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Immunogenic cell death (ICD) refers to a form of regulated cell death that activates adaptive immunity, forming long‐term immunological memory. Using chemotherapeutic drugs to induce ICD in cancer cells can help create an inflamed, immunogenic tumor environment, key for optimal immune checkpoint blockade (ICB) therapy response. ICB targets immune checkpoints such as cytotoxic T‐lymphocyte‐associated antigen 4 (CTLA4) and programmed death 1 (PD‐1). Durable responses and better quality of life in ICB patients compared with many other treatments has prompted additional investigation into its therapeutic potential and possible approaches, in an effort to further understand the functions of the costimulatory molecules and how new treatments may be designed. In this review, we will summarize ICD induction, including stress responses, damage‐associated molecular patterns, and various assays by which immunogenicity is evaluated in dying cells. In addition, the mechanisms and biomarkers underlying the CTLA4 and PD‐1 pathways of checkpoint blockade will be covered. Finally, we will review the synergistic effects of ICD induction combined with ICB therapy, as well as combination blockade therapies involving the use of multiple drugs.

Defect in DNA damage response (DDR) is a common feature of cancer cells, which regulates tumor growth and therapeutic response. Recently, the approval of immune checkpoint blockade (ICB) for tumors with defective mismatch repair has paved the way for investigating the role of other DDR defects in sensitizing cancer to ICB therapy. Despite great progress in understanding DDR pathways, the mechanisms that link DDR defects and ICB response remain incompletely understood. Further, the clinical activity of ICB in patients with DDR defective tumors has not been well described. Here, we discuss recent studies demonstrating that biomarkers in DDR pathways may serve as potential predictors to guide the selection of patients for ICB therapy. A better understanding of the relationship between deficiency in DDR and response to ICB would facilitate efforts in optimizing the efficacy of immunotherapy.

A non-immunogenic tumor microenvironment (TME) is a significant barrier to immune checkpoint blockade (ICB) response. The impact of Polybromo-1 (PBRM1) on TME and response to ICB in renal cell carcinoma (RCC) remains to be resolved. Here we show that PBRM1/Pbrm1 deficiency reduces the binding of brahma-related gene 1 (BRG1) to the IFNγ receptor 2 (Ifngr2) promoter, decreasing STAT1 phosphorylation and the subsequent expression of IFNγ target genes. An analysis of 3 independent patient cohorts and of murine pre-clinical models reveals that PBRM1 loss is associated with a less immunogenic TME and upregulated angiogenesis. Pbrm1 deficient Renca subcutaneous tumors in mice are more resistance to ICB, and a retrospective analysis of the IMmotion150 RCC study also suggests that PBRM1 mutation reduces benefit from ICB. Our study sheds light on the influence of PBRM1 mutations on IFNγ-STAT1 signaling and TME, and can inform additional preclinical and clinical studies in RCC., PBRM1, encoding for a subunit of the SWI/SNF complex, is the second most frequently mutated gene in clear cell renal cell carcinoma (ccRCC). Here, the authors show that PBRM1 loss reduces IFNγ-mediated signalling resulting in a less immunogenic tumor microenvironment and that PBRM1 mutations correlate with lack of response to checkpoint inhibitor therapy in ccRCC patients..

Alterations in immune-related pathways are common hallmarks of cancer. A comprehensive understanding of how cancer mutations rewire immune signaling networks and functional output across cancer types is instrumental to realize the full potential of immunotherapy. Here, we systematically interrogated somatic mutations involved in immune signaling that alter immune responses in patients with cancer. To do so, we developed a Network-based Integrative model to Prioritize Potential immune respondER genes (NIPPER). Identified mutations were enriched in essential protein domains and genes identified by NIPPER were associated with responsiveness to multiple immunotherapy modalities. These genes were used to devise an interactome network propagation framework integrated with drug-associated gene signatures to identify potential immunomodulatory drug candidates. Together, our systems-level analysis results help interpret the heterogeneous immune responses among patients and serve as a resource for future functional studies and targeted therapeutics. Significance: This study demonstrates that integration of multi-omics data can help identify critical molecular determinants for effective targeted therapeutics.

Poly (ADP-ribose) polymerase (PARP) inhibitors have demonstrated great promise in the treatment of patients with deficiencies in homologous recombination (HR) DNA repair, such as those with loss of BRCA1 or BRCA2 function. However, emerging studies suggest that PARP inhibition can also target HR-competent cancers, such as non-small-cell lung cancer (NSCLC), and that the therapeutic effect of PARP inhibition may be improved by combination with chemotherapy agents. In our study, it was found that PARP inhibitors talazoparib (BMN-673) and olaparib (AZD-2281) both had synergistic activity with the common first-line chemotherapeutic gemcitabine in a panel of lung cancer cell lines. Furthermore, the combination demonstrated significant in vivo antitumor activity in an H23 xenograft model of NSCLC compared to either agent as monotherapy. This synergism occurred without loss of HR repair efficiency. Instead, the combination induced synergistic single-strand DNA breaks, leading to accumulation of toxic double-strand DNA lesions in vitro and in vivo. Our study elucidates the underlying mechanisms of synergistic activity of PARP inhibitors and gemcitabine, providing a strong motivation to pursue this combination as an improved therapeutic regimen.

Summary Extensive spontaneous DNA damage from oncogene-induced replication stress is ubiquitous in precancerous lesions. While this damage induces differentiation, senescence, or apoptosis in normal cells, defects in DNA replication stress response (RSR) allow cells to continue proliferating, ultimately leading to early tumorigenesis. Using systems-level approaches, we developed a replication stress response defect gene signature that predicted risk of cancer development from hyperplastic lesions. Intriguingly, we found that replication stress response defects rewire non-malignant cells into a cancer stem cell (CSC)-like state, and analysis of CSCs indicated that they inherently harbor replication stress response defects. High-throughput drug screening to elucidate molecules required for survival of replication stress response defective cells identified a dependence on MEK/ERK signaling. Inhibition of this signaling cascade restored oncogene-induced senescence through a p53-independent MDM2/p21 axis. Moreover, MEK/ERK inhibition also depleted CSC populations. Together, these findings provide insights into the role of replication stress response defects in CSCs and an actionable pathway for therapeutic targeting.

To realize the full potential of immunotherapy, it is critical to understand the drivers of tumor infiltration by immune cells. Previous studies have linked immune infiltration with tumor neoantigen levels, but the broad applicability of this concept remains unknown. Here, we find that while this observation is true across cancers characterized by recurrent mutations, it does not hold for cancers driven by recurrent copy number alterations, such as breast and pancreatic tumors. To understand immune invasion in these cancers, we developed an integrative multi-omics framework, identifying the DNA damage response protein ATM as a driver of cytokine production leading to increased immune infiltration. This prediction was validated in numerous orthogonal datasets, as well as experimentally in vitro and in vivo by cytokine release and immune cell migration. These findings demonstrate diverse drivers of immune cell infiltration across cancer lineages and may facilitate the clinical adaption of immunotherapies across diverse malignancies., Neoantigen load has been associated with tumour immune infiltration. Here, the authors show that while this is true for tumours with recurrent mutations, cancers with recurrent CNAs show neoantigen-independent infiltration driven by cytokine production downstream of the DNA damage sensor ATM.

Despite rapid advancement in generation of large-scale microarray gene expression datasets, robust multigene expression signatures that are capable of guiding the use of specific therapies have not been routinely implemented into clinical care. We have developed an iterative resampling analysis to predict sensitivity algorithm to generate gene expression sensitivity profiles that predict patient responses to specific therapies. The resultant signatures have a robust capacity to accurately predict drug sensitivity as well as the identification of synergistic combinations. Here, we apply this approach to predict response to PARP inhibitors, and show it can greatly outperforms current clinical biomarkers, including BRCA1/2 mutation status, accurately identifying PARP inhibitor-sensitive cancer cell lines, primary patient-derived tumor cells, and patient-derived xenografts. These signatures were also capable of predicting patient response, as shown by applying a cisplatin sensitivity signature to ovarian cancer patients. We additionally demonstrate how these drug-sensitivity signatures can be applied to identify novel synergizing agents to improve drug efficacy. Tailoring therapeutic interventions to improve patient prognosis is of utmost importance, and our drug sensitivity prediction signatures may prove highly beneficial for patient management., Personalized medicine: Signature-guided cancer therapy Personalized cancer therapy is one of the holy grails of oncology, as the ability to determine what treatment would best benefit a patient would serve not only to improve outcomes, but also mitigate side effects from less effective treatments. Here, we develop algorithms to predict what patients will respond to a given therapeutic modality, as well as ways to specifically target any observed phenotype, by integrating large scale data sets that profile cancer cell line gene expression and sensitivity to hundreds of drugs. Furthermore, we show how these gene expression signatures can be used to predict novel synergizing agents to further enhance the efficacy of these therapeutics. Taken together, this work stands to advance the era of personalized medicine by enabling precision medicine approaches in the clinic.

Hepatocellular carcinomas (HCC) are adapted to survive extreme genomic stress conditions imposed by hyperactive DNA replication and genotoxic drug treatment. The underlying mechanisms remain unclear, but may involve intensified DNA damage response/repair programs. Here, we investigate a new role of nucleostemin (NS) in allowing HCC to survive its own malignancy, as NS was previously shown to promote liver regeneration via a damage repair mechanism. We first established that a higher NS transcript level correlates with high-HCC grades and poor prognostic signatures, and is an independent predictor of shorter overall and progression-free survival specifically for HCC and kidney cancer but not for others. Immunostaining confirmed that NS is most abundantly expressed in high-grade and metastatic HCCs. Genome-wide analyses revealed that NS is coenriched with MYC target and homologous recombination (HR) repair genes in human HCC samples and functionally intersects with those involved in replication stress response and HR repair in yeasts. In support, NS-high HCCs are more reliant on the replicative/oxidative stress response pathways, whereas NS-low HCCs depend more on the mTOR pathway. Perturbation studies showed NS function in protecting human HCC cells from replication- and drug-induced DNA damage. Notably, NS depletion in HCC cells increases the amounts of physical DNA damage and cytosolic double-stranded DNA, leading to a reactive increase of cytokines and PD-L1. This study shows that NS provides an essential mechanism for HCC to adapt to high genomic stress for oncogenic maintenance and propagation. NS deficiency sensitizes HCC cells to chemotherapy but also triggers tumor immune responses. Implications: HCC employs a novel, nucleostemin (NS)-mediated-mediated adaptive mechanism to survive high genomic stress conditions, a deficiency of which sensitizes HCC cells to chemotherapy but also triggers tumor immune responses.

Transcription factors are direct effectors of altered signaling pathways in cancer and frequently determine clinical outcomes in cancer patients. To uncover new transcription factors that would determine clinical outcomes in breast cancer, we systematically analyzed gene expression data from breast cancer patients. Our results revealed that Forkhead box protein M1 (FOXM1) is the top-ranked survival-associated transcription factor in patients with triple-negative breast cancer. Surprisingly, silencing FOXM1 expression led breast cancer cells to become more sensitive to doxorubicin (Dox). We found that FOXM1-dependent resistance to Dox is mediated by regulating DNA repair genes. We further demonstrated that NFκB1 interacts with FOXM1 in the presence of Dox to protect breast cancer cells from DNA damage. Finally, silencing FOXM1 expression in breast cancer cells in a mouse xenograft model significantly sensitized the cells to Dox. Our systematic approaches identified an unexpected role of FOXM1 in Dox resistance by regulating DNA repair genes, and our findings provide mechanistic insights into how FOXM1 mediates resistance to Dox and evidence that FOXM1 may be a promising therapeutic target for sensitizing breast cancer cells to Dox.

Deficient DNA mismatch repair (dMMR) induces a hypermutator phenotype that can lead to tumorigenesis; however, the functional impact of the high mutation burden resulting from this phenotype remains poorly explored. Here, we demonstrate that dMMR-induced destabilizing mutations lead to proteome instability in dMMR tumors, resulting in an abundance of misfolded protein aggregates. To compensate, dMMR cells utilize a Nedd8-mediated degradation pathway to facilitate clearance of misfolded proteins. Blockade of this Nedd8 clearance pathway with MLN4924 causes accumulation of misfolded protein aggregates, ultimately inducing immunogenic cell death in dMMR cancer cells. To leverage this immunogenic cell death, we combined MLN4924 treatment with PD1 inhibition and found the combination was synergistic, significantly improving efficacy over either treatment alone.

Post-translation modifications of proteins alter their functional activity and thus are key contributors of tumor initiation and progression. Glycosylation, one of the most common post-translational modifications of proteins, has been associated with tumorigenesis for decades. However, due to complexity in analysis of the functional effects of glycosylation, definitive information on the role of altered glycosylation in cancer is lacking. Importantly, imputing changes in glycosylation in proteins from analysis of DNA mutations has not been attempted globally. It is thus critical to elucidate the role of glycosylation in tumor pathophysiology as well as potential roles of altered glycosylation as cancer biomarkers and therapeutic targets. In this review, we summarize the evidence that glycosylation regulates functions of a set of frequently mutated oncogenes and tumor suppressors. Moreover, we explore the potential that protein sequence changes engendered by genomic mutations broadly alter glycosylation and thus promote tumor initiation and progression.

We demonstrate that concurrent administration of poly(ADP-ribose) polymerase (PARP) and WEE1 inhibitors is effective in inhibiting tumor growth but poorly tolerated. Concurrent treatment with PARP and WEE1 inhibitors induces replication stress, DNA damage, and abrogates the G2 DNA damage checkpoint in both normal and malignant cells. Following cessation of monotherapy with PARP or WEE1 inhibitors, effects of these inhibitors persist suggesting that sequential administration of PARP and WEE1 inhibitors could maintain efficacy while ameliorating toxicity. Strikingly, while sequential administration mirrored concurrent therapy in cancer cells that have high basal replication stress, low basal replication stress in normal cells protected them from DNA damage and toxicity, thus improving tolerability while preserving efficacy in ovarian cancer xenograft and patient-derived xenograft models.

Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition.

Summary Poly(ADP-ribose) polymerase inhibitors (PARPi) are selectively active in cells with homologous recombination (HR) deficiency (HRD) caused by mutations in BRCA1 , BRCA2 , and other pathway members. We sought small molecules that induce HRD in HR-competent cells to induce synthetic lethality with PARPi and extend the utility of PARPi. We demonstrated that inhibition of bromodomain containing 4 (BRD4) induced HRD and sensitized cells across multiple tumor lineages to PARPi regardless of BRCA1/2 , TP53 , RAS , or BRAF mutation status through depletion of the DNA double-stand break resection protein CtIP (C-terminal binding protein interacting protein). Importantly, BRD4 inhibitor (BRD4i) treatment reversed multiple mechanisms of resistance to PARPi. Furthermore, PARPi and BRD4i are synergistic in multiple in vivo models.

This work presents a new preclinical platform for implementation of selected targeted therapeutics for breast cancer patients., We previously demonstrated that altered activity of lysophosphatidic acid in murine mammary glands promotes tumorigenesis. We have now established and characterized a heterogeneous collection of mouse-derived syngeneic transplants (MDSTs) as preclinical platforms for the assessment of personalized pharmacological therapies. Detailed molecular and phenotypic analyses revealed that MDSTs are the most heterogeneous group of genetically engineered mouse models (GEMMs) of breast cancer yet observed. Response of MDSTs to trametinib, a mitogen-activated protein kinase (MAPK) kinase inhibitor, correlated with RAS/MAPK signaling activity, as expected from studies in xenografts and clinical trials providing validation of the utility of the model. Sensitivity of MDSTs to talazoparib, a poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) inhibitor, was predicted by PARP1 protein levels and by a new PARP sensitivity predictor (PSP) score developed from integrated analysis of drug sensitivity data of human cell lines. PSP score–based classification of The Cancer Genome Atlas breast cancer suggested that a subset of patients with limited therapeutic options would be expected to benefit from PARP-targeted drugs. These results indicate that MDSTs are useful models for studies of targeted therapies, and propose novel potential biomarkers for identification of breast cancer patients likely to benefit from personalized pharmacological treatments.

AXL receptor tyrosine kinase is overexpressed in a number of solid tumor types including triple-negative breast cancer (TNBC). AXL is considered an important regulator of epithelial-to-mesenchymal transition (EMT) and a potential therapeutic target for TNBC. In this work, we used microPET/CT with 64Cu-labeled anti-human AXL antibody (64Cu-anti-hAXL) to noninvasively interrogate the degradation of AXL in vivo in response to 17-allylamino-17-demethoxygeldanamycin (17-AAG), a potent inhibitor of HSP90. 17-AAG treatment caused significant decline in AXL expression in orthotopic TNBC MDA-MB-231 tumors, inhibited EMT, and delayed tumor growth in vivo, resulting in significant reduction in tumor uptake of 64Cu-anti-hAXL as clearly visualized by microPET/CT. Our data indicate that 64Cu-anti-hAXL can be useful for monitoring anti-AXL therapies and for assessing inhibition of HSP90 molecular chaperone using AXL as a molecular surrogate.

In humans, the gene encoding the BRCA1 C terminus-repeat inhibitor of human telomerase expression 1 (BRIT1) protein is located on chromosome 8p23.1, a region implicated in the development of several malignancies, including breast cancer. Previous studies by our group and others suggested that BRIT1 might function as a novel tumor suppressor. Thus, identifying the molecular mechanisms that underlie BRIT1's tumor suppressive function is important to understand cancer etiology and to identify effective therapeutic strategies for BRIT1-deficient tumors. We thus investigated the role of BRIT1 as a tumor suppressor in breast cancer by using genetic approaches. We discovered that BRIT1 functions as a post-transcriptional regulator of p53 expression. BRIT1 regulates p53 protein stability through blocking murine double minute 2-mediated p53 ubiquitination. To fully demonstrate the role of BRIT1 as a tumor suppressor, we depleted BRIT1 in normal breast epithelial cells. We found that knockdown of BRIT1 caused the oncogenic transformation of normal mammary epithelial cells. Furthermore, ectopic expression of BRIT1 effectively suppressed breast cancer cell proliferation and colony formation in vitro and tumor growth in vivo. Taken together, our study provides new insights into the biological functions of BRIT1 as a tumor suppressor in human breast cancer.

Genome-wide sequencing studies in breast cancer have recently identified frequent mutations in the zinc finger protein 668 (ZNF668), the function of which is undefined. Here, we report that ZNF668 is a nucleolar protein that physically interacts with and regulates p53 and its negative regulator MDM2. Through MDM2 binding, ZNF668 regulated autoubiquitination of MDM2 and its ability to mediate p53 ubiquitination and degradation. ZNF668 deficiency also impaired DNA damage–induced stabilization of p53. RNA interference–mediated knockdown of ZNF668 was sufficient to transform normal mammary epithelial cells. ZNF668 effectively suppressed breast cancer cell proliferation in vitro and tumorigenicity in vivo. Taken together, our studies identify ZNF668 as a novel breast tumor suppressor gene that functions in regulating p53 stability. Cancer Res; 71(20); 6524–34. ©2011 AACR.

Defects in the DNA damage response often lead to an increased susceptibility to cancer, and so the DDR presents an interesting set of novel therapeutic targets. The maintenance of genomic integrity by the DDR has also been found to be involved in the process of organismal ageing. While the removal of cells containing damaged DNA can be beneficial in the prevention of cancer, it may contribute to both normal and pathological ageing.

Jun activation domain-binding protein 1 (JAB1) is a multifunctional protein that participates in the control cell proliferation and the stability of multiple proteins. JAB1 overexpression has been implicated in the pathogenesis of human cancer. JAB1 regulates several key proteins and thereby produces varied effects on cell cycle progression, genome stability, and cell survival. However, the biological significance of JAB1 activity in these cellular signaling pathways is unclear. Therefore, we developed mice that were deficient in Jab1 and analyzed the null embryos and heterozygous cells. This disruption of Jab1 in mice resulted in early embryonic lethality due to accelerated apoptosis. Loss of Jab1 expression sensitized both mouse primary embryonic fibroblasts and osteosarcoma cells to gamma radiation–induced apoptosis, with an increase in spontaneous DNA damage and homologous recombination (HR) defects, both of which correlated with reduced levels of the DNA repair protein Rad51 and elevated levels of p53. Furthermore, the accumulated p53 directly binds to Rad51 promoter, inhibited its activity, and represent a major mechanism underlying the HR repair defect in Jab1-deficient cells. These results indicate that Jab1 is essential for efficient DNA repair and mechanistically link Jab1 to the maintenance of genome integrity and to cell survival.

Background: To date, there are few reports on gene products contributing to colon cancer progression. Methods: We used a gene trap comprised of an enhanced retroviral mutagen (ERM) cassette that includes a tetracycline-responsive promoter upstream of a haemagglutinin (HA) tag and a splice donor site. Integration of the ERM within an endogenous gene yields a tetracycline-regulated HA-tagged transcript. We transduced RKO colon cancer cells expressing a tetracycline trans-activator-off with the ERM-encoding retrovirus and screened for enhanced migration. Results: One clone showed fivefold enhanced migration with tetracycline withdrawal. Rapid amplification of cDNA ends identified the trapped gene as the chloride channel 4 (CLCN4) exchanger. Stable expression of a CLCN4 cDNA enhanced motility, whereas cells knocked down or null for this transcript showed reduced migration/invasion. CLCN4-overexpressing RKO colon cancer cells were more resistant than controls to proton load-induced cytotoxicity, consistent with the H+-extruding function of this antiporter. Intra-splenic delivery of RKO-CLCN4 transfectants, but not controls, yielded liver metastases, and transcript levels were higher in colon cancer metastases to the liver when compared with primary tumours. Conclusions: CLCN4 is a novel driver of colon cancer progression.

Mammalian cells are frequently at risk of DNA damage from multiple sources. Accordingly, cells have evolved the DNA damage response (DDR) pathways to monitor the integrity of their genome. Conceptually, DDR pathways contain three major components (some with overlapping functions): sensors, signal transducers, and effectors. At the level of sensors, ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3-related) are proximal kinases that act as the core sensors of and are central to the entire DDR. These two kinases function to detect various forms of damaged DNA and trigger DNA damage response cascades. If cells harbor DDR defects and fail to repair the damaged DNA, it would cause genomic instability and, as a result, lead to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers. Interestingly, this property of cancer also provides a great opportunity for cancer therapy. For example, by using a synthetic lethality model to search for the effective drugs, ChK1 inhibitors have been shown to selectively target the tumor cells with p53 mutations. In addition, the inhibitors of poly(ADP-ribose) polymerase (PARP-1) showed selectively killing effects on the cells with defects of homologous recombination (HR), particularly in the context of BRCA1/2 mutations. Since Brit1 is a key regulator in DDR and HR repair, we believe that we can develop a similar strategy to target cancers with Brit1 deficiency. Currently, we are conducting a high-throughput screening to identify novel compounds that specifically target the Brit1-deficient cancer which will lead to development of effective personalized drugs to cure cancer in clinic.

Purpose: Triple-negative breast cancer (TNBC) is a highly heterogeneous disease and has the worst outcome among all subtypes of breast cancers. Although PARP inhibitors represent a promising treatment in TNBC with BRCA1/BRCA2 mutations, there is great interest in identifying drug combinations that can extend the use of PARP inhibitors to a majority of TNBC patients with wild-type BRCA1/BRCA2. Here we explored whether mTOR inhibitors, through modulating homologous recombination (HR) repair, would provide therapeutic benefit in combination with PARP inhibitors in preclinical models of BRCA-proficient TNBC. Experimental Design: We have studied the effects of mTOR inhibitors on HR repair following DNA double-strand breaks (DSB). We further demonstrated the in vitro and in vivo activities of combined treatment of mTOR inhibitors with PARP inhibitors in BRCA-proficient TNBC. Moreover, microarray analysis and rescue experiments were used to investigate the molecular mechanisms of action. Results: We found that mTOR inhibitors significantly suppressed HR repair in two BRCA-proficient TNBC cell lines. mTOR inhibitors and PARP inhibitors in combination exhibited strong synergism against these TNBC cell lines. In TNBC xenografts, we observed enhanced efficacy of everolimus in combination with talazoparib (BMN673) compared with either drug alone. We further identified through microarray analysis and by rescue assays that mTOR inhibitors suppressed HR repair and synergized with PARP inhibitors through regulating the expression of SUV39H1 in BRCA-proficient TNBCs. Conclusions: Collectively, these findings strongly suggest that combining mTOR inhibitors and PARP inhibitors would be an effective therapeutic approach to treat BRCA-proficient TNBC patients. Clin Cancer Res; 22(7); 1699–712. ©2015 AACR.

Over the past few decades, major strides have advanced the techniques for early detection and treatment of cancer. However, metastatic tumor growth still accounts for the majority of cancer-related deaths worldwide. In fact, breast cancers are notorious for relapsing years or decades after the initial clinical treatment, and this relapse can vary according to the type of breast cancer. In estrogen receptor-positive breast cancers, late tumor relapses frequently occur whereas relapses in estrogen receptor-negative cancers or triple negative tumors arise early resulting in a higher mortality risk. One of the main causes of metastasis is tumor dormancy in which cancer cells remain concealed, asymptomatic, and untraceable over a prolonged period of time. Under certain conditions, dormant cells can re-enter into the cell cycle and resume proliferation leading to recurrence. However, the molecular and cellular regulators underlying this transition remain poorly understood. To date, three mechanisms have been identified to trigger tumor dormancy including cellular, angiogenic, and immunologic dormancies. In addition, recent studies have suggested that DNA repair mechanisms may contribute to the survival of dormant cancer cells. In this article, we summarize the recent experimental and clinical evidence governing cancer dormancy. In addition, we will discuss the role of DNA repair mechanisms in promoting the survival of dormant cells. This information provides mechanistic insight to explain why recurrence occurs, and strategies that may enhance therapeutic approaches to prevent disease recurrence.

BRCA1 expression is lost frequently in breast cancers in which it promotes malignant development. In the present study, we performed a global expression analysis of breast cancer cells in which the tumor-suppressor candidate gene TUSC4 was silenced to gain insights into its function. TUSC4 silencing affected genes involved in cell cycle and cell death, which have broad reaching influence on cancer development. Most importantly, we found a cluster pattern of gene-expression profiles in TUSC4-silenced cells that defined a homologous recombination (HR) repair defect signature. Mechanistic investigations indicated that TUSC4 protein could physically interact with the E3 ligase Herc2, which prevents BRCA1 degradation through the ubiquitination pathway. TUSC4 silencing enhanced BRCA1 polyubiquitination, leading to its degradation and a marked reduction in HR repair efficiency. Notably, ectopic expression of TUSC4 suppressed the proliferation, invasion, and colony formation of breast cancer cells in vitro and tumorigenesis in vivo. Furthermore, TUSC4 silencing was sufficient to transform normal mammary epithelial cells and to enhance sensitivity to PARP inhibitors. Our results provide a set of genetic and biologic proofs that TUSC4 functions as a bona fide tumor suppressor by regulating the protein stability and function of BRCA1 in breast cancer. Cancer Res; 75(2); 378–86. ©2014 AACR.

In this award, we have been focusing on the role of cellular dormancy in promoting cancer aggressiveness and drug resistance in recurred breast cancer. We aimed to determine the impact of dormancy on breast cancer phenotype, and to identify the molecular determinants that mediate breast cancer dormancy. During this two-year of grant period, we have successfully established a reliable in vitro breast cancer dormancy cell model. Using this model, we tested and confirmed the hypothesis that we originally proposed in our grant proposal. As what we hypothesized, we found that ER+ but not ER- dormant breast cancer cells repaired double strand DNA breaks likely through the increased activity of the error-prone NHEJ in cells, which led to higher gene mutations and transcriptome reprogramming. After exited from dormancy, these ER+ but not ER- breast cancer cells became more drug-resistant and more malignant compared to the parental cells. More importantly, our work on whole exome sequencing and transcriptome analysis revealed several potential molecules involved in dormancy-mediated recurrence and malignancy which may serve as important diagnosis biomarkers and therapeutic targets for breast cancer recurrence prevention and therapy in the future.

Telomerase expression is repressed in most somatic cells but is observed in stem cells and a high percentage of human cancers and has been hypothesized to contribute to tumorigenesis and maintenance of stem cell states. To explore telomerase regulation, we employed a general genetic screen to identify negative regulators of hTERT. We discovered three tumor suppressor/oncogene pathways involved in hTERT repression. One, the Mad1/c-Myc pathway, had been previously implicated in hTERT regulation. The second, SIP1, a transcriptional target of the TGF-β pathway, mediates the TGF-β regulated repression of hTERT. The third, the tumor suppressor Menin, is a direct repressor of hTERT. Depleting Menin immortalizes primary human fibroblasts and causes a transformation phenotype when coupled with expression of SV40 Large and Small T antigen and oncogenic ras. These studies suggest that multiple tumor suppressor/oncogene pathways coordinately repress hTERT expression and imply that telomerase is reactivated in human tumors through oncogenic mutations.

Sp1 sites can mediate growth/cell cycle induction of dihydrofolate reductase in late G1 (Jensen, D. E., Black, A. R. Swick, A. G., and Azizkhan, J. C. (1997) J. Cell. Biochem. 67, 24-31). To investigate mechanisms underlying this induction, effects of serum stimulation on regulation of Sp1 were examined. In Balb/c 3T3 cells, serum stimulation did not affect Sp1 synthesis or the relative binding of Sp1 family members to DNA; however, it did result in a rapid, approximately 2-fold increase in Sp1 levels and an approximately 3-fold increase in specific Sp1 phosphorylation in mid-G1. In normal human diploid fibroblasts, serum stimulation also increased Sp1 phosphorylation in mid-G1 but did not affect Sp1 levels. Therefore, Sp1 phosphorylation is regulated in a growth/cell cycle-dependent manner which correlates temporally with induction of dihydrofolate reductase transcription. Further studies revealed a kinase activity specifically associated with Sp1 in a growth-regulated manner. This activity is distinct from purified kinases previously shown to phosphorylate Sp1 in vitro and phosphorylates Sp1 between amino acids 612 and 678 in its C terminus, a region also phosphorylated in mid-G1 in vivo. Therefore, this study indicates that phosphorylation of the C terminus of Sp1 may play a role in the cell cycle regulation of its transcriptional activity.

Topoisomerase II (TOP2)-targeting poisons such as anthracyclines and etoposide are commonly used for cancer chemotherapy and kill tumor cells by causing accumulation of DNA double-strand breaks (DSB). Several lines of evidence indicate that overexpression of TOP2A, the gene encoding topoisomerase IIα, increases sensitivity of tumor cells to TOP2 poisons, but it is not clear why some TOP2A-overexpressing (TOP2A-High) tumors respond poorly to these drugs. In this study, we identified that TOP2A expression is induced by DLX4, a homeoprotein that is overexpressed in breast and ovarian cancers. Analysis of breast cancer datasets revealed that TOP2A-high cases that also highly expressed DLX4 responded more poorly to anthracycline-based chemotherapy than TOP2A-high cases that expressed DLX4 at low levels. Overexpression of TOP2A alone in tumor cells increased the level of DSBs induced by TOP2 poisons. In contrast, DLX4 reduced the level of TOP2 poison-induced DSBs irrespective of its induction of TOP2A. DLX4 did not stimulate homologous recombination–mediated repair of DSBs. However, DLX4 interacted with Ku proteins, stimulated DNA-dependent protein kinase activity, and increased erroneous end-joining repair of DSBs. Whereas DLX4 did not reduce levels of TOP2 poison-induced DSBs in Ku-deficient cells, DLX4 stimulated DSB repair and reduced the level of TOP2 poison–induced DSBs when Ku was reconstituted in these cells. Our findings indicate that DLX4 induces TOP2A expression but reduces sensitivity of tumor cells to TOP2 poisons by stimulating Ku-dependent repair of DSBs. These opposing activities of DLX4 could explain why some TOP2A-overexpressing tumors are not highly sensitive to TOP2 poisons. Cancer Res; 73(2); 1000–10. ©2012 AACR.

During the fifth year of this project, we have successfully verified AXL protein as a reliable membrane marker for replication stress response defect (RSRD) breast cancer cells and we demonstrated the potential to use isotope-labeled anti-human AXL antibody to detect RSRD breast cancer cells in vivo. In addition, we have clearly demonstrated the in vivo therapeutic effects of MEK inhibitor, AZD6244, and ERK inhibitor, SCH772984 on targeting RSRD breast cancer cells in two xenograft mouse models

Transcription factors are direct effectors of altered signaling pathways in cancer and frequently determine clinical outcomes in cancer patients. To uncover new transcription factors that would determine clinical outcomes in breast cancer, we systematically analyzed gene expression data from breast cancer patients. Our results revealed that Forkhead box protein M1 (FOXM1) is the top-ranked survival-associated transcription factor in patients with triple-negative breast cancer. Surprisingly, silencing FOXM1 expression led breast cancer cells to become more sensitive to doxorubicin (Dox). We found that FOXM1-dependent resistance to Dox is mediated by regulating DNA repair genes. We further demonstrated that NFκB1 interacts with FOXM1 in the presence of Dox to protect breast cancer cells from DNA damage. Finally, silencing FOXM1 expression in breast cancer cells in a mouse xenograft model significantly sensitized the cells to Dox. Our systematic approaches identified an unexpected role of FOXM1 in Dox resistance by regulating DNA repair genes, and our findings provide mechanistic insights into how FOXM1 mediates resistance to Dox and evidence that FOXM1 may be a promising therapeutic target for sensitizing breast cancer cells to Dox.

The cells throughout the human body are constantly subjected to both internal forces and external insults that cause damage to their DNA. This damage to the DNA can be harmful to the overall integrity of the cell and the ability for replication. Accurate transmission of genetic information from one cell to its progeny is dependent upon mechanisms within the cell to monitor any defects within its genome and to repair these deficiencies so as not to pass them to subsequent generations. These mechanisms are mainly mediated through an array of DNA damage response proteins including DNA damage sensors, signal transducers and effectors. Sensors, such as ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3-related), have the ability to recognize areas of damage and activate signal transducers, which either activate or inactivate effectors. Effector proteins trigger cell cycle checkpoint and the cell may successfully repair the damage or proceed towards apoptosis if these damages are irreparable. These molecules are not only necessary for surveillance of occasional non-lethal DNA damage, but are also important for the survival of the cell and the organism. Moreover, mutations to these DNA damage response proteins may contribute to an unstable genome and the development of cancer. In this chapter we will briefly review the cell cycle and relevant checkpoint proteins. We will also discuss in detail the DNA damage response signal transduction pathway and associated proteins: ATM, ATR, ChK1, Chk2, p53, BRCA-1, PARP-1, and BRIT-1,. Finally, we will discuss the future strategy in targeting the defects of these proteins in the treatment of breast cancer.

Double strand breaks (DSBs) are the most deleterious of the DNA lesions that initiate genomic instability and promote tumorigenesis. Cells have evolved a complex protein network to detect, signal, and repair DSBs. In mammalian cells, a key component in this network is H2AX, which becomes rapidly phosphorylated at Ser(139) (γ-H2AX) at DSBs. Here we show that monoubiquitination of H2AX mediated by the RNF2-BMI1 complex is critical for the efficient formation of γ-H2AX and functions as a proximal regulator in DDR (DNA damage response). RNF2-BMI1 interacts with H2AX in a DNA damage-dependent manner and is required for monoubiquitination of H2AX at Lys(119)/Lys(120). As a functional consequence, we show that the H2AX K120R mutant abolishes H2AX monoubiquitination, impairs the recruitment of p-ATM (Ser(1981)) to DSBs, and thereby reduces the formation of γ-H2AX and the recruitment of MDC1 to DNA damage sites. These data suggest that monoubiquitination of H2AX plays a critical role in initiating DNA damage signaling. Consistent with these observations, impairment of RNF2-BMI1 function by siRNA knockdown or overexpression of the ligase-dead RNF2 mutant all leads to significant defects both in accumulation of γ-H2AX, p-ATM, and MDC1 at DSBs and in activation of NBS1 and CHK2. Additionally, the regulatory effect of RNF2-BMI1 on γ-H2AX formation is dependent on ATM. Lacking their ability to properly activate the DNA damage signaling pathway, RNF2-BMI1 complex-depleted cells exhibit impaired DNA repair and increased sensitivity to ionizing radiation. Together, our findings demonstrate a distinct monoubiquitination-dependent mechanism that is required for H2AX phosphorylation and the initiation of DDR.

Cyclin E is aberrantly expressed in many types of cancer including breast cancer. High levels of the full length as well as the low molecular weight isoforms of cyclin E are associated with poor prognosis of breast cancer patients. Notably, cyclin E overexpression is also correlated with triple-negative basal-like breast cancers, which lack specific therapeutic targets. In this study, we used siRNA to target cyclin E overexpression and assessed its ability to suppress breast cancer growth in nude mice. Our results revealed that cyclin E siRNA could effectively inhibit overexpression of both full length and low molecular weight isoforms of cyclin E. We found that depletion of cyclin E promoted apoptosis of cyclin E-overexpressing cells and blocked their proliferation and transformation phenotypes. Significantly, we further demonstrated that administration of cyclin E siRNA could inhibit breast tumor growth in nude mice. In addition, we found that cyclin E siRNA synergistically enhanced the cell killing effects of doxorubicin in cell culture and this combination greatly suppressed the tumor growth in mice. In conclusion, our results indicate that cyclin E, which is overexpressed in 30% of breast cancer, may serve as a novel and effective therapeutic target. More importantly, our study clearly demonstrates a very promising therapeutic potential of cyclin E siRNA for treating the cyclin E-overexpressing breast cancers, including the very malignant triple-negative breast cancers.

Protein tyrosine kinases have been implicated in the regulation of many cellular events such as cellular proliferation, differentiation and development. Deregulation of protein tyrosine kinase activity has been shown to result in human cancer. The majority of the protein tyrosine kinases studied to date localize to the cell membrane, where they function as components of signal transduction pathways. However, small group of nuclear tyrosine kinases has been identified that includes Rak. Our recent investigations demonstrated that Rak functions as a potent tumor suppressor by regulating PTEN protein stability and function. Rak also effectively suppresses phenotypes associated with in vitro transformation in breast cancer cells and tumorigenicity in vivo. Moreover, depletion of Rak is sufficient to induce tumorigenicity in mammary epithelial cells. However, the mechanisms by which Rak and its substrates function in cancer remain largely unexplored, leaving many potential therapeutic targets yet undiscovered. Therefore, fully elucidating the biological functions of Rak may contribute to effective therapeutic approaches for Rak-defective cancers.

MCPH1, also known as BRIT1, has recently been identified as a novel key regulatory gene of the DNA damage response pathway. MCPH1 is located on human chromosome 8p23.1, where human cancers frequently show loss of heterozygosity. As such, MCPH1 is aberrantly expressed in many malignancies, including breast and ovarian cancers, and the function of MCPH1 has been implicated in tumor suppression. However, it remains poorly understood whether MCPH1 deficiency leads to tumorigenesis. Here, we generated and studied both Mcph1−/− and Mcph1−/−p53−/− mice; we showed that Mcph1−/− mice developed tumors with long latency, and that primary lymphoma developed significantly earlier in Mcph1−/−p53−/− mice than in Mcph11+/+p53−/− and Mcph1+/−p53−/- mice. The Mcph1−/−p53−/− lymphomas and derived murine embryonic fibroblasts (MEFs) were both more sensitive to irradiation. Mcph1 deficiency resulted in remarkably increased chromosome and chromatid breaks in Mcph1−/−p53−/− lymphomas and MEFs, as determined by metaphase spread assay and spectral karyotyping analysis. Additionally, Mcph1 deficiency significantly enhanced aneuploidy as well as abnormal centrosome multiplication in Mcph1−/−p53−/− cells. Meanwhile, Mcph1 deficiency impaired double strand break (DSB) repair in Mcph1−/−p53−/− MEFs as demonstrated by neutral Comet assay. Compared with Mcph1+/+p53−/− MEFs, homologous recombination and non-homologous end joining activities were significantly decreased in Mcph1−/−p53−/− MEFs. Notably, reconstituted MCPH1 rescued the defects of DSB repair and alleviated chromosomal aberrations in Mcph1−/−p53−/- MEFs. Taken together, our data demonstrate MCPH1 deficiency promotes genomic instability and increases cancer susceptibility. Our study using knockout mouse models provides convincing genetic evidence that MCPH1 is a bona fide tumor suppressor gene. Its deficiency leading to defective DNA repair in tumors can be utilized to develop novel targeted cancer therapies in the future. Citation Format: Yulong Liang, Hong Gao, Shiaw-Yih Lin, John A. Goss, Chunying Du, Kaiyi Li. Mcph1/Brit1 deficiency promotes genomic instability and tumor formation in a mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2029. doi:10.1158/1538-7445.AM2015-2029

BRIT1 [BRCT-repeat inhibitor of hTERT expression], a repressor of human telomerase function, is implicated in cellular immortalization. Here, we find that BRIT1 acts as a regulator of both the intra-S and G 2 /M checkpoints. When BRIT1 expression is depleted, cells lose the ionizing radiation (IR)-induced cell cycle arrest and become IR sensitive. BRIT1 is a chromatin-associated protein that forms irradiation-induced nuclear foci that colocalize with γ-H2AX foci. BRIT1 is also required for the expression of both BRCA1 and the checkpoint kinase Chk1 and phosphorylation of Nbs1. Thus, the checkpoint defects in the absence of BRIT1 are likely to result from its regulation of Nbs1, BRCA1, and Chk1. BRIT1 is identical to the recently discovered MCPH1 gene, found mutant in patients with primary microcephaly. The ataxia telangiectasia mutated-Rad3 related (ATR)–Chk1 pathway is defective in Seckel syndrome, another microcephaly disorder. We propose that the microcephaly observed in patients with MCPH1 deficiencies is due to disruption of the ATR–BRCA1–Chk1 signaling pathway that is also disrupted in Seckel syndrome patients.

Breast cancer is the most prevalent cancer in women and early diagnosis of breast cancer greatly improves successful treatment and survival rate. Therefore, it is essential to expand current understanding in breast tumorigenesis in order to find specific treatment to target breast cancer in its precancerous or earliest stage. To date, two important observations have been made in terms of precancerous and early breast caner development. First, replication stress response (RSR), also referred to as intra-S checkpoint, serves as a barrier to prevent the occurrence of precancerous lesions and tumorigenesis. When normal cells encounter DNA damages resulting from oncogenic stress, these damages will trigger RSR and provoke cell cycle arrest for repairing damaged DNA or force damaged cells to undergo senescence or apoptosis. However, when RSR is defective, the cells with DNA damage will be able to break the barrier and transform to cancer cells. Second, tumor-initiating cells, which possess stem cell-like properties, are also required for tumorigenesis. Tumor-initiating cells are on the top of the tumor development hierarchy. Therefore, they form in the early stage and then differentiate themselves into nontumorgenic cancer cell to give the heterogeneity of tumor. Even though both processes strongly regulate tumorigenesis, it remains unclear whether defective RSR is associated with tumor-initiating cell formation. To gain insight into breast tumorigenesis from this aspect, RSR-intact cells were given oncogenic stress by exogenous expression of cyclin E, a major oncogene in breast cancer. Once the cells encounter oncogenic-stress-induced DNA damage, several key RSR genes, such as ataxia telangiectasia and Rad3 related (ATR), are then suppressed in order to allow damaged cells to escape from senescence. By measuring the expression changes of transcriptional landscape in RSR-deficient model system, the molecular profile of RSR defective cells are more similar to breast mammary stem cell and breast tumor-initiating cells. Furthermore, the RSR defective cells increase ALDH1, a marker of normal and malignant human mammary stem cell, and increase the ability of mammosphere formation, an indicator of self-renewal property of mammary epithelial stem cells.Importantly, we have also identified EGFR/MEK1 signaling as a key pathway that renders RSR defective cells the ability to escape from senescence or evade apoptosis and acquire tumor-initiating properties. Our findings not only provide insight into the RSR defective function network and the association between RSR defect and tumor-initiating cell formation in early breast cancer development, but also identify the key pathway for breast cancer target therapy and prevention. Citation Format: Curtis Chun-Jen Lin, Hui Dai, Shiaw-Yih Lin. The replication stress response defect is associated with tumor-initiating cell formation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 354. doi:10.1158/1538-7445.AM2014-354

Homologous recombination (HR) repair deficiency predisposes to cancer development, but also sensitizes cancer cells to DNA-damage-inducing therapeutics. Here we identify an HR-defect (HRD) gene signature, which can be used to functionally assess HR repair status without interrogating individual genetic alterations in cells. By using this HRD gene signature as a functional network analysis tool, we discover that simultaneous loss of two major tumor suppressors BRCA1 and PTEN extensively rewire the HR repair-deficient phenotype, which is found in cells with defects in either BRCA1 or PTEN alone. Moreover, the HRD gene signature serves as an effective drug discovery platform to identify agents targeting HR repair as potential chemo/radio-sensitizers. More importantly, this HRD gene signature is able to predict clinical outcomes across multiple cancer lineages. Our findings, therefore, provide a molecular profile of HR repair to assess its status at a functional network level, which can provide both biological insights and have clinical implications in cancer.

β-Catenin can function as an oncogene when it is translocated to the nucleus, binds to T cell factor or lymphoid enhancer factor family members, and transactivates its target genes. In this study, we demonstrate that cyclin D1 is one of the targets of β-catenin in breast cancer cells. Transactivation of β-catenin correlated significantly with cyclin D1 expression both in eight breast cell lines in vitro and in 123 patient samples. More importantly, we found that high β-catenin activity significantly correlated with poor prognosis of the patients and was a strong and independent prognostic factor in breast cancer. Our studies, therefore, indicated that β-catenin can be involved in breast cancer formation and/or progression and may serve as a target for breast cancer therapy.

The adenovirus E1A protein has been implicated in increasing cellular susceptibility to apoptosis induced by tumor necrosis factor (TNF); however, its mechanism of action is still unknown. Since activation of nuclear factor kappaB (NF-kappaB) has been shown to play an anti-apoptotic role in TNF-induced apoptosis, we examined apoptotic susceptibility and NF-kappaB activation induced by TNF in the E1A transfectants and their parental cells. Here, we reported that E1A inhibited activation of NF-kappaB and rendered cells more sensitive to TNF-induced apoptosis. We further showed that this inhibition was through suppression of IkappaB kinase (IKK) activity and IkappaB phosphorylation. Moreover, deletion of the p300 and Rb binding domains of E1A abolished its function in blocking IKK activity and IkappaB phosphorylation, suggesting that these domains are essential for the E1A function in down-regulating IKK activity and NF-kappaB signaling. However, the role of E1A in inhibiting IKK activity might be indirect. Nevertheless, our results suggest that inhibition of IKK activity by E1A is an important mechanism for the E1A-mediated sensitization of TNF-induced apoptosis.

This project is to study the M-phase specific regulation of EGF receptor (EGFR) in breast epithelial cells and to study how EGFR overexpressed cells can escape this regulation. In addition, we plan to develop a therapeutic strategy to specifically target at the EGFR overexpressors by the combination of EGF-Psuedomonas-Exotoxin (EGF-PE) with Taxol. So far, We have observed the specific phosphorylation spots of EGFR in M phase. We are currently purifying the spots and try to identify those serine/threonine phosphorylation sites and to generate the EGFR mutant with those sites mutated. The identification and mutation of those M-phase specific sites of EGFR will be very helpful for us to understand how the activity of this receptor is cell-cycle regulated. In addition, we have completed the first step in generating the tetracycline- inducible cell line for our future studies. Finally, we have basically completed our aim 3a. We found that EGF-PE could specifically target at EGFR overexpressed cells when nocodazole or taxol was combined to enrich the M-phase population of cells. The combination of EGF-PE with the M-phase specific chemodrug, such as taxol may be a potential new therapeutic approach to treat the breast cancer patients with EGFR-overexpressing tumors in the future.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and there are no safe and effective therapeutic agents yet to treat HCC after diagnosis. However, in HCC, genomic instability is the major remarkable hallmark, especially in the tumors at the late stage and metastatic tumors. Thus, we wonder if targeting genomic instability can inhibit tumor growth in HCC. BRIT1/MCPH1 has been identified to be one of the key DNA damage and repair proteins, which harbors three BRCT domains, conserved in BRCA1 and some other proteins involved in DNA damage response pathways. Our previous studies and other reports have illustrated BRIT1’s essential roles in mitotic and meiotic homologous recombination DNA repair and maintaining genomic stability, which functions through interacting with BRCA2/Rad51 and/or chromatin structure remodeling protein SWI/SNF. Recently, we identified that BRIT1 is aberrantly expressed in ∼25% of HCC samples (n=28). In these samples, the locus of BRIT2 gene showed notable loss of heterogeneity (nearly 50%). We also found a somatic mutation in BRIT1, which occurred in the splicing donor site of intro 10 in the BRIT1 gene, leading to a truncated protein. Functional analysis showed that this mutation can cause failure of foci formation of BRIT1 under irradiation. Importantly, given that BRIT1-deficient cells exhibited defective DNA repair described above, our in vitro studies showed that the BRIT1-low expressing HCC cells HepG2 and Hep3B are more sensitive to olaparib, the inhibitor of poly(ADP-ribose) polymerase (PARPi), compared to the BRIT1-proficient cell SNU-449, which was determined by WST-1 and colony formation assays. Furthermore, we also test the inhibitory effect of olaparib in the xenograft mouse model, and found that PARPi can significantly suppress tumor growth of Hep3B xenografts. Collectively, our results clearly demonstrate that BRIT1 is mutated or aberrantly expressed in HCC, and targeting BRIT1 deficiency by PARP inhibitors may lead to novel and effective targeted therapies to treat BRIT1-deficient HCC. Citation Format: Yulong Liang, Lihou Yu, Dongxiao Zhang, Hong Gao, Shiaw-Yih Lin, Kaiyi Li. Characterization and targeting of BRIT1 deficiency in liver cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3342. doi:10.1158/1538-7445.AM2013-3342 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

DNA repair pathways play a fundamental role to maintain genomic stability. Defects in specific DNA repair pathways predispose to cancer development, but also provide therapeutic opportunities: for example, poly-ADP ribose polymerase (PARP) inhibitors are synthetically lethal with cancer cells, which are defective in homologous recombination (HR)-mediated DNA repair, such as BRCA1/BRCA2-deficient tumors. However, recent clinical trials of PARP inhibitors have shown rather disappointing results, particularly in the treatment of triple-negative breast cancer (TNBC), which has a molecular phenotype similar to that of BRCA-deficient breast cancer. As a targeted therapy, the clinical success of PARP inhibitors is dependent on identifying cancer cells with HR repair deficiency. Thus the observations of PARP inhibitors from clinical trials raise a key question: Can we identify a molecular signature of HR repair deficiency in cancer cells? To answer this question, we utilized the gene expression profiling approach to measure the cellular transcriptional reprograming in HR-deficient cell lines. By analyzing the molecular changes of transcriptional landscape, we identified an HR-defect gene signature that predicted HR repair deficiency in cells, predicted clinical outcomes of multiple human cancers, and predicted sensitivity of human cancer cells to PARP inhibitors. HR repair deficiency leads to genomic instability and often promotes accumulation of additional mutations. Our gene signature analysis further revealed that combinatorial effects of co-existing genetic changes such as loss of both BRCA1 and PTEN may extensively rewire the HR repair network and confer resistance to PARP inhibitor treatment. Moreover, to combat PARP inhibitor resistance, we successfully used our HR-defect gene signature as a drug discovery tool and found that TTK, mTOR and PI3K inhibitors could induce HR repair deficiency and sensitize cancer cells to PARP inhibitor treatment. Collectively, our findings demonstrate that transcriptional signature can be used to define the functional status of the HR repair network in cells. In summary, our study identifies a gene signature of HR repair deficiency, which provides a novel network view of the DNA repair process. In addition to using our gene signature as a prognostic marker for multiple cancers including breast, ovarian and lung cancer, our findings potentially have a significant impact on predicting responsiveness and overcoming resistance to targeted therapeutic regimens with DNA repair inhibitors such as PARP inhibitors. Citation Format: Guang Peng, Gordon Mills, Shiaw-Yih Lin. A gene signature that predicts deficiency of homologous recombination DNA repair. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-217. doi:10.1158/1538-7445.AM2013-LB-217

Transcription factors (TFs) are direct effectors of altered signaling pathways in cancer. To uncover new TFs that would determine clinical outcomes in breast cancer patients, we systematically analyzed gene expression data from breast cancer patients. Our results revealed that forkhead box protein M1 (FOXM1) is the top-ranked survival-associated TF with triple-negative breast cancer. Surprisingly, silencing FOXM1 expression led breast cancer cells to become more sensitive to doxorubicin (Dox). We found that FOXM1-dependent resistance to Dox is mediated by regulating DNA repair genes. We further demonstrated that NF-κB1 interacts with FOXM1 in the presence of Dox to protect breast cancer cells from DNA damage. Finally, silencing FOXM1 expression in a mouse xenograft model significantly sensitized the cells to Dox. Our systematic approaches identified an unexpected role of FOXM1 in Dox resistance by regulating DNA repair genes and FOXM1 may be a promising therapeutic target for sensitizing breast cancer cells to Dox. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2962. doi:1538-7445.AM2012-2962

Chromatin remodeling complexes use the energy of ATM hydrolysis to dynamically regulate the structure of chromatin. Growing evidence indicates that these complexes have a widespread role in tumor suppression. However, the mechanisms by which aberrations in these complexes drive tumorigenesis remain largely unknown. In addition to their well-established role in gene transcriptional control, our recent studies provide a novel linkage between chromatin remodeling and homologous recombination (HR)-mediated DNA repair in the maintenance of genomic stability. Our results showed that an evolutionarily conserved chromatin remodeling complex SWI/SNF is targeted to DNA damage lesions via an interaction with BRIT1, an early DNA damage responsive protein. Chromatin remodeling activity is required for relaxing highly condensed chromatin and facilitating the loading of HR repair proteins to conduct efficient HR repair. Furthermore, our unpublished data identified that another remodeling complex CHD4 functions as an upstream regulator of HR repair by recruiting BRIT1 and BRCA1 to DNA lesions. As a functional consequence of impaired HR repair, our data indicated that cells with depletions of chromatin remodeling complexes SWI/SNF and CHD4 show hypersensitivity to a new class of drugs poly ADP ribose polymerase (PARP) inhibitors via a synthetic lethal interaction. In conclusion, our studies reveal the interplay between epigenetic regulation and genome maintenance. Chromatin remodeling affects HR repair process, which may lead to the changes in the DNA coding sequencing that are inheritable through cell division. Moreover, Deficient HR repair has been well known to predispose to cancer development, which is highlighted by mutations of HR repair genes BRCA1/BRCA2 in breast and ovarian cancers. Our findings may have important clinical implications in cancer prevention. Chromatin remodeling complexes, such as SWI/SNF and CHD4 might be used as epigenetic markers for prognosis, and prediction in early stage of tumorigenesis, and also for targeted preventive intervention by using PARP inhibitors. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B53.

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. We identified BRIT1 mutations or aberrant expression in cancer patients including breast cancer and ovarian cancer. Our recent in vitro studies also suggest that BRIT1/MCPH1 functions as a novel key regulator in the DDR pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1−/− mice and identified its essential roles in both mitotic and meiotic homologous recombination (HR) DNA repair and in maintaining genomic stability. BRIT1−/− mice were infertile and meiotic HR was impaired with reduced foci formation of RAD51 or BRCA2. In addition, both BRIT1−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to gamma-irradiation. BRIT1−/− MEFs exhibited severe chromatid breaks. Notably, BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site and as a result, protect the genome from DNA damage. Furthermore, we found that BRIT1 deficiency could promote tumorigenesis in mice. We observed incidence of ovarian tumors in BRIT1−/− mice (1.5 to 2 year-old). By administering the carcinogen DMBA, BRIT1−/− mice were prone to develop breast tumors. Moreover, BRIT1 deficiency enhanced tumor susceptibility of p53−/− mice by generation of the double null (BRIT1−/−p53−/−) mice. BRIT1−/−p53−/− MEFs exhibited more aberrant chromatids than p53−/− MEFs and ectopic BRIT1 could rescue this aberration. IR-induced foci formation (IRIF) of RAD51/BRCA2 was impaired in the double null MEFs and ectopic BRIT1 restored IRIF of RAD51/BRCA2 in BRIT1−/−p53−/− MEFs, indicating BRIT1 participates in DNA repair via recruitment of the complex RAD51/BRCA2. Finally, given that BRIT1−/− cells exhibited defective HR DNA repair described above, we exploited PARP inhibitors to target the BRIT1-deficient cancers, and found that PARP inhibitor, olaparib could selectively kill the BRIT1-deficient breast cancer cells and MEFs. Currently, we are using mouse models to assess if the BRIT1-deficient tumor can be selectively suppressed by PARP inhibitors in vivo. Collectively, our unique knockout mouse model clearly demonstrates that BRIT1 is essential for maintaining genomic stability through mediating recruitment of RAD51/BRCA2 to the DNA damage site, and its deficiency causes defective HR DNA repair and promotes cancer development. Our data further indicate that targeting BRIT1 deficiency using PARP inhibitors may lead to novel and effective targeted therapies to treat BRIT1-deficient cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2807. doi:10.1158/1538-7445.AM2011-2807