Long Noncoding RNAs CUPID1 and CUPID2 Mediate Breast Cancer Risk at 11q13 by Modulating the Response to DNA Damage.

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.

Exosomes derived from mesenchymal non-small cell lung cancer cells promote chemoresistance.

Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mRNA, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mRNA, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mRNA in exosomes.

Quinazolinone derivatives as inhibitors of homologous recombinase RAD51.

RAD51 is a vital component of the homologous recombination DNA repair pathway and is overexpressed in drug-resistant cancers, including aggressive triple negative breast cancer (TNBC). A proposed strategy for improving therapeutic outcomes for patients is through small molecule inhibition of RAD51, thereby sensitizing tumor cells to DNA damaging irradiation and/or chemotherapy. Here we report structure-activity relationships for a library of quinazolinone derivatives. A novel RAD51 inhibitor (17) displays up to 15-fold enhanced inhibition of cell growth in a panel of TNBC cell lines compared to compound B02, and approximately 2-fold increased inhibition of irradiation-induced RAD51 foci formation. Additionally, compound 17 significantly inhibits TNBC cell sensitivity to DNA damage, implying a potentially targeted therapy for cancer treatment.

High content screening application for cell-type specific behaviour in heterogeneous primary breast epithelial subpopulations.

BACKGROUND: The complex interaction between multiple cell types and the microenvironment underlies the diverse pathways to carcinogenesis and necessitates sophisticated approaches to in vitro hypotheses testing. The combination of mixed culture format with high content immunofluorescence screening technology provides a powerful platform for observation of cell type specific behavior. METHODS: We have developed a versatile, high-throughput method for assessing cell-type specific responses. In addition to the specificity and sensitivity offered traditionally by immunofluorescent detection in flow cytometry, the 'in-cell' analysis method we describe provides the added benefits of higher throughput and the ability to analyse protein subcellular localisation in situ. Furthermore, elimination of the cell dissociation step allows for more immediate analysis of responses to specific extrinsic stimuli. We applied this method to investigate ionising radiation treatment response in normal breast epithelial cells, measuring growth rate, cell cycle response and double-strand DNA breaks. RESULTS: The 'in-cell' analysis approach elucidated several interesting donor and cell-type specific differences. Notably, in response to ionizing radiation we observed differential expression in luminal and basal-like cells of a member of the APOBEC enzyme family, recently identified as a critical driver of an oncogenic signature. Our findings suggest that this enzyme is active in the normal breast epithelium during DNA damage response. CONCLUSIONS: We demonstrate the practical application of a new method for assessing cell-type specific change in mixed cultures, especially the analysis of normal primary cultures, overcoming a major technical issue of dissecting the response of multiple cell types in a heterogeneous population.

Current challenges in metastasis research and future innovation for clinical translation.

While immense strides have been made in understanding tumor biology and in developing effective treatments that have substantially improved the prognosis of cancer patients, metastasis remains the major cause of cancer-related death. Improvements in the detection and treatment of primary tumors are contributing to a growing, detailed understanding of the dynamics of metastatic progression. Yet challenges remain in detecting metastatic dissemination prior to the establishment of overt metastases and in predicting which patients are at the highest risk of developing metastatic disease. Further improvements in understanding the mechanisms governing metastasis have great potential to inform the adaptation of existing therapies and the development of novel approaches to more effectively control metastatic disease. This article presents a forward-looking perspective on the challenges that remain in the treatment of metastasis, and the exciting emerging approaches that promise to transform the treatment of metastasis in cancer patients.

Defining the target specificity of ABT-737 and synergistic antitumor activities in combination with histone deacetylase inhibitors.

The apoptotic and therapeutic activities of the histone deacetylase inhibitor (HDACi) vorinostat are blocked by overexpression of Bcl-2 or Bcl-X(L). Herein, we used the small molecule inhibitor ABT-737 to restore sensitivity of Emu-myc lymphomas overexpressing Bcl-2 or Bcl-X(L) to vorinostat and valproic acid (VPA). Combining low-dose ABT-737 with vorinostat or VPA resulted in synergistic apoptosis of these cells. ABT-737 was ineffective against Emu-myc/Mcl-1 and Emu-myc/A1 cells either as a single agent or in combination with HDACi. However, in contrast to the reported binding specificity data, Emu-myc/Bcl-w lymphomas were insensitive to ABT-737 used alone or in combination with HDACi, indicating that the regulatory activity of ABT-737 is restricted to Bcl-2 and Bcl-X(L). Emu-myc lymphomas that expressed Bcl-2 throughout the tumorigenesis process were especially sensitive to ABT-737, while those forced to overexpress Mcl-1 were not. This supports the notion that tumor cells "addicted" to ABT-737 target proteins (ie, Bcl-2 or Bcl-X(L)) are likely to be the most sensitive target cell population. Our studies provide important preclinical data on the binding specificity of ABT-737 and its usefulness against primary hematologic malignancies when used as a single agent and in combination with HDACi.

Correction to 'Genome instability and pressure on non-homologous end joining drives chemotherapy resistance via a DNA repair crisis switch in triple negative breast cancer'.

[This corrects the article DOI: 10.1093/nar/zcab022.].

Genome instability and pressure on non-homologous end joining drives chemotherapy resistance via a DNA repair crisis switch in triple negative breast cancer.

Chemotherapy is used as a standard-of-care against cancers that display high levels of inherent genome instability. Chemotherapy induces DNA damage and intensifies pressure on the DNA repair pathways that can lead to deregulation. There is an urgent clinical need to be able to track the emergence of DNA repair driven chemotherapy resistance and tailor patient staging appropriately. There have been numerous studies into chemoresistance but to date no study has elucidated in detail the roles of the key DNA repair components in resistance associated with the frontline clinical combination of anthracyclines and taxanes together. In this study, we hypothesized that the emergence of chemotherapy resistance in triple negative breast cancer was driven by changes in functional signaling in the DNA repair pathways. We identified that consistent pressure on the non-homologous end joining pathway in the presence of genome instability causes failure of the key kinase DNA-PK, loss of p53 and compensation by p73. In-turn a switch to reliance on the homologous recombination pathway and RAD51 recombinase occurred to repair residual double strand DNA breaks. Further we demonstrate that RAD51 is an actionable target for resensitization to chemotherapy in resistant cells with a matched gene expression profile of resistance highlighted by homologous recombination in clinical samples.

Small molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes MCL-1-mediated resistance to apoptosis.

Elevated expression of members of the BCL-2 pro-survival family of proteins can confer resistance to apoptosis in cancer cells. Small molecule obatoclax (GX15-070), which is predicted to occupy a hydrophobic pocket within the BH3 binding groove of BCL-2, antagonizes these members and induces apoptosis, dependent on BAX and BAK. Reconstitution in yeast confirmed that obatoclax acts on the pathway and overcomes BCL-2-, BCL-XL-, BCL-w-, and MCL-1-mediated resistance to BAX or BAK. The compound potently interfered with the direct interaction between MCL-1 and BAK in intact mitochondrial outer membrane and inhibited the association between MCL-1 and BAK in intact cells. MCL-1 has been shown to confer resistance to the BCL-2/BCL-XL/BCL-w-selective antagonist ABT-737 and to the proteasome inhibitor bortezomib. In both cases, this resistance was overcome by obatoclax. These findings support a rational clinical development opportunity for the compound in cancer indications or treatments where MCL-1 contributes to resistance to cell killing.

Circulating Tumor Cells in Metastatic Breast Cancer: From Genome Instability to Metastasis.

The emergence of clinical resistance in repeatedly treated cancers extends from the primary tumor's capability to exploit genome instability to adapt, escape, and progress. Triple negative breast cancer serves as a good example of such a response demonstrating poor clinical outcome due to a high rate of cellular heterogeneity resulting in metastatic relapse. The capability to effectively track the emergence of therapeutic resistance in real-time and adapt the clinical response is the holy grail for precision medicine and has yet to be realized. In this review we present liquid biopsy using CTCs and ctDNA as a potential replacement and/or addition to the current diagnostic tests to deliver personalized therapies to patients with advanced breast cancer. We outline current uses of liquid biopsy in the metastatic breast cancer setting and discuss their limitations. In addition, we provide a detailed overview of common genome instability events in patients with metastatic breast cancer and how these can be tracked using liquid biopsy.

Identifying Therapies to Combat Epithelial Mesenchymal Plasticity-Associated Chemoresistance to Conventional Breast Cancer Therapies Using An shRNA Library Screen.

BACKGROUND: Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse. METHODS: We undertook a cell polarity/epithelial mesenchymal plasticity (EMP)-enriched short hairpin RNA (shRNA) screen in MDA-MB-468 breast cancer cells to identify factors underpinning heterogeneous responses to three chemotherapeutic agents used clinically in breast cancer: Doxorubicin, docetaxel, and eribulin. shRNA-transduced cells were treated for 6 weeks with the EC10 of each drug, and shRNA representation assessed by deep sequencing. We first identified candidate genes with depleted shRNA, implying that their silencing could promote a response. Using the Broad Institute's Connectivity Map (CMap), we identified partner inhibitors targeting the identified gene families that may induce cell death in combination with doxorubicin, and tested them with all three drug treatments. RESULTS: In total, 259 shRNAs were depleted with doxorubicin treatment (at p < 0.01), 66 with docetaxel, and 25 with eribulin. Twenty-four depleted hairpins overlapped between doxorubicin and docetaxel, and shRNAs for TGFB2, RUNX1, CCDC80, and HYOU1 were depleted across all the three drug treatments. Inhibitors of MDM/TP53, TGFBR, and FGFR were identified by CMap as the top pharmaceutical perturbagens and we validated the combinatorial benefits of the TGFBR inhibitor (SB525334) and MDM inhibitor (RITA) with doxorubicin treatment, and also observed synergy between the inhibitor SB525334 and eribulin in MDA-MB-468 cells. CONCLUSIONS: Taken together, a cell polarity/EMP-enriched shRNA library screen identified relevant gene products that could be targeted alongside current chemotherapeutic agents for the treatment of invasive BC.

Simultaneous targeting of DNA replication and homologous recombination in glioblastoma with a polyether ionophore.

BACKGROUND: Despite significant endeavor having been applied to identify effective therapies to treat glioblastoma (GBM), survival outcomes remain intractable. The greatest nonsurgical benefit arises from radiotherapy, though tumors typically recur due to robust DNA repair. Patients could therefore benefit from therapies with the potential to prevent DNA repair and synergize with radiotherapy. In this work, we investigated the potential of salinomycin to enhance radiotherapy and further uncover novel dual functions of this ionophore to induce DNA damage and prevent repair. METHODS: In vitro primary GBM models and ex vivo GBM patient explants were used to determine the mechanism of action of salinomycin by immunoblot, flow cytometry, immunofluorescence, immunohistochemistry, and mass spectrometry. In vivo efficacy studies were performed using orthotopic GBM animal xenograft models. Salinomycin derivatives were synthesized to increase drug efficacy and explore structure-activity relationships. RESULTS: Here we report novel dual functions of salinomycin. Salinomycin induces toxic DNA lesions and prevents subsequent recovery by targeting homologous recombination (HR) repair. Salinomycin appears to target the more radioresistant GBM stem cell-like population and synergizes with radiotherapy to significantly delay tumor formation in vivo. We further developed salinomycin derivatives which display greater efficacy in vivo while retaining the same beneficial mechanisms of action. CONCLUSION: Our findings highlight the potential of salinomycin to induce DNA lesions and inhibit HR to greatly enhance the effect of radiotherapy. Importantly, first-generation salinomycin derivatives display greater efficacy and may pave the way for clinical testing of these agents.

EGFR and Prion protein promote signaling via FOXO3a-KLF5 resulting in clinical resistance to platinum agents in colorectal cancer.

Epidermal growth factor receptor (EGFR) supports colorectal cancer progression via oncogenic signaling. Anti-EGFR therapy is being investigated as a clinical option for colorectal cancer, and an observed interaction between EGFR and Prion protein has been detected in neuronal cells. We hypothesized that PrPC expression levels may regulate EGFR signaling and that detailed understanding of this signaling pathway may enable identification of resistance mechanisms and new actionable targets in colorectal cancer. We performed molecular pathway analysis following knockdown of PrPC or inhibition of EGFR signaling via gefitinib to identify changes in expression of key signaling proteins that determine cellular sensitivity or resistance to cisplatin. Expression of these proteins was examined in matched primary and metastatic patient samples and was correlated for resistance to therapy and progression of disease. Utilizing three colorectal cancer cell lines, we observed a correlation between high expression of PrPC and resistance to cisplatin. Investigation of molecular signaling in a resistant cell line revealed that PrPC contributed to signaling via colocalization with EGFR, which could be overcome by targeting p38 mitogen-activated protein kinases (p38 MAPK). We revealed that the level of Krüppel-like factor 5 (KLF5), a target downstream of p38 MAPK, was predictive for cell line and patient response to platinum agents. Further, high KLF5 expression was observed in BRAF-mutant colorectal cancer. Our study indicates that the EGFR to KLF5 pathway is predictive of patient progression on platinum-based therapy.

Secreted cellular prion protein binds doxorubicin and correlates with anthracycline resistance in breast cancer.

Anthracyclines are amongst the most effective chemotherapeutics ever developed, but they produce grueling side-effects, serious adverse events and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular Prion protein (PrPC), blocking their cytotoxic activity. This effect was dose-dependent using either cell line-conditioned medium or human serum as a source of PrPC. Genetic depletion of PrPC or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pre-treatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly amongst doxorubicin-treated patients with residual disease at surgery (p=2.8E-08). These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrPC protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by immunohistochemistry, particularly soluble isoforms released into the extracellular environment by shedding (p=0.015). These findings have important potential clinical implications for frontline regimen decision-making. We suggest there is warranted utility for prognostic PrPC/PRNP assays to guide chemo-sensitization strategies that exploit an understanding of PrPC-anthracycline-copper ion complexes.

Breast Cancer-Derived Exosomes Alter Macrophage Polarization via gp130/STAT3 Signaling.

Tumor-derived exosomes are being recognized as essential mediators of intercellular communication between cancer and immune cells. It is well established that bone marrow-derived macrophages (BMDMs) take up tumor-derived exosomes. However, the functional impact of these exosomes on macrophage phenotypes is controversial and not well studied. Here, we show that breast cancer-derived exosomes alter the phenotype of macrophages through the interleukin-6 (IL-6) receptor beta (glycoprotein 130, gp130)-STAT3 signaling pathway. Addition of breast cancer-derived exosomes to macrophages results in the activation of the IL-6 response pathway, including phosphorylation of the key downstream transcription factor STAT3. Exosomal gp130, which is highly enriched in cancer exosomes, triggers the secretion of IL-6 from BMDMs. Moreover, the exposure of BMDMs to cancer-derived exosomes triggers changes from a conventional toward a polarized phenotype often observed in tumor-associated macrophages. All of these effects can be inhibited through the addition of a gp130 inhibitor to cancer-derived exosomes or by blocking BMDMs exosome uptake. Collectively, this work demonstrates that breast cancer-derived exosomes are capable of inducing IL-6 secretion and a pro-survival phenotype in macrophages, partially via gp130/STAT3 signaling.

Monitoring DNA Repair Consequences of ATM Signaling Using Simultaneous Fluorescent Readouts.

The repair of deleterious DNA double strand breaks is required to maintain genome integrity. The efficacy in which this occurs relies upon the available machinery and is guided by factors that include cell cycle status, availability of donor template, and the local chromosome structure. Therefore at a single DNA breakpoint there are different outcomes that can occur. The Traffic light reporter (TLR) assay protocol is a dual fluorescent readout that has the ability to monitor simultaneous homologous recombination and non-homologous end joining activity in response to DNA damage. This provides insight to determine the upstream functionality of either pathway mediated through ATM.

Defects in recombination activity caused by somatic and germline mutations in the multimerization/BRCA2 binding region of human RAD51 protein.

The human RAD51 recombinase possesses DNA pairing and strand exchange activities that are essential for the error-free, homology-directed repair of DNA double-strand breaks. The recombination activities of RAD51 are activated upon its assembly into presynaptic filaments on single-stranded DNA at resected DSB ends. Defects in filament assembly caused by mutations in RAD51 or its regulators such as BRCA2 are associated with human cancer. Here we describe two novel RAD51 missense variants located in the multimerization/BRCA2 binding region of RAD51. F86L is a breast tumor-derived somatic variant that affects the interface between adjacent RAD51 protomers in the presynaptic filament. E258A is a germline variant that maps to the interface region between the N-terminal and RecA homology domains of RAD51. Both variants exhibit abnormal biochemistry including altered DNA strand exchange activity. Both variants inhibit the DNA strand exchange activity of wild-type RAD51, suggesting a mechanism for negative dominance. The inhibitory effect of F86L on wild-type RAD51 is surprising since F86L alone exhibits robust DNA strand exchange activity. Our findings indicate that even DNA strand exchange-proficient variants can have negative functional interactions with wild-type RAD51. Thus heterozygous F86L or E258 mutations in RAD51 could promote genomic instability, and thereby contribute to tumor progression.

Prion protein scrapie and the normal cellular prion protein.

Prions are infectious proteins and over the past few decades, some prions have become renowned for their causative role in several neurodegenerative diseases in animals and humans. Since their discovery, the mechanisms and mode of transmission and molecular structure of prions have begun to be established. There is, however, still much to be elucidated about prion diseases, including the development of potential therapeutic strategies for treatment. The significance of prion disease is discussed here, including the categories of human and animal prion diseases, disease transmission, disease progression and the development of symptoms and potential future strategies for treatment. Furthermore, the structure and function of the normal cellular prion protein (PrP(C)) and its importance in not only in prion disease development, but also in diseases such as cancer and Alzheimer's disease will also be discussed.

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

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

Targeting homologous recombination, new pre-clinical and clinical therapeutic combinations inhibiting RAD51.

The DNA damage response (DDR) is essential for maintaining genomic stability and cell survival. However, when tumour cells with deficiencies in HR are faced with radio- and chemotherapies they are forced to rely on error-prone, alternative repair pathways or aberrant HR for survival; threatening genome integrity and driving further mutation. Accurate therapeutic targeting of the key drivers of DNA repair can circumvent survival pathways and avoid aggressive therapy resistant mutants. Several studies have identified that stabilization of the cancer genome in HR deficient cells can be achieved by overexpression of the recombinase RAD51. Radio- and chemotherapeutic resistance is associated with overactive HR repair mechanisms. However no clinical trials have directly targeted RAD51, despite RAD51 displaying synergy in several drug screens against multiple cancer types. Currently synthetic lethality targeting the DDR pathways and HR deficiency has had clinical success with BRCA1 functional loss and PARP inhibition. In this review we suggest that clinical outcomes could be improved by additionally targeting RAD51. We examine the latest developments in directly and indirectly targeting RAD51. We scrutinize the potential treatment efficacy and future clinical applications of RAD51 inhibitors as single agents and in combination with other therapies and consider the best therapeutic options.