Dasatinib attenuates overexpression of Src signaling induced by the combination treatment of veliparib plus carboplatin in triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) has a poor prognosis because of limited treatment options. The combination of a poly ADP ribose polymerase (PARP) inhibitor with a DNA-damaging agent has shown promise in treating TNBC; however, not all patients respond to this combination. The Src protein kinase modulates multiple cancer cell properties and plays a key role in tumorigenic processes. However, Src inhibitors as single agents have shown limited effects in solid tumors. Here, we examined the antitumor effects of the Src inhibitor dasatinib, the PARP inhibitor veliparib, and the DNA-damaging agent carboplatin in TNBC models to try and identify the combination with the most clinical potential. METHODS: Dasatinib, veliparib and carboplatin were tested in TNBC cells in vitro and in xenograft tumors in vivo. RESULTS: Surprisingly, treatment with the combination of veliparib plus carboplatin led to an increase in Src phosphorylation. Importantly, dasatinib attenuated Src overexpression induced by veliparib plus carboplatin and further inhibited the downstream signaling of Src. In xenograft models, the triple combination of dasatinib with veliparib plus carboplatin showed greater tumor growth inhibitory effects compared with single agents or double combinations. No systemic toxicity was observed in mice treated with the triple combination. CONCLUSIONS: This study emphasizes the merit of evaluating the triple combination therapy, dasatinib with veliparib plus carboplatin, in TNBC clinical trials.

Enriched transcription factor signatures in triple negative breast cancer indicates possible targeted therapies with existing drugs.

PURPOSE: Triple negative (TN) breast cancers which lack expression of the estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors convey a poor prognosis due in part to a lack of targeted therapies. METHODS: To identify viable targets for the treatment of TN disease, we have conducted a gene set enrichment analysis (GSEA) on seven different breast cancer whole genome gene expression cohorts comparing TN vs. ER + HER2 - to identify consistently enriched genes that share a common promoter motif. The seven cohorts were profiled on three different genome expression platforms (Affymetrix, Illumina and RNAseq) consisting in total of 2088 samples with IHC metadata. RESULTS: GSEA identified enriched gene expression patterns in TN samples that share common promoter motifs associated with SOX9, E2F1, HIF1A, HMGA1, MYC BACH2, CEBPB, and GCNF/NR6A1. Unexpectedly, NR6A1 an orphan nuclear receptor normally expressed in germ cells of gonads is highly expressed in TN and ER + HER2 - samples making it an ideal drug target. CONCLUSION: With the increasing number of large sample size breast cancer cohorts, an exploratory analysis of genes that are consistently enriched in TN sharing common promoter motifs allows for the identification of possible therapeutic targets with extensive validation in patient derived data sets.

Doubling down on the PI3K-AKT-mTOR pathway enhances the antitumor efficacy of PARP inhibitor in triple negative breast cancer model beyond BRCA-ness.

Phosphoinositide 3-kinase (PI3K) pathway, in addition to its pro-proliferative and antiapoptotic effects on tumor cells, contributes to DNA damage repair (DDR). We hypothesized that GDC-0980, a dual PI3K-mammalian target of rapamycin (mTOR) inhibitor, would induce an efficient antitumor effect in BRCA-competent triple negative breast cancer (TNBC) model when combined with ABT888 and carboplatin. Mechanism-based in vitro studies demonstrated that GDC-0980 treatment alone or in combination led to DNA damage (increased pγH2AX(S139); Western blot, immunofluorescence), gain in poly ADP-ribose (PAR), and a subsequent sensitization of BRCA-competent TNBC cells to ABT888 plus carboplatin with a time-dependent 1) decrease in proliferation signals (pAKTT308/S473, pP70S6KT421/S424, pS6RPS235/236), PAR/poly(ADP-ribose) polymerase (PARP) ratios, PAR/pγH2AX ratios, live/dead cell ratios, cell cycle progression, and three-dimensional clonogenic growths and 2) increase in apoptosis markers (cleaved caspases 3 and 9, a pro-apoptotic BH3-only of Bcl-2 family (BIM), cleaved PARP, annexin V). The combination was effective in vitro in BRCA-wild-type PIK3CA-H1047R-mutated BT20 and PTEN-null HCC70 cells. The combination blocked the growth of established xenograft tumors by 80% to 90% with a concomitant decrease in tumor Ki67, CD31, phosphorylated vascular endothelial growth factor receptor, pS6RPS235/236, and p4EBP1T37/46 as well as an increase in cleaved caspase 3 immunohistochemistry (IHC) levels. Interestingly, a combination with GDC-0941, a pan-PI3K inhibitor, failed to block the tumor growth in MDA-MB231. Results demonstrate that the dual inhibition of PI3K and mTOR regulates DDR. In a BRCA-competent model, GDC-0980 enhanced the antitumor activity of ABT888 plus carboplatin by inhibiting both tumor cell proliferation and tumor-induced angiogenesis along with an increase in the tumor cell apoptosis. This is the first mechanism-based study to demonstrate the integral role of the PI3K-AKT-mTOR pathway in DDR-mediated antitumor action of PARP inhibitor in TNBC.

Wnt signaling in triple negative breast cancer is associated with metastasis.

BACKGROUND: Triple Negative subset of (TN) Breast Cancers (BC), a close associate of the basal-like subtype (with limited discordance) is an aggressive form of the disease which convey unpredictable, and poor prognosis due to limited treatment options and lack of proven effective targeted therapies. METHODS: We conducted an expression study of 240 formalin-fixed, paraffin-embedded (FFPE) primary biopsies from two cohorts, including 130 TN tumors, to identify molecular mechanisms of TN disease. RESULTS: The annotation of differentially expressed genes in TN tumors contained an overrepresentation of canonical Wnt signaling components in our cohort and others. These observations were supported by upregulation of experimentally induced oncogenic Wnt/ß-catenin genes in TN tumors, recapitulated using targets induced by Wnt3A. A functional blockade of Wnt/ß-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, sulidac sulfide, or ß-catenin (functional read out of Wnt/ß-catenin pathway) SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-directed migration, F-actin organization, and invasion in TNBC cells. A classifier, trained on microarray data from ß-catenin transfected mammary cells, identified a disproportionate number of TNBC breast tumors as compared to other breast cancer subtypes in a meta-analysis of 11 studies and 1,878 breast cancer patients, including the two cohorts published here. Patients identified by the Wnt/ß-catenin classifier had a greater risk of lung and brain, but not bone metastases. CONCLUSION: These data implicate transcriptional Wnt signaling as a hallmark of TNBC disease associated with specific metastatic pathways.

Protein-coding and microRNA biomarkers of recurrence of prostate cancer following radical prostatectomy.

An important challenge in prostate cancer research is to develop effective predictors of tumor recurrence following surgery to determine whether immediate adjuvant therapy is warranted. To identify biomarkers predictive of biochemical recurrence, we isolated the RNA from 70 formalin-fixed, paraffin-embedded radical prostatectomy specimens with known long-term outcomes to perform DASL expression profiling with a custom panel that we designed of 522 prostate cancer-relevant genes. We identified a panel of 10 protein-coding genes and two miRNA genes (RAD23B, FBP1, TNFRSF1A, CCNG2, NOTCH3, ETV1, BID, SIM2, LETMD1, ANXA1, miR-519d, and miR-647) that could be used to separate patients with and without biochemical recurrence (P < 0.001), as well as for the subset of 42 Gleason score 7 patients (P < 0.001). We performed an independent validation analysis on 40 samples and found that the biomarker panel was also significant at prediction of biochemical recurrence for all cases (P = 0.013) and for a subset of 19 Gleason score 7 cases (P = 0.010), both of which were adjusted for relevant clinical information including T-stage, prostate-specific antigen, and Gleason score. Importantly, these biomarkers could significantly predict clinical recurrence for Gleason score 7 patients. These biomarkers may increase the accuracy of prognostication following radical prostatectomy using formalin-fixed specimens.

Proposals for uniform collection of biospecimens from neoadjuvant breast cancer clinical trials: timing and specimen types.

In this Personal View, we outline proposals for uniform collection of biospecimens obtained in neoadjuvant breast cancer trials undertaken by the Breast International Group (BIG) and the National Cancer Institute-sponsored North American Breast Cancer Group (NABCG). These proposals aim to standardise collection of high-quality specimens, with respect to both type and timing, to enhance and allow integration of results obtained from neoadjuvant trials done by several groups. They should be considered in parallel with recommendations for tissue-specimen collection and handling previously developed by BIG and NABCG. We propose that tumour tissue (formalin-fixed, paraffin-embedded and samples dedicated for molecular studies) should be taken at baseline, 1-3 weeks after the start of treatment, and at definitive surgery, with clear prioritisation in the study protocol of number, order, and preservation of samples to be gathered. This step should be accompanied by blood collection (plasma, serum, and whole blood) whenever possible. We advocate strongly a move towards one diagnostic and research biopsy procedure in all women with breast cancers potentially suitable for neoadjuvant treatment. If possible, patients should be referred at the outset to specialised centres to give them the opportunity to participate in neoadjuvant clinical trials, thereby avoiding several biopsy procedures.

Recommendations for collection and handling of specimens from group breast cancer clinical trials.

Recommendations for specimen collection and handling have been developed for adoption across breast cancer clinical trials conducted by the Breast International Group (BIG)-sponsored Groups and the National Cancer Institute (NCI)-sponsored North American Cooperative Groups. These recommendations are meant to promote identifiable standards for specimen collection and handling within and across breast cancer trials, such that the variability in collection/handling practices that currently exists is minimized and specimen condition and quality are enhanced, thereby maximizing results from specimen-based diagnostic testing and research. Three working groups were formed from the Cooperative Group Banking Committee, BIG groups, and North American breast cancer cooperative groups to identify standards for collection and handling of (1) formalin-fixed, paraffin-embedded (FFPE) tissue; (2) blood and its components; and (3) fresh/frozen tissue from breast cancer trials. The working groups collected standard operating procedures from multiple group specimen banks, administered a survey on banking practices to those banks, and engaged in a series of discussions from 2005 to 2007. Their contributions were synthesized into this document, which focuses primarily on collection and handling of specimens to the point of shipment to the central bank, although also offers some guidance to central banks. Major recommendations include submission of an FFPE block, whole blood, and serial serum or plasma from breast cancer clinical trials, and use of one fixative and buffer type (10% neutral phosphate-buffered formalin, pH 7) for FFPE tissue across trials. Recommendations for proper handling and shipping were developed for blood, serum, plasma, FFPE, and fresh/frozen tissue.