BackgroundInhibitor of differentiation 4 (Id4), a member of the Id gene family is also a dominant negative regulator of basic helix loop helix (bHLH) transcription factors. Some of the functions of Id4 appear to be unique as compared to its other family members Id1, Id2 and Id3. Loss of Id4 gene expression in many cancers in association with promoter hypermethylation has led to the proposal that Id4 may act as a tumor suppressor. In this study we provide functional evidence that Id4 indeed acts as a tumor suppressor and is part of a cancer associated epigenetic re-programming.MethodsData mining was used to demonstrate Id4 expression in prostate cancer. Methylation specific polymerase chain reaction (MSP) analysis was performed to understand molecular mechanisms associated with Id4 expression in prostate cancer cell lines. The effect of ectopic Id4 expression in DU145 cells was determined by cell cycle analysis (3H thymidine incorporation and FACS), expression of androgen receptor, p53 and cyclin dependent kinase inhibitors p27 and p21 by a combination of RT-PCR, real time-PCR, western blot and immuno-cytochemical analysis.ResultsId4 expression was down-regulated in prostate cancer. Id4 expression was also down-regulated in prostate cancer line DU145 due to promoter hyper-methylation. Ectopic Id4 expression in DU145 prostate cancer cell line led to increased apoptosis and decreased cell proliferation due in part by an S-phase arrest. In addition to S-phase arrest, ectopic Id4 expression in PC3 cells also resulted in prolonged G2/M phase. At the molecular level these changes were associated with increased androgen receptor (AR), p21, p27 and p53 expression in DU145 cells.ConclusionThe results suggest that Id4 acts directly as a tumor suppressor by influencing a hierarchy of cellular processes at multiple levels that leads to a decreased cell proliferation and change in morphology that is possibly mediated through induction of previously silenced tumor suppressors.

PARP inhibitors (PARPi) benefit only a fraction of breast cancer patients. Several of those patients exhibit intrinsic/acquired resistance mechanisms that limit efficacy of PARPi monotherapy. Here we show how the efficacy of PARPi in triple-negative breast cancers (TNBC) can be expanded by targeting MYC-induced oncogenic addiction. In BRCA-mutant/sporadic TNBC patients, amplification of the MYC gene is correlated with increased expression of the homologous DNA recombination enzyme RAD51 and tumors overexpressing both genes are associated with worse overall survival. Combining MYC blockade with PARPi yielded synthetic lethality in MYC-driven TNBC cells. Using the cyclin-dependent kinase inhibitor dinaciclib, which downregulates MYC expression, we found that combination with the PARPi niraparib increased DNA damage and downregulated homologous recombination, leading to subsequent downregulation of the epithelial-mesenchymal transition and cancer stem-like cell phenotypes. Notably, dinaciclib resensitized TBNC cells, which had acquired resistance to niraparib. We found that the synthetic lethal strategy employing dinaciclib and niraparib was also highly efficacious in ovarian, prostate, pancreatic, colon, and lung cancer cells. Taken together, our results show how blunting MYC oncogene addiction can leverage cancer cell sensitivity to PARPi, facilitating the clinical use of c-myc as a predictive biomarker for this treatment. Dual targeting of MYC-regulated homologous recombination and PARP-mediated DNA repair yields potent synthetic lethality in triple-negative breast tumors and other aggressive tumors characterized by MYC overexpression. .

Epithelial to mesenchymal transition (EMT) is associated with a wide range of changes in cancer cells, including stemness, chemo- and radio-resistance and metastasis. The mechanistic role of upstream mediators of EMT has not yet been well characterized. Recently, we showed that non-small cell lung cancers (NSCLCs) that have undergone EMT overexpress AXL, a receptor tyrosine kinase. AXL is also overexpressed in a subset of triple-negative breast cancers (TNBCs) and head and neck squamous cell carcinomas (HNSCCs) and its overexpression has been associated with more aggressive tumor behavior and linked to resistance to chemotherapy, radiation, and targeted therapy. Since the DNA repair pathway is also altered in patient tumor specimens overexpressing AXL, it is hypothesized that modulation of AXL in cells that have undergone EMT will sensitize them to agents targeting the DNA repair pathway. Downregulation or inhibition of AXL directly reversed the EMT phenotype, led to decreased expression of DNA repair genes and diminished efficiency of homologous recombination (HR) and RAD51 foci formation. As a result, AXL inhibition caused a state of HR-deficiency in the cells, making them sensitive to inhibition of the DNA repair protein, PARP1. AXL inhibition synergized with PARP inhibition, leading to apoptotic cell death. AXL expression also associated positively with markers of DNA repair across TNBC, HNSCC and NSCLC patient cohorts.

Purpose: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are currently used in combination with endocrine therapy to treat advanced hormone receptor-positive, HER2-negative breast cancer. Although this treatment doubles time to progression compared with endocrine therapy alone, about 25%-35% of patients do not respond, and almost all patients eventually acquire resistance. Discerning the mechanisms of resistance to CDK4/6 inhibition is crucial in devising alternative treatment strategies.Experimental Design: Palbociclib-resistant cells (MCF-7 and T47D) were generated in a step-wise dose-escalading fashion. Whole-exome sequencing, genome-wide expression analysis, and proteomic analysis were performed in both resistant and parental (sensitive) cells. Pathway alteration was assessed mechanistically and pharmacologically. Biomarkers of altered pathways were examined in tumor samples from patients with palbociclib-treated breast cancer whose disease progressed while on treatment.Results: Palbociclib-resistant cells are cross-resistant to other CDK4/6 inhibitors and are also resistant to endocrine therapy (estrogen receptor downregulation). IL6/STAT3 pathway is induced, whereas DNA repair and estrogen receptor pathways are downregulated in the resistant cells. Combined inhibition of STAT3 and PARP significantly increased cell death in the resistant cells. Matched tumor samples from patients with breast cancer who progressed on palbociclib were examined for deregulation of estrogen receptor, DNA repair, and IL6/STAT3 signaling, and results revealed that these pathways are all altered as compared with the pretreatment tumor samples.Conclusions: Palbociclib resistance induces endocrine resistance, estrogen receptor downregulation, and alteration of IL6/STAT3 and DNA damage response pathways in cell lines and patient samples. Targeting IL6/STAT3 activity and DNA repair deficiency using a specific STAT3 inhibitor combined with a PARP inhibitor could effectively treat acquired resistance to palbociclib.