Mariska Miranda

Meta-analysis of the global gene expression profile of triple-negative breast cancer identifies genes for the prognostication and treatment of aggressive breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking expression of estrogen and progesterone receptors (ER/PR) and HER2, thus limiting therapy options. We hypothesized that meta-analysis of TNBC gene expression profiles would illuminate mechanisms underlying the aggressive nature of this disease and identify therapeutic targets. Meta-analysis in the Oncomine database identified 206 genes that were recurrently deregulated in TNBC compared with non-TNBC and in tumors that metastasized or led to death within 5 years. This ‘aggressiveness gene list’ was enriched for two core functions/metagenes: chromosomal instability (CIN) and ER signaling metagenes. We calculated an ‘aggressiveness score’ as the ratio of the CIN metagene to the ER metagene, which identified aggressive tumors in breast cancer data sets regardless of subtype or other clinico-pathological indicators. A score calculated from six genes from the CIN metagene and two genes from the ER metagene recapitulated the aggressiveness score. By multivariate survival analysis, we show that our aggressiveness scores (from 206 genes or the 8 representative genes) outperformed several published prognostic signatures. Small interfering RNA screen revealed that the CIN metagene holds therapeutic targets against TNBC. Particularly, the inhibition of TTK significantly reduced the survival of TNBC cells and synergized with docetaxel in vitro. Importantly, mitosis-independent expression of TTK protein was associated with aggressive subgroups, poor survival and further stratified outcome within grade 3, lymph node-positive, HER2-positive and TNBC patients. In conclusion, we identified the core components of CIN and ER metagenes that identify aggressive breast tumors and have therapeutic potential in TNBC and aggressive breast tumors. Prognostication from these metagenes at the mRNA level was limited to ER-positive tumors. However, we provide evidence that mitosis-independent expression of TTK protein was prognostic in TNBC and other aggressive breast cancer subgroups, suggesting that protection of CIN/aneuploidy drives aggressiveness and treatment resistance.

Treatment of Triple-Negative Breast Cancer Using Anti-EGFR Directed Radioimmunotherapy Combined with Radiosensitizing Chemotherapy and PARP Inhibitor

Triple-negative breast cancer (TNBC) is associated with poor survival. Chemotherapy is the only standard treatment for TNBC. The prevalence of BRCA1 inactivation in TNBC has rationalized clinical trials of poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors. Similarly, the overexpression of epidermal growth factor receptor (EGFR) rationalized anti-EGFR therapies in this disease. However, clinical trials using these 2 strategies have not reached their promise. In this study, we used EGFR as a target for radioimmunotherapy and hypothesized that EGFR-directed radioimmunotherapy can deliver a continuous lethal radiation dose to residual tumors that are radiosensitized by PARP inhibitors and chemotherapy. Methods: We analyzed EGFR messenger RNA in published gene expression array studies and investigated EGFR protein expression by immunohistochemistry in a cohort of breast cancer patients to confirm EGFR as a target in TNBC. Preclinically, using orthotopic and metastatic xenograft models of EGFR-positive TNBC, we investigated the effect of the novel combination of 177Lu-labeled anti-EGFR monoclonal antibody, chemotherapy, and PARP inhibitors on cell death and the survival of breast cancer stem cells. Results: In this first preclinical study of anti-EGFR radioimmunotherapy in breast cancer, we found that anti-EGFR radioimmunotherapy is safe and that TNBC orthotopic tumors and established metastases were eradicated in mice treated with anti-EGFR radioimmunotherapy combined with chemotherapy and PARP inhibitors. We showed that the superior response to this triple-agent combination therapy was associated with apoptosis and eradication of putative breast cancer stem cells. Conclusion: Our data support further preclinical investigations toward the development of combination therapies using systemic anti-EGFR radioimmunotherapy for the treatment of recurrent and metastatic TNBC.

Essential developmental, genomic stability, and tumour suppressor functions of the mouse orthologue of hSSB1/NABP2.

Single-stranded DNA binding proteins (SSBs) regulate multiple DNA transactions, including replication, transcription, and repair. We recently identified SSB1 as a novel protein critical for the initiation of ATM signaling and DNA double-strand break repair by homologous recombination. Here we report that germline Ssb1−/− embryos die at birth from respiratory failure due to severe rib cage malformation and impaired alveolar development, coupled with additional skeletal defects. Unexpectedly, Ssb1 −/− fibroblasts did not exhibit defects in Atm signaling or γ-H2ax focus kinetics in response to ionizing radiation (IR), and B-cell specific deletion of Ssb1 did not affect class-switch recombination in vitro. However, conditional deletion of Ssb1 in adult mice led to increased cancer susceptibility with broad tumour spectrum, impaired male fertility with testicular degeneration, and increased radiosensitivity and IR–induced chromosome breaks in vivo. Collectively, these results demonstrate essential roles of Ssb1 in embryogenesis, spermatogenesis, and genome stability in vivo.

The CIMP Phenotype in BRAF Mutant Serrated Polyps from a Prospective Colonoscopy Patient Cohort

Colorectal cancers arising via the serrated pathway are often associated with BRAF V600E mutation, CpG island methylator phenotype (CIMP), and microsatellite instability. Previous studies have shown a strong association between BRAF V600E mutation and serrated polyps. This study aims to evaluate CIMP status of all the serrated polyp subtypes and its association with functionally important genes such as MLH1, p16, and IGFBP7. CIMP status and methylation were evaluated using the real-time based MethyLight assay in 154 serrated polyps and 63 conventional adenomas. Results showed that CIMP-high serrated polyps were strongly associated with BRAF mutation and proximal colon. CIMP-high was uncommon in conventional adenomas (1.59%), occurred in 8.25% of hyperplastic polyps (HPs), and became common in sessile serrated adenomas (SSAs) (51.43%). MLH1 methylation was mainly observed in the proximal colon and was significantly associated with BRAF mutation and CIMP-high. The number of samples methylated for p16 and IGFBP7 was the highest in SSAs. The methylation panel we used to detect CIMP is highly specific for CIMP-high cancers. With this panel, we demonstrate that CIMP-high is much more common in SSAs than HPs. This suggests that CIMP-high correlates with increased risk of malignant transformation which was also observed in methylation of functionally important genes.

Gemcitabine and CHK1 Inhibition Potentiate EGFR-Directed Radioimmunotherapy against Pancreatic Ductal Adenocarcinoma

Purpose: To develop effective combination therapy against pancreatic ductal adenocarcinoma (PDAC) with a combination of chemotherapy, CHK1 inhibition, and EGFR-targeted radioimmunotherapy. Experimental Design: Maximum tolerated doses were determined for the combination of gemcitabine, the CHK1 inhibitor PF-477736, and Lutetium-177 (177Lu)–labeled anti-EGFR antibody. This triple combination therapy was investigated using PDAC models from well-established cell lines, recently established patient-derived cell lines, and fresh patient-derived xenografts. Tumors were investigated for the accumulation of 177Lu-anti-EGFR antibody, survival of tumor-initiating cells, induction of DNA damage, cell death, and tumor tissue degeneration. Results: The combination of gemcitabine and CHK1 inhibitor PF-477736 with 177Lu-anti-EGFR antibody was tolerated in mice. This triplet was effective in established tumors and prevented the recurrence of PDAC in four cell line–derived and one patient-derived xenograft model. This exquisite response was associated with the loss of tumor-initiating cells as measured by flow cytometric analysis and secondary implantation of tumors from treated mice into treatment-naïve mice. Extensive DNA damage, apoptosis, and tumor degeneration were detected in the patient-derived xenograft. Mechanistically, we observed CDC25A stabilization as a result of CHK1 inhibition with consequent inhibition of gemcitabine-induced S-phase arrest as well as a decrease in canonical (ERK1/2 phosphorylation) and noncanonical EGFR signaling (RAD51 degradation) as a result of EGFR inhibition. Conclusions: Our study developed an effective combination therapy against PDAC that has potential in the treatment of PDAC. Clin Cancer Res; 20(12); 3187–97. ©2014 AACR.

Effects of silibinin on growth and invasive properties of human ovarian carcinoma cells through suppression of heregulin/HER3 pathway

Epithelial ovarian cancer (EOC) is the most fatal gynecological malignancy due to its high proliferative and invasive capacities. A heregulin (HRG)/HER3 autocrine loop increases proliferative and metastatic properties of EOC cells, suggesting that modulators of this signaling pathway may prove effective to trammel growth and motility of these cells. This study aimed to evaluate the effects of multi-tyrosine kinase inhibitor silibinin on proliferative and invasive characteristics of EOC cell lines OVCAR8 and SKOV3 through suppression of the HRG/HER3 pathway. To achieve this, the effects of silibinin on proliferation, DNA synthesis, clonogenicity, cell cycle progression, cathepsin B enzymatic activity, and migration and invasion were explored in vitro. Silibinin suppressed proliferation, DNA synthesis, and clonogenic abilities of OVCAR8 and SKOV3 cells through inhibition of the autocrine HRG/HER3 circuit. Silibinin-mediated attenuation of the HER3 signaling disabled the HER3/AKT/survivin axis and thereby, induced G1/S cell cycle arrest. Furthermore, silibinin reduced invasive potentials of the EOC cells through quelling the HRG/HER3 pathway and suppression of cathepsin B activity. Altogether, these results suggest that silibinin is a potential anti-cancer drug to inhibit proliferative and invasive characteristics of the EOC cells that exhibit an autocrine HRG/HER3 pathway.

RAD51 inhibition in triple negative breast cancer cells is challenged by compensatory survival signaling and requires rational combination therapy

The molecular rationale to induce synthetic lethality, by targeting defective homologous recombination repair in triple negative breast cancer (TNBC), has proven to have several shortcomings. Not meeting the expected minimal outcomes in clinical trials has highlighted common clinical resistance mechanisms including; increased expression of the target gene PARP1, increased expression or reversion mutation of BRCA1, or up-regulation of the compensatory homologous recombination protein RAD51. Indeed, RAD51 has been demonstrated to be an alternative synthetic lethal target in BRCA1-mutated cancers. To overcome selective pressure on DNA repair pathways, we examined new potential targets within TNBC that demonstrate synthetic lethality in association with RAD51 depletion. We confirmed complementary targets of PARP1/2 and DNA-PK as well as a new synthetic lethality combination with p38. p38 is considered a relevant target in breast cancer, as it has been implicated in resistance to chemotherapy, including tamoxifen. We show that the combination of targeting RAD51 and p38 inhibits cell proliferation both in vitro and in vivo, which was further enhanced by targeting of PARP1. Analysis of the molecular mechanisms revealed that depletion of RAD51 increased ERK1/2 and p38 signaling. Our results highlight a potential compensatory mechanism via p38 that limits DNA targeted therapy.

Abstract P6-09-47: The development of personalized diagnostic tests and therapeutic strategies in breast cancer

Despite some improvement in the overall survival rates of breast cancer, it remains as a leading cause of cancer-related deaths in women. Currently, we are unable to accurately predict patients9 response to therapies and their long-term outcome. We developed and patented a 275-gene signature based on in silico meta-analysis of global transcriptome profiles (approximately 10,000 cases) that can predict which patients suffer from aggressive disease and succumb to their disease within 5 years of diagnosis. This test, the integrated Breast Cancer Recurrence (iBCR) score outperformed every clinicopathological indicator available in three independent, large cohorts of breast cancer. The iBCR can also predict the likelihood of response to standard treatments and which emerging targeted therapies should be added to an individual9s treatment regime to improve outcomes. In addition, 21 of the genes in this signature are novel potential drug targets that have not previously been described in aggressive breast tumours. We performed a pilot study using the NanoString nCounter Dx platform to measure the expression of the top 125 genes within the signature in a cohort of 48 patients. We have validated with 100% accuracy the prognostic power of the iBCR in the Queensland Follow Up (QFU) cohort with 25 years of follow up (p In vitro siRNA screening of the 21 novel genes revealed that at least 10 of these genes are required for breast cancer cell survival. We have started validation of the top 4 hits and these studies confirm the requirement of these genes in breast cancer progression. These data will pave the way towards the study of these genes as new drug targets. Collectively, our test addresses the significant issue of heterogeneous responses to breast cancer treatment. The iBCR platform aims to improve both patients9 clinical outcome and quality of life by directing more appropriate treatment with greater likelihood of success, and preventing overtreatment for those with a less aggressive tumor type. Citation Format: Kutasovic JR, Rozali E, Miranda M, Lakhani SR, Al-Ejeh F. The development of personalized diagnostic tests and therapeutic strategies in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-47.

Abstract P6-10-06: Rational combination therapy against triple-negative breast cancer

Background: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with higher incidence of recurrence, more distant metastasis, and poorer survival. This subtype is also characterized by complex genomes where little of their genomes remain at normal copy number but without high, focal copy number amplifications. At the transciptome level, the majority of TNBC (∼75%) are classified as basal-like breast cancer (BLBC) according to the five intrinsic subtypes. Despite considerable genomic and gene expression characterization of TNBC, proteomic and phospho-proteomic investigations of this disease are limited with no available targeted therapies in clinical use. Methods & Results: We used the Kinex™ antibody array (http://www.kinexus.ca/) to interrogate protein/phosphoproteins levels in 43 primary breast cancer biopsies (16 TNBC, 16 ER/PR positive and 11 HER2-positive) and 16 breast cancer cell lines. Unsupervised hierarchical clustering of protein/phosphoprotein levels revealed two subgroups of TNBC in comparison to other subtypes. Western blotting and Proteome Profiler™ Arrays (RD we developed EGFR-targeted radioimmunotherapy (RIT) strategy to systemically deliver cytotoxic loads of beta particles ( 177 Lu) that would kill targeted cells and surrounding cells by crossfire effect. The combination of EGFR-directed RIT with chemotherapy and PARP inhibition successfully treated orthotopic and metastatic TNBC models established from cell lines and patient-derived xenografts. The superior efficacy of this triple-agent combination therapy is explained by enhanced DNA damage and reduced DNA repair response, higher apoptotic cell death and the elimination of putative breast cancer stem cells. Conclusion: Proteomic analysis of TNBC provides a powerful tool to elucidate druggable signaling networks with therapeutic potential. TNBC utilizes complex interacting signaling networks and rational combination therapies are required for effective therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-10-06.