Murugan Kalimutho

Targeted Therapies for Triple-Negative Breast Cancer: Combating a Stubborn Disease

Triple-negative breast cancers (TNBCs) constitute a heterogeneous subtype of breast cancers that have a poor clinical outcome. Although no approved targeted therapy is available for TNBCs, molecular-profiling efforts have revealed promising molecular targets, with several candidate compounds having now entered clinical trials for TNBC patients. However, initial results remain modest, thereby highlighting challenges potentially involving intra- and intertumoral heterogeneity and acquisition of therapy resistance. We present a comprehensive review on emerging targeted therapies for treating TNBCs, including the promising approach of immunotherapy and the prognostic value of tumor-infiltrating lymphocytes. We discuss the impact of pathway rewiring in the acquisition of drug resistance, and the prospect of employing combination therapy strategies to overcome challenges towards identifying clinically-viable targeted treatment options for TNBC.

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.

Natural killer cells are essential for the ability of BRAF inhibitors to control BRAFV600E-mutant metastatic melanoma.

BRAF(V600E) is a major oncogenic mutation found in approximately 50% of human melanoma that confers constitutive activation of the MAPK pathway and increased melanoma growth. Inhibition of BRAF(V600E) by oncogene targeting therapy increases overall survival of patients with melanoma, but is unable to produce many durable responses. Adaptive drug resistance remains the main limitation to BRAF(V600E) inhibitor clinical efficacy and immune-based strategies could be useful to overcome disease relapse. Tumor microenvironment greatly differs between visceral metastasis and primary cutaneous melanoma, and the mechanisms involved in the antimetastatic efficacy of BRAF(V600E) inhibitors remain to be determined. To address this question, we developed a metastatic BRAF(V600E)-mutant melanoma cell line and demonstrated that the antimetastatic properties of BRAF inhibitor PLX4720 (a research analogue of vemurafenib) require host natural killer (NK) cells and perforin. Indeed, PLX4720 not only directly limited BRAF(V600E)-induced tumor cell proliferation, but also affected NK cell functions. We showed that PLX4720 increases the phosphorylation of ERK1/2, CD69 expression, and proliferation of mouse NK cells in vitro. NK cell frequencies were significantly enhanced by PLX4720 specifically in the lungs of mice with BRAF(V600E) lung metastases. Furthermore, PLX4720 also increased human NK cell pERK1/2, CD69 expression, and IFNγ release in the context of anti-NKp30 and IL2 stimulation. Overall, this study supports the idea that additional NK cell-based immunotherapy (by checkpoint blockade or agonists or cytokines) may combine well with BRAF(V600E) inhibitor therapy to promote more durable responses in melanoma.

AXL Is a Key Regulator of Inherent and Chemotherapy-Induced Invasion and Predicts a Poor Clinical Outcome in Early-Stage Colon Cancer

Purpose: Despite the use of 5-fluorouracil (5-FU)–based adjuvant treatments, a large proportion of patients with high-risk stage II/III colorectal cancer will relapse. Thus, novel therapeutic strategies are needed for early-stage colorectal cancer. Residual micrometastatic disease from the primary tumor is a major cause of patient relapse. Experimental Design: To model colorectal cancer tumor cell invasion/metastasis, we have generated invasive (KRASMT/KRASWT/+chr3/p53-null) colorectal cancer cell subpopulations. Receptor tyrosine kinase (RTK) screens were used to identify novel proteins that underpin the migratory/invasive phenotype. Migration/invasion was assessed using the XCELLigence system. Tumors from patients with early-stage colorectal cancer (N = 336) were examined for AXL expression. Results: Invasive colorectal cancer cell subpopulations showed a transition from an epithelial-to-mesenchymal like phenotype with significant increases in migration, invasion, colony-forming ability, and an attenuation of EGF receptor (EGFR)/HER2 autocrine signaling. RTK arrays showed significant increases in AXL levels in all invasive sublines. Importantly, 5-FU treatment resulted in significantly increased migration and invasion, and targeting AXL using pharmacologic inhibition or RNA interference (RNAi) approaches suppressed basal and 5-FU–induced migration and invasion. Significantly, high AXL mRNA and protein expression were found to be associated with poor overall survival in early-stage colorectal cancer tissues. Conclusions: We have identified AXL as a poor prognostic marker and important mediator of cell migration/invasiveness in colorectal cancer. These findings provide support for the further investigation of AXL as a novel prognostic biomarker and therapeutic target in colorectal cancer, in particular in the adjuvant disease in which EGFR/VEGF–targeted therapies have failed. Clin Cancer Res; 20(1); 164–75. ©2013 AACR.

Adenosine 2B Receptor Expression on Cancer Cells Promotes Metastasis

Adenosine plays an important role in inflammation and tumor development, progression, and responses to therapy. We show that an adenosine 2B receptor inhibitor (A2BRi) decreases both experimental and spontaneous metastasis and combines with chemotherapy or immune checkpoint inhibitors in mouse models of melanoma and triple-negative breast cancer (TNBC) metastasis. Decreased metastasis upon A2BR inhibition is independent of host A2BR and lymphocytes and myeloid cells. Knockdown of A2BR on mouse and human cancer cells reduces their metastasis in vivo and decreases their viability and colony-forming ability, while transiently delaying cell-cycle arrest in vitro The prometastatic activity of adenosine is partly tumor A2BR dependent and independent of host A2BR expression. In humans, TNBC cell lines express higher A2BR than luminal and Her2(+) breast cancer cell lines, and high expression of A2BR is associated with worse prognosis in TNBC. Collectively, high A2BR on mouse and human tumors promotes cancer metastasis and is an ideal candidate for therapeutic intervention. Cancer Res; 76(15); 4372-82. ©2016 AACR.

Beyond cytokinesis: The emerging roles of CEP55 in tumorigenesis

CEP55 was initially identified as a pivotal component of abscission, the final stage of cytokinesis, serving to regulate the physical separation of two daughter cells. Over the past 10 years, several studies have illuminated additional roles for CEP55 including regulating the PI3K/AKT pathway and midbody fate. Concurrently, CEP55 has been studied in the context of cancers including those of the breast, lung, colon and liver. CEP55 overexpression has been found to significantly correlate with tumor stage, aggressiveness, metastasis and poor prognosis across multiple tumor types and therefore has been included as part of several prognostic ‘gene signatures’ for cancer. Here by discussing in depth the functions of CEP55 across different effector pathways, and also its roles as a biomarker and driver of tumorigenesis, we assemble an exhaustive review, thus commemorating a decade of research on CEP55.

RNF43 and ZNRF3 are commonly altered in serrated pathway colorectal tumorigenesis

Serrated pathway colorectal cancers (CRCs) are characterised by a BRAF mutation and half display microsatellite instability (MSI). The Wnt pathway is commonly upregulated in conventional CRC through APC mutation. By contrast, serrated cancers do not mutate APC. We investigated mutation of the ubiquitin ligases RNF43 and ZNRF3 as alternate mechanism of altering the Wnt signal in serrated colorectal neoplasia. RNF43 was mutated in 47/54(87%) BRAF mutant/MSI and 8/33(24%) BRAF mutant/microsatellite stable cancers compared to only 3/79(4%) BRAF wildtype cancers (p<0.0001). ZNRF3 was mutated in 16/54(30%) BRAF mutant/MSI and 5/33(15%) BRAF mutant/microsatellite stable compared to 0/27 BRAF wild type cancers (p=0.004). An RNF43 frameshift mutation (X659fs) occurred in 80% BRAF mutant/MSI cancers. This high rate was verified in a second series of 25/35(71%) BRAF mutant/MSI cancers. RNF43 and ZNRF3 had lower transcript expression in BRAF mutant compared to BRAF wildtype cancers and less cytoplasmic protein expression in BRAF mutant/MSI compared to other subtypes. Treatment with a porcupine inhibitor reduced RNF43/ZNRF3 mutant colony growth by 50% and synergised with a MEK inhibitor to dramatically reduce growth. This study suggests inactivation of RNF43 and ZNRF3 is important in serrated tumorigenesis and has identified a potential therapeutic strategy for this cancer subtype.

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.

Germline polymorphisms in an enhancer of PSIP1 are associated with progression-free survival in epithelial ovarian cancer

Women with epithelial ovarian cancer (EOC) are usually treated with platinum/taxane therapy after cytoreductive surgery but there is considerable inter-individual variation in response. To identify germline single-nucleotide polymorphisms (SNPs) that contribute to variations in individual responses to chemotherapy, we carried out a multi-phase genome-wide association study (GWAS) in 1,244 women diagnosed with serous EOC who were treated with the same first-line chemotherapy, carboplatin and paclitaxel. We identified two SNPs (rs7874043 and rs72700653) in TTC39B (best P=7×10−5, HR=1.90, for rs7874043) associated with progression-free survival (PFS). Functional analyses show that both SNPs lie in a putative regulatory element (PRE) that physically interacts with the promoters of PSIP1, CCDC171 and an alternative promoter of TTC39B. The C allele of rs7874043 is associated with poor PFS and showed increased binding of the Sp1 transcription factor, which is critical for chromatin interactions with PSIP1. Silencing of PSIP1 significantly impaired DNA damage-induced Rad51 nuclear foci and reduced cell viability in ovarian cancer lines. PSIP1 (PC4 and SFRS1 Interacting Protein 1) is known to protect cells from stress-induced apoptosis, and high expression is associated with poor PFS in EOC patients. We therefore suggest that the minor allele of rs7874043 confers poor PFS by increasing PSIP1 expression.

Understanding the functional impact of copy number alterations in breast cancer using a network modeling approach

UNLABELLED Copy number alterations (CNAs) are thought to account for 85% of the variation in gene expression observed among breast tumours. The expression of cis-associated genes is impacted by CNAs occurring at proximal loci of these genes, whereas the expression of trans-associated genes is impacted by CNAs occurring at distal loci. While a majority of these CNA-driven genes responsible for breast tumourigenesis are cis-associated, trans-associated genes are thought to further abet the development of cancer and influence disease outcomes in patients. Here we present a network-based approach that integrates copy-number and expression profiles to identify putative cis- and trans-associated genes in breast cancer pathogenesis. We validate these cis- and trans-associated genes by employing them to subtype a large cohort of breast tumours obtained from the METABRIC consortium, and demonstrate that these genes accurately reconstruct the ten subtypes of breast cancer. We observe that individual breast cancer subtypes are driven by distinct sets of cis- and trans-associated genes. Among the cis-associated genes, we recover several known drivers of breast cancer (e.g. CCND1, ERRB2, MDM2 and ZNF703) and some novel putative drivers (e.g. BRF2 and SF3B3). siRNA-mediated knockdown of BRF2 across a panel of breast cancer cell lines showed significant reduction in cell viability for ER-/HER2+ (MDA-MB-453) cells, but not in normal (MCF10A) cells thereby indicating that BRF2 could be a viable therapeutic target for estrogen receptor-negative/HER2-enriched (ER-/HER2+) cancers. Among the trans-associated genes, we identify modules of immune response (CD2, CD19, CD38 and CD79B), mitotic/cell-cycle kinases (e.g. AURKB, MELK, PLK1 and TTK), and DNA-damage response genes (e.g. RFC4 and FEN1). siRNA-mediated knockdown of RFC4 significantly reduced cell proliferation in ER-negative normal breast and cancer lines, thereby indicating that RFC4 is essential for both normal and cancer cell survival but could be a useful biomarker for aggressive (ER-negative) breast tumours. AVAILABILITY under NetStrat.