Breast Cancer-Derived Microparticles Reduce Cancer Cell Adhesion, an Effect Augmented by Chemotherapy.

Tumor cell heterogeneity is primarily dictated by mutational changes, sometimes leading to clones that undergo a metastatic switch. However, little is known about tumor heterogeneity following chemotherapy perturbation. Here we studied the possible involvement of tumor-derived extracellular vesicles, often referred to as tumor-derived microparticles (TMPs), as mediators of the metastatic switch in the tumor microenvironment by hindering cell adhesion properties. Specifically, we show that highly metastatic or chemotherapy-treated breast cancer cells shed an increased number of TMPs compared to their respective controls. We found that these TMPs substantially reduce cell adhesion and disrupt actin filament structure, therefore increasing their biomechanical force pace, further implicating tumor cell dissemination as part of the metastatic cascade. Our results demonstrate that these pro-metastatic effects are mediated in part by CD44 which is highly expressed in TMPs obtained from highly metastatic cells or cells exposed to chemotherapy when compared to cells with low metastatic potential. Consequently, when we inhibited CD44 expression on TMPs by a pharmacological or a genetic approach, increased tumor cell adhesion and re-organized actin filament structure were observed. We also demonstrated that breast cancer patients treated with paclitaxel chemotherapy exhibited increased CD44-expressing TMPs. Overall, our study provides further insights into the role of TMPs in promoting metastasis, an effect which is augmented when tumor cells are exposed to chemotherapy.

WDR62 mediates TNFα-dependent JNK activation via TRAF2-MLK3 axis.

The mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes. The three main MAPK cascades are the extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and p38 kinases. A typical MAPK cascade is composed of MAP3K-MAP2K-MAPK kinases that are held by scaffold proteins. Scaffolds function to assemble the protein tier and contribute to the specificity and efficacy of signal transmission. WD repeat domain 62 (WDR62) is a JNK scaffold protein, interacting with JNK, MKK7, and several MAP3Ks. The loss of WDR62 in human leads to microcephaly and pachygyria. Yet the role of WDR62 in cellular function is not fully studied. We used the CRISPR/Cas9 and short hairpin RNA approaches to establish a human breast cancer cell line MDA-MB-231 with WDR62 loss of function and studied the consequence to JNK signaling. In growing cells, WDR62 is responsible for the basal expression of c-Jun. In stressed cells, WDR62 specifically mediates TNFα-dependent JNK activation through the association with both the adaptor protein, TNF receptor-associated factor 2 (TRAF2), and the MAP3K protein, mixed lineage kinase 3. TNFα-dependent JNK activation is mediated by WDR62 in HCT116 and HeLa cell lines as well. MDA-MB-231 WDR62-knockout cells display increased resistance to TNFα-induced cell death. Collectively, WDR62 coordinates the TNFα receptor signaling pathway to JNK activation through association with multiple kinases and the adaptor protein TRAF2.

Clinically significant sub-clonality for common drivers can be detected in 26% of KRAS/EGFR mutated lung adenocarcinomas.

Genetic sub-clonality has been described in multiple malignancies, however the presence of sub-clonality for major drivers in lung adenocarcinoma and its clinical significance is a subject under debate. Using molecular and morphometric approach, 347 lung adenocarcinoma samples were analyzed for KRAS and EGFR sub-clonality, which was further correlated with clinical and pathological variables.KRAS and EGFR mutations were identified in 100 (29%) and 82 (23%) cases, respectively. One hundred and forty four KRAS or EGFR positive cases were also available for morphometric analysis, among which 37 (26%) were defined as sub-clonal. The presence of sub-clonality was associated with shorter survival time (p=0.02). Interestingly, cases with sub-clonality were also associated with earlier disease stage (89% vs 66% stage I disease in sub-clonal vs clonal cases, respectively, p=0.01) and less lymph node involvement (8% vs 25% in sub-clonal vs clonal cases, respectively, p=0.02). Our findings demonstrate the presence of sub-clonality for mutations in common drivers in lung adenocarcinoma and link it both to earlier disease stage and to poor survival. These findings are in line with the different evolutionary models that can present with genetic sub-clonality.

Variation in KRAS driver substitution distributions between tumor types is determined by both mutation and natural selection.

Different tumor types vary greatly in their distribution of driver substitutions. Here, we analyzed how mutation and natural selection contribute to differences in the distribution of KRAS driver substitutions between lung, colon and pancreatic adenocarcinomas. We were able to demonstrate that both differences in mutation and differences in selection drive variation in the distribution of KRAS driver substitutions between tumor types. By accounting for the effects of mutation on the distribution of KRAS driver substitutions, we could identify specific KRAS driver substitutions that are more favored by selection in specific tumor types. Such driver substitutions likely improve fitness most when they occur within the context of the tumor type in which they are preferentially favored. Fitting with this, we found that driver substitutions that are more favored by natural selection in a specific type of tumor tend to associate with worse clinical outcomes specifically in that type of tumor.

Subclonality for BRAF Mutation in Papillary Thyroid Carcinoma Is Associated With Earlier Disease Stage.

CONTEXT: The presence of driver mutations only in a subset of tumor cells within a single lesion, defined as subclonality, is being appreciated as a clinically significant factor. BRAF mutation is the most common driver mutation in papillary thyroid carcinoma (PTC). There are conflicting data in the literature regarding the presence of BRAF mutation subclonality in PTC and its clinical significance. OBJECTIVE: The purpose of the present study was to use a molecular and morphometric approach to determine BRAF clonality status and its clinical-pathological correlates. DESIGN: Fifty-nine cases of PTC were studied. DNA extracted from the tumors underwent deep sequencing to determine the percentage of BRAF mutant allele copies. Additionally, we used computerized morphometry to determine the fraction of tumor cells in each sample. Using both variables, we were able to determine the presence or absence of subclonality for BRAF mutation, which was further correlated with clinical, pathological, and prognostic data. RESULTS: BRAF mutation was found in 49 (83%) cases. The average percentage of tumor cells and of BRAF mutant alleles in the samples were 68.1 ± 9.8 and 26 ± 6.7, respectively. Based on the molecular and morphometric analysis, 11 (24%) cases were found to be subclonal for BRAF mutation. Tumors with subclonal BRAF mutations were significantly smaller compared to tumors with clonal mutation (0.82 ± 0.38 cm vs 1.37 ± 0.57 cm, P = .005) and were less likely to have lymph node metastasis (0% vs 32%, P = .03). CONCLUSIONS: In PTC, subclonality for BRAF mutation is associated with earlier stage. Molecular-morphometric analysis of PTC can provide clonality information with potential clinical significance.

Intratumor Heterogeneity of KRAS Mutation Status in Pancreatic Ductal Adenocarcinoma Is Associated With Smaller Lesions.

OBJECTIVES: Recent studies have demonstrated intratumor heterogeneity (ITH) for genetic mutations in various tumors and suggest that ITH might have significant clinical impact. Because KRAS is the most commonly mutated oncogene in pancreatic ductal adenocarcinoma and has an important role in pancreatic carcinogenesis, the purpose of this study was to evaluate ITH for KRAS gene mutation and its clinical significance. METHODS: Deep sequencing of 47 pancreatic ductal adenocarcinoma cases was used to determine the fraction of KRAS mutant alleles. In addition, computerized morphometry was used to calculate the fraction of tumor cells. After analysis of both results, cases were classified as ITH or as having amplification of mutant KRAS. The presence of ITH was correlated with clinical and pathological factors. RESULTS: KRAS mutation was found in 38 (81%) cases, of which 12 (32%) showed ITH and 9 (23%) were found to have KRAS mutant allele amplification. The presence of ITH was associated with smaller tumors, whereas amplification was associated with higher tumor diameter. CONCLUSIONS: In pancreatic ductal adenocarcinoma, ITH for KRAS gene mutation was associated with smaller tumors. It is possible that, as the tumor progresses, more cells carry this mutation, which leads to more aggressive tumor features.

Adenoma and carcinoma components in colonic tumors show discordance for KRAS mutation.

Activating mutations in KRAS are common events in the pathogenesis of colorectal carcinoma and predict response to treatment with anti-EGFR antibodies. Molecular pathology testing for KRAS mutations has become the standard of practice for patients with metastatic colorectal carcinoma. Despite the known histologic and molecular differences between adenomas and carcinomas, the concordance of KRAS mutation between adenomas and carcinomas has not been established leaving some open questions regarding the appropriate choice of tissue for KRAS mutation analysis and correct interpretation of the test results. To address these questions, we analyzed the concordance of KRAS mutation in 70 tumors that contained both adenoma and carcinoma components (2 cases of intramucosal carcinoma, 66 cases with invasion of the submucosa, and 2 invading the muscularis propria). For each case, DNA was separately isolated from the adenoma and the carcinoma component and analyzed for KRAS mutation using direct sequencing. Overall, 30 (43%) of the adenoma cases and 36 (51%) of the carcinoma cases were positive for KRAS mutation. Of the 70 cases, 16 (23%) showed discordant results. Interestingly, the fraction of discordant cases went down as the depth of carcinoma invasion increased. In summary, we identified significant KRAS mutation discordance between the adenoma and carcinoma component of the lesion. Our results suggest that effort should be made to analyze only the invasive component of the lesion and that caution should be taken when interpreting a result based on DNA extracted from noninvasive elements.