mRNA coexpression patterns of Wnt pathway components and their clinicopathological associations in breast and colorectal cancer.

Aberrant Wnt signaling is implicated in carcinogenesis triggering efforts for the development of new therapeutic agents, many of which have entered clinical trials. We extend our previous analysis of WNT3, FZD7, LEF1 expression levels in breast and colorectal cancer including WNT2, FZD4 and ß-catenin expression, in an effort to delineate their relative expression levels along with concurrent expression patterns and possible prognostic value. We analyzed 82 breast and 102 colorectal carcinomas for relative mRNA expression levels of the investigated genes by RT-PCR relative quantification with the ΔΔCt method. Statistical analysis was performed in order to determine associations of relative mRNA expression and linear correlations. ß-catenin expression was determined by immunochemistry. Regarding breast carcinomas, decreased relative mRNA expression levels of WNT2, FZD4 were found frequently and WNT2 expression was correlated with ER/ PR status (p = 0.045/p = 0.028), whereas ß-catenin with grade (p = 0.026). In colorectal carcinomas, increased relative mRNA expression levels of WNT2 and FZD4 were found in 59% and 32% of cases respectively, whereas ß-catenin showed decreased mRNA expression levels in 57% of cases and a correlation with pN-category (p = 0.037). Linear correlations were observed between WNT2/FZD4 (R=0.542, p < 0.001), WNT2/ß-catenin (R=0.254, p = 0.010), FZD4/ß-catenin (R=0.406, p < 0.001) expression and a correlation between mRNA expression and membranous/cytoplasmic ß-catenin emerged (p = 0.039/0.046). Our results suggest a possible clinical significance for Wnt pathway gene expression levels in both tumour types. The concurrent expression of the investigated genes as well as the different expression profiles, underlines the complexity of this pathway and the necessity of patient selection in order to maximize the efficacy of drugs targeting Wnt pathway.

Concurrent Wnt pathway component expression in breast and colorectal cancer.

Wnt signaling pathway regulates important cell functions such as proliferation and migration and is frequently deregulated in colorectal and breast cancer. Thus, it constitutes an attractive therapeutic target with many drugs being investigated in clinical trials. Eighty-two breast and 102 colorectal carcinomas were analyzed for: relative mRNA expression levels of Wnt pathway components namely Wnt3 ligand, Frizzled 7 receptor and LEF1 transcriptional factor, their concurrent expression patterns and their correlation with clinicopathological features. Regarding breast carcinomas, increased relative mRNA expression levels of WNT3 were found in 54 % of cases whereas decreased relative mRNA expression levels were observed in FZD7 and LEF1 in 82 % and 43 % of cases, respectively. Expression levels of WNT3 were significantly correlated with tumour grade (p = 0.021) in breast cancer. As far as colorectal carcinomas are concerned, increased relative mRNA expression levels of WNT3, FZD7 and LEF1 were found in 60 %, 37 % and 48 % of cases respectively. A statistically significant correlation emerged between LEF1expression levels and pT-category (p = 0.027), suggesting a possible association with tumour aggressiveness in colorectal carcinomas. Statistically significant linear correlations were observed between the expression of WNT3/LEF1 (R = 0.233, p = 0.035) and FZD7/LEF1 (R = 0.359, p = 0.001) in breast carcinomas as well as in colorectal carcinomas (R = 0.536, p < 0.01 and R = 0.210, p = 0.034) respectively. Our results demonstrate a possible clinical significance of Wnt pathway gene expression levels in both tumour types. The distinct expression patterns and simultaneous expression of the investigated genes underscore the complexity of this pathway in breast and colorectal carcinogenesis and highlights the necessity of patient selection with regard to the effectiveness of Wnt pathway inhibitors.

Autophagy-related Proteins as a Prognostic Factor of Patients With Colorectal Cancer.

OBJECTIVES: Autophagy plays a dual role in tumorigenesis. In the initial stages, it promotes cell survival and suppresses carcinogenesis, whereas in cancer development, it induces cancer cell survival. In this study, we investigate the role of autophagy as a protective or tumor suppressor mechanism in colorectal cancer (CRC) cell lines and evaluate its role as a potential biomarker in human tumor samples. MATERIALS AND METHODS: The data of 68 patients with CRC treated at our Department from January 1 to December 31, 2016 were analyzed. Immunohistochemistry evaluation of p62, LC3B, Beclin-1, and Rab-7 in formalin-fixed paraffin-embedded tissue samples was performed and their expression was correlated with clinicopathologic characteristics, mutation status, and therapeutic approach. The χ was used to test an association among categorical variables. Survival curves were estimated using the Kaplan-Meier method and differences were assessed using the log-rank test. Colo-205, HT29, SW-480, and Caco-2 cell lines were also used so as to test the autophagy markers with oxaliplatin, irinotecan, hydroxychloroquine, and 3-methyladenine. RESULTS: Overexpression of Beclin-1 is associated with poor survival (P=0.001) in patients with CRC treated with chemotherapy, irrespective of the stage and mutational status. Rab-7 is also correlated with progression-free survival (PFS) (P=0.088). Oxaliplatin (10 and 20 µΜ) and irinotecan (10 and 20 µΜ) inhibit autophagy in microsatellite stable (MSS) CRC cell lines. The inhibition of autophagy in MSS CRC cell lines after treatment with oxaliplatin and irinotecan is further identified through monodancylcadaverine staining. Moreover, inhibition of autophagy with molecules such as hydroxychloroquine (20 µΜ) and 3-methyladenine (5 mM) was identified by the accumulation of p62 and LC3B. CONCLUSIONS: Beclin-1 is an independent prognostic factor of overall survival and PFS. Also, Rab-7 is identified as an independent prognostic factor of PFS. Besides, several chemotherapeutic drugs such as oxaliplatin and irinotecan inhibit autophagy in MSS CRC cell lines in a similar way like hydroxychloroquine and 3-methyladenine. Thus, in MSS patients who develop chemoresistance, a combination of other therapies that include an autophagy inhibitor could be more beneficial. Further clinical trials are needed to investigate these therapeutic strategies.

CHEK2 c.1100delC allele is rarely identified in Greek breast cancer cases.

The CHEK2 gene encodes a protein kinase that plays a crucial role in maintenance of genomic integrity and the DNA repair mechanism. CHEK2 germline mutations are associated with increased risk of breast cancer and other malignancies. From a clinical perspective, the most significant mutation identified is the c.1100delC mutation, which is associated with an approximately 25% lifetime breast cancer risk. The distribution of this mutation shows wide geographical variation; it is more prevalent in the Northern European countries and less common, or even absent, in Southern Europe. In order to estimate the frequency of the CHEK2 c.1100delC mutation in Greek breast cancer patients, we genotyped 2,449 patients (2,408 females and 41 males), which was the largest series ever tested for c.1100delC. The mean age of female and male breast cancer diagnosis was 49 and 59 years, respectively. All patients had previously tested negative for the Greek BRCA1 founder and recurrent mutations. The CHEK2 c.1100delC mutation was detected in 0.16% (4 of 2,408) of females, all of whom were diagnosed with breast cancer before the age of 50 years. Only one c.1100delC carrier was reported with breast cancer family history. The present study indicates that the CHEK2 c.1100delC mutation does not contribute substantially to hereditary breast cancer in patients of Greek descent.