzed the correlations between those measurements and their associations with clinical and pathological characteristics. RESULTS: Positive expressions of Tcf-4, MMP7 and survivin mRNA were found in both cancer and adjacent mucosa tissues, despite a higher level of expression in the cancer tissues (P < 0.01). Expressed proteins were detected in cancer tissues of 69.00% (69/100) of those with a positive Tcf-4 expression, 77.00% (77/100) of those with a positive MMP7 expression, and 65.00% (65/100) of those with a positive survivin expression. Compared with cancer tissues, lower levels of protein expression were found in normal mucosa tissues [16.67% (10/60) for Tcf-4, 13.33% (8/60) for MMP7 and 15.00% (9/60) for survivin, P < 0.01]. The expressions of Tcf-4, MMP7 and survivin were all associated with lymphatic metastasis and Dukes staging (P < 0.05). MMP7 expression was associated with depth of tumor invasion (P < 0.05). Survivin expression was associated with tumor differentiation. The Spearman rank correlation analyses showed that protein expressions in colorectal cancer tissues in those with a positive Tcf-4 were correlated with those with a positive MMP7 (r = 0.302) and those with a positive survivin (r = 0.279) (P < 0.01), but not in those with a positive MMP7 and those with a positive survivin (r = 0.097, P > 0.05). CONCLUSION: The expression levels of Tcf-4, MMP7 and survivin are high in colorectal cancer, all being linked to lymph node metastasis and Dukes stages of patients. This suggests that they may be involved in the occurrence, development, malignant growth and clinical progression of colorectal cancer.

Grape seed proanthocyanidins prevent DOCA-salt hypertension-induced renal injury and its mechanisms in rats.

Renal dysfunction is one of the major effects of DOCA (deoxycorticosterone acetate)-salt hypertension and there is an increasing amount of evidence that oxidative stress damages the function of the kidney. Grape seed proanthocyanidins (GSPE) have been reported to be potent anti-oxidants and free radical scavengers. The present study sought to investigate the ability of GSPE to prevent renal injury in DOCA-salt hypertensive rats and to explore the molecular mechanisms underlying its protective effects. A total of 54 Sprague Dawley (SD) rats were randomly divided into 7 groups: Sham group (n = 7), UnX-sham group (n = 8), DOCA-salt group (n = 8), GSPE150 group (150 mg kg(-1), n = 7), GSPE240 group (240 mg kg(-1), n = 8), GSPE384 group (384 mg kg(-1), n = 8) and ALM (amlodipine besylate tablets) group (5 mg kg(-1), n = 8), and treated for 4 weeks. Compared to sham group rats, renal injury was observed in DOCA-salt hypertensive group rats as the urine protein, KW/BW (kidney weight/body weight), degree of renal fibrosis, renal MDA (malondialdehyde) and Hyp (hydroxyproline) contents significantly increased (P < 0.01). Moreover, SOD (Superoxide Dismutase) activities decreased in the model group (P < 0.01). In contrast, DOCA-salt hypertensive rats treated with different dose of GSPE or ALM showed a significant improvement of renal injury with decreased urine protein, KW/BW, degree of renal fibrosis, renal total MDA and Hyp contents compared to the untreated group. In addition, SOD activities increased in the treatment group. Since the experimental modeling time was short, kidney damage occurs to a lesser extent. BUN (Blood Urea Nitrogen), Scr (Serum Creatinine) and UA (Uric Acid) contents did not appear significantly changed in all groups. Finally, the activation of JNK and p38 kinases in the kidney was suppressed in rats treated with GSPEs or ALM compared to the untreated group, suggesting that the inhibition of these kinase pathways by GSPE contributes to the improvement of renal function. Taking these results together, we conclude that the anti-hypertensive and anti-oxidative stress beneficial effects of GSPE on renal injury in rats with DOCA-salt hypertension occur via the attenuation of JNK and p38 activity.

[Expression of secreted frizzled related protein 1, ß-catenin and E-cadherin in colorectal carcinoma and its clinicopathological significances].

OBJECTIVE: To investigate the expression of secreted frizzled-related protein 1 (SFRP1), ß-catenin and E-cadherin in colorectal carcinoma and its clinicopathological significance. METHODS: The expression of SFRP1, ß-catenin and E-cadherin mRNA and protein in tumor and pericancerous tissue samples from 60 cases of colorectal cancer was assayed by reverse-transcription PCR and immunohistochemistry, respectively. The correlation of their expression with clinicopathological factors of colorectal cancer was analyzed. RESULTS: In 52/60 cases the relative mRNA expression of SFRP1 in cancer tissue and pericancerous tissue was 0.4837±0.1532 and 0.7170 ±0.1830; for ß-catenin was 0.9293± 0.3705 and 0.6469±0.3166; and for E-cadherin was 0.5556±0.2535 and 0.9422±0.2372 (P<0.01), respectively. SFRP1 mRNA expression was associated with lymphatic metastasis (P<0.05). The positive rate of SFRP1 in colorectal cancer was 31.67% (19/60), and was significantly lower than that in pericancerous colorectal mucosa (75.00%, 45/60). No relationship between SFRP1 protein expression and clinical pathology was found. Abnormal expression rates of ß-catenin and E-cadherin in colorectal cancer were 75.00% (45/60) and 58.33% (35/60), respectively, which were significantly higher than that in pericancerous colorectal mucosa (1.67% and 6.67%), respectively. Abnormal ß-catenin and E-cadherin expression was associated with tumor differentiation, lymphatic metastasis and Duke's staging. SFRP1 protein expression was negatively correlated with ß-catenin and E-cadherin expression (r=-0.517, -0.442, Ps<0.01). CONCLUSION: Down-regulation of SFRP1 in colorectal cancer may cause abnormal Wnt signaling and induce abnormal ß-catenin and E-cadherin expression, indicating that SFRP1 might be involved in the development and progression of colorectal cancer, and could be a novel therapeutic target for colorectal cancer.