Aberrant plasma levels of circulating miR-16, miR-107, miR-130a and miR-146a are associated with lymph node metastasis and receptor status of breast cancer patients.

Within the multicenter SUCCESS trial, we investigated the association of plasma microRNAs with different subtypes of invasive breast cancer.Six miRs (miR-16, miR-27a, miR-107, miR-130a, miR-132 and miR-146a) were selected from microarray profiling and further validated in plasma of 111 breast cancer patients before and after chemotherapy and 46 healthy women by quantitative real-time PCR.Plasma levels of miR-16 (p = 0.0001), miR-27a (p = 0.039) and miR-132 (p = 0.020) were higher in breast cancer patients before chemotherapy than healthy women. With the exception of miR-16, the increased levels of miR-27a (p = 0.035) and miR-132 (p = 0.025) decreased after chemotherapy to those observed in healthy women. Levels of miR-16 (p = 0.019), miR-107 (p = 0.036), miR-130a (p = 0.027) and miR-146a (p = 0.047) were different between lymph node -positive and -negative patients, while the levels of miR-130a (p = 0.001) and miR-146a (p = 0.025) also differed between HER2-positive and -negative status. Estrogen-receptor negative tumors displayed higher concentrations of circulating miR-107 than their counterparts (p = 0.035). However, overexpression of miR-107 in MCF-7 cells did not downregulate estrogen receptor protein. Altered expression levels of miR-107 influenced the migration and invasion behavior of MCF-7 and MDA-MB-231 cells.Our data indicate differential concentrations of plasma miR-16, miR-107, miR-130a and miR-146a in different breast cancer subtypes, suggesting a potential role of these miRs in breast cancer biology and tumor progression.

Increased serum levels of circulating exosomal microRNA-373 in receptor-negative breast cancer patients.

In this study, we compared the blood serum levels of circulating cell-free and exosomal microRNAs, and their involvement in the molecular subtypes of breast cancer patients. Our analyses on cell-free miR-101, miR-372 and miR-373 were performed in preoperative blood serum of 168 patients with invasive breast cancer, 19 patients with benign breast diseases and 28 healthy women. MicroRNAs were additionally quantified in exosomes of 50 cancer patients and 12 healthy women from the same cohort. Relative concentrations were measured by quantitative TaqMan MicroRNA assays and correlated to clinicopathological risk factors. The concentrations of cell-free miR-101 (p=0.013) and miR-373 (p=0.024) were significantly different between patients with breast cancer and benign tumors. A prevalence of miR-101, miR-372 and miR-373 were found in exosomes. The levels of circulating exosomal (but not cell-free) miR-373 were higher in triple negative than luminal carcinomas (p=0.027). Also, estrogen-negative (p=0.021) and progesterone-negative (p=0.01) tumors displayed higher concentrations of exosomal miR-373 than patients with hormone-receptor positive tumors. Overexpression of miR-373 by transfection of MCF-7 cells showed downregulated protein expression of the estrogen receptor, and inhibition of apoptosis induced by camptothecin. Our data indicate that serum levels of exosomal miR-373 are linked to triple negative and more aggressive breast carcinomas.

Low levels of cell-free circulating miR-361-3p and miR-625* as blood-based markers for discriminating malignant from benign lung tumors.

The high mortality rate of lung cancer patients is mainly due to the late stage at which lung cancer is diagnosed. For effective cancer prevention programs and early diagnosis, better blood-based markers are needed. Hence, blood-based microarray profiling of microRNA (miR) expression was performed in preoperative serum of 21 non-small cell lung cancer (NSCLC) patients and 11 healthy individuals by microfluid biochips containing 1158 different miRs. Two out of the 30 most dysregulated miRs were further validated in serum of 97 NSCLC patients, 20 patients with benign lung diseases and 30 healthy individuals by TaqMan MicroRNA Assays. Microarray profiling showed that miR-361-3p and miR-625* were significantly down-regulated in serum of lung cancer patients. Their further evaluation by quantitative RT-PCR showed that the levels of miR-361-3p and miR-625* were lower in NSCLC than in benign disease (p = 0.0001) and healthy individuals (p = 0.0001, p = 0.0005, respectively). Moreover, the levels of miR-625* were significantly lower in patients with large cell lung cancer (LCLC, p = 0.014) and smoking patients (p = 0.030) than in patients with adenocarcinoma and non-smoking patients, respectively. A rise in the levels of both miRs was observed in the postoperative samples compared with the preoperative levels (p = 0.0001). Functional analyses showed that Smad2 and TGFß1 are not dysregulated by miR-361-3p and miR-625* in the lung cell line A549, respectively. Our present pilot study suggests that miR-361-3p and miR-625* might have a protective influence on the development of NSCLC, and the quantitative assessment of these miRs in blood serum might have diagnostic potential to detect NSCLC, in particular in smokers.

Identifying genotype-dependent efficacy of single and combined PI3K- and MAPK-pathway inhibition in cancer.

In cancer, genetically activated proto-oncogenes often induce "upstream" dependency on the activity of the mutant oncoprotein. Therapeutic inhibition of these activated oncoproteins can induce massive apoptosis of tumor cells, leading to sometimes dramatic tumor regressions in patients. The PI3K and MAPK signaling pathways are central regulators of oncogenic transformation and tumor maintenance. We hypothesized that upstream dependency engages either one of these pathways preferentially to induce "downstream" dependency. Therefore, we analyzed whether downstream pathway dependency segregates by genetic aberrations upstream in lung cancer cell lines. Here, we show by systematically linking drug response to genomic aberrations in non-small-cell lung cancer, as well as in cell lines of other tumor types and in a series of in vivo cancer models, that tumors with genetically activated receptor tyrosine kinases depend on PI3K signaling, whereas tumors with mutations in the RAS/RAF axis depend on MAPK signaling. However, efficacy of downstream pathway inhibition was limited by release of negative feedback loops on the reciprocal pathway. By contrast, combined blockade of both pathways was able to overcome the reciprocal pathway activation induced by inhibitor-mediated release of negative feedback loops and resulted in a significant increase in apoptosis and tumor shrinkage. Thus, by using a systematic chemo-genomics approach, we identify genetic lesions connected to PI3K and MAPK pathway activation and provide a rationale for combined inhibition of both pathways. Our findings may have implications for patient stratification in clinical trials.

Predicting drug susceptibility of non-small cell lung cancers based on genetic lesions.

Somatic genetic alterations in cancers have been linked with response to targeted therapeutics by creation of specific dependency on activated oncogenic signaling pathways. However, no tools currently exist to systematically connect such genetic lesions to therapeutic vulnerability. We have therefore developed a genomics approach to identify lesions associated with therapeutically relevant oncogene dependency. Using integrated genomic profiling, we have demonstrated that the genomes of a large panel of human non-small cell lung cancer (NSCLC) cell lines are highly representative of those of primary NSCLC tumors. Using cell-based compound screening coupled with diverse computational approaches to integrate orthogonal genomic and biochemical data sets, we identified molecular and genomic predictors of therapeutic response to clinically relevant compounds. Using this approach, we showed that v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations confer enhanced Hsp90 dependency and validated this finding in mice with KRAS-driven lung adenocarcinoma, as these mice exhibited dramatic tumor regression when treated with an Hsp90 inhibitor. In addition, we found that cells with copy number enhancement of v-abl Abelson murine leukemia viral oncogene homolog 2 (ABL2) and ephrin receptor kinase and v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) kinase family genes were exquisitely sensitive to treatment with the SRC/ABL inhibitor dasatinib, both in vitro and when it xenografted into mice. Thus, genomically annotated cell-line collections may help translate cancer genomics information into clinical practice by defining critical pathway dependencies amenable to therapeutic inhibition.