Targeting PRMTs Creates Vulnerability of DNA Double-Stand Break Repair Pathways, and Potentiates Chemotherapy Efficacy in TNBC.

Purpose Recent studies re-classified the c.5339T->C; p.Leu1780Pro (L1780P) BRCA1 missense mutation as “likely pathogenic” in ACMG classification, which shows high prevalence in Korean population. This study aims to reveal the molecular mechanisms and therapeutic relevance of BRCA1 L1780P mutation in DNA damaging response of triple-negative breast cancer (TNBC) cells. Method We established MDA-MB 231 and HCC1937 TNBC cell lines expressing BRCA1 wild-type and BRCA1 L1780P by stable transfection of plasmid DNAs. We performed cell proliferation assays of the TNBC cell lines with treatment of olaparib and carboplatin. We compared the protein and mRNA expression level of RAD51 in BRCA1 wild-type and L1780 transfected TNBC cell lines by western blot and RT-PCR, and BRCA1-mediated DNA damaging responses were evaluated by immunofluorescence staining of RAD51 upon olaparib and carboplatin treatment. The growth inhibitory potential of RAD51 inhibitor, RI-1 was tested in BRCA1 L1780P transfected TNBC cell lines. Results BRCA1 L1780P transfected TNBC cells showed higher migration and invasion capacity, as well as increased sensitivity to olaparib and carboplatin compared to BRCA1 wild-type transfected cells. The induction of RAD51 expression on carboplatin treatment was significantly decreased in BRCA1 L1780P TNBC cells compared with BRCA1 wild-type cells. BRCA1 L1780P transfected TNBC cells showed also showed reduced nuclear RAD51 foci formation on carboplatin and olaparib treatment. The molecular interaction between p-BRCA1 and ATM was decreased by BRCA1 L1780P introduction in TNBC cells, suggesting BRCA1 L1780P mutation disrupts binding of p-BRCA1-ATM. In a cell proliferation assay, BRCA1 L1780P transfected TNBC cells were highly sensitive to RAD51 inhibitor (IC50 = 4.15μM). Conclusion This study shows BRCA1 L1780P mutation causes defects in DNA damage responses of TNBC cells by disrupting molecular interaction between p-BRCA1 and ATM, resulting in RAD51 downregulation. The combinatory inhibition of RAD51 and PARP is a candidate strategy for TNBCs with BRCA1 L1780P mutation. Keywords: Triple-negative Breast Cancer; BRCA1 L1780 Mutation; Homologous Recombination Deficiency; RAD51 Citation Format: Jeong Dong Lee, Min Hwan Kim, Yeon A Choi, Won-Ji Ryu, Hyun Ju Han, Ae Ran Choi, Tae Yeong Kim, Joo Hyuk Sohn. Molecular characterization of BRCA1c.5339T>C missense mutation in DNA damage response of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2053.

Background: Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype. Despite the early response to chemotherapy, high incidence of recurrence leads to short overall survival and poor prognosis. Native hyaluronic acid (HA) interacts with CD44 and receptors for hyaluronan mediated motility (RHAMM) overexpressed in TNBC mediating chemo-resistance and metastasis. In contrast, oligomeric HA (oHA) can disrupt HA-CD44/RHAMM interactions and attenuate oncogenic and pro-metastatic pathways, such as the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway, reducing downstream DNA repair and drug resistance markers. Our previous work demonstrated that oHA can exert synergistic anti-tumor and anti-metastatic effects when combined with doxorubicin (DOX) and co-loaded in an integrin-targeted iRGD-modified nanoparticle system (iRGD-DOX-oHA-PLNs). This study aims to investigate the inhibitory effect of iRGD-DOX-oHA-PLNs on both DNA single-strand break (SSB) and double-strand break (DSB) repair proteins and drug efflux pumps responsible for multidrug resistance to enhance DOX efficacy in breast cancer gene 1 (BRCA1) mutant and non-mutant TNBC. Methods: The cytotoxicity of DOX, oHA and their combinations in free solution or in nanoparticles was evaluated by clonogenic assay in human MDA-MB-231-luc-D3H2LN and MDA-MB-436 (BRCA1 mutant) TNBC cells. The in vitro expression of a DNA DSB marker, DNA repair markers, and drug efflux pump P-glycoprotein (P-gp) were measured by Western blot. The in vivo expression level of BRCA1 and Rad51 in an orthotopic TNBC mouse model was determined by immunohistochemical staining. Results: The combination of DOX-oHA showed synergism against both BRCA1 mutant and non-mutant TNBC cells. The iRGD-DOX-oHA-PLNs induced great increases in DNA DSBs demonstrated by the highest γH2AX level compared to other treatment groups. These nanoparticles also showed inhibitory effects on the expression of both DNA SSB repair protein (poly (ADP-ribose) polymerase) and DNA DSB repair proteins (Rad50 and Rad51), contributing to the enhanced efficacy of chemotherapy. The immunohistochemical staining of tumor tissues indicated lower levels of BRCA1 and Rad51 after iRGD-DOX-oHA-PLN treatment than the formulation without oHA, attributable to the effect of intracellularly delivered oHA on limiting the MAPK signaling. Additionally, iRGD-DOX-oHA-PLNs reduced the expression of the drug efflux pump P-gp as compared to DOX treatment groups without oHA. Conclusion: The co-delivery of oHA and DOX in the iRGD-DOX-oHA-PLNs efficiently blocked DNA damage repair and down-regulated the drug efflux pump P-gp, thus improving the efficacy of DOX. Collectively, this nanoparticle system could be a promising option for metastatic TNBC treatment. Citation Format: Pei Zhi, Ibrahim Alradwan, Tian Zhang, HoYin Lip, Abdulmutalib Zetrini, Chunsheng He, Jeffery Henderson, Andrew Michael Rauth, Xiao Yu Wu. Synergistic combination nanomedicine of doxorubicin and oligo hyaluronic acid inhibits DNA damage repair and overcomes drug resistance in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5399.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) have shown efficacy in treatment of breast and ovarian cancers with hereditary deletions of BRCA1/2, but the high promise of these drugs has not yet been realized in sporadic cancers. We present here strong preclinical data for a novel, mechanistically based, combinatorial approach to using DNA methyltransferase inhibitors (DNMTi’s), such as decitabine (DAC) and 5-Azacytidine (5-AZA), with PARP inhibitors (PARPi’s) as a treatment strategy for acute myelogenous leukemias (AML) and estogen-, progesterone- and HER2-receptor negative, or triple negative breast cancer (TNBC). We have previously demonstrated that low doses of 5-AZA and DAC alone show efficacy in AML and TNBC, and propose treatment with PARPi's to enhance sensitivity of cancer cells to DNMTis. The mechanistic rationale for our approach is based upon: 1) data from our group and others showing DNMT1 and PARP1 associate in a complex, and this association increases with DNA damage; 2) the fact that 5-AZA and DAC trap DNMT's led us to hypothesize that these drugs might also increase PARP trapping at DNA damage sites; and 3) the cytotoxicity of the most potent PARPi's (e.g. Talazoparib) appears to correlate with the degree of trapping of PARP1 in chromatin. We find that in cultured human AML and TNBC cells, the DNMTi's (5 to 20 nM DAC or 100 to 200nM 5-AZA) and PARPi's (1 to 10 nM Talazoparib) alone trap PARP into chromatin, and this effect is enhanced when the drugs are combined. In addition, the PARPi-DNMTi combination treatment in TNBC MDA-MB-231 and AML MOLM-14 cell lines resulted in significantly increased DNA double strand breaks (DSBs) and enhanced retention of PARP1 and DNMT1 at laser microirradiation DNA damage sites. Compared with non-tumorigenic MCF10A cells, in TNBC cell lines (N = 4), the combined doses resulted in significant (p<0.05) increases in colony survival, when compared to single drug treatments. Similarly, AML cell lines (N = 3) as well as primary cells (N = 8) showed dramatic decreases (p<0.05) in colonies in combination vs single agent drug treatments. In the most important translational implications, mouse xenograft experiments, using BRCA mutant TNBC cell line SUM149PT and MDA-MB-231 with intact BRCA1, both models demonstrated that the combination treatment has a significant (p<0.05) survival advantage, compared to control (vehicle), AZA (0.5mg/kg) or BMN (0.3 mg/kg) alone. Likewise in AML xenografts of MOLM14 and MV411 cell lines with poor prognosis marker, FMS-like 3 internal tandem duplication (FLT3/ITD), the drug combination show (p<0.05) significantly decreased leukemia burden, as measured by luciferase imaging. Our data suggest a novel use of both DNMTi's and PARPi's in a compelling therapeutic strategy for TNBCs independent of BRCA mutations and poor prognosis AML; the latter will be investigated in a clinical trial funded by Van Andel-SU2C. Citation Format: Feyruz V. Rassool, Nidal Muvarak, Khadiza Chowdury, Carine Robert, Limin Xia, Eun Yong Choi, Yi Cai, Marina Bellani, Ying Zou, Michael Seidman, Søren Bentzen, Maria Baer, Rena Lapidus, Stephen B. Baylin. Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for poor prognosis acute myeloid leukemia and triple-negative breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-205.

Non-homologous end joining (NHEJ) is a principal pathway of DNA double-strand break (DSB) repair in mammalian cells,1 but is also involved in assembly of antigen receptor genes and telomere maintenance.2,3 Loss of NHEJ function can result in chromosome instability promoting malignant transformation, in particular when other cellular safeguards are compromised. Inherited NHEJ defects underlie radiosensitive severe combined immunodeficiency. NHEJ is initiated by binding of Ku70–Ku80 heterodimers to both ends of a DNA break, followed by recruitment of catalytic subunits of DNA-dependent protein kinase (DNA-PKcs) to form juxtaposed DNA-PK holoenzymes which join the ends in a synaptic complex. Cohesive blunt DNA ends can be joined directly by the XFL-XRCC4-DNA ligase IV complex, but in many cases DNA ends contain damaged bases or DNA backbone sugars that require processing before ligation. End processing may involve nucleases such as Artemis, DNA polymerases and polynucleotide kinase among other enzymes.4,5

lncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

Background. Triple negative breast cancer (TNBC) is a challenging both in the choice of therapies and clinical outcomes. In the present study, we investigated the potential prospect of a Chinese medicine formation, ShenLingLan, shown to have benefit to patients with cancer and able to influence the biological behaviour of cancer cells, on breast cancer cells in particular the differential response from TNBC and non-TNBC cells and, on the discovery that TNBC cells were particularly sensitive to the medicine, we went on to determine the signalling and mechanism of action. Methods. A panel of three TNBC (MDA MB-231, BT20 and BT549) and a panel of three non-TNBC (MCF-7, ZR 75-1 and T47D) cells were used. A soluble extract from ShenLingLan, designated as SLDM, was utilised during this study. The proliferation, cellular migration and adhesiveness were tested using conventional and biophysical methods. Signalling profiling was conducted using a protein kinase array platform (Kinexustm). Metabolic profiling was conducted using the Seahorse platform. Expression of insulin receptor (IR) and insulin-like growth factor receptor (IGFR) gene transcripts (quantitative transcript analysis) and proteins (IHC) were conducted using a fresh breast cancer cohort and tissue array, respectively. Results. SLDM had little effects on the growth of breast cancer cells. However, it had profound inhibitory effects on the migration of both TNBC and non-TNBC cells in a concentration dependent manner. Interestingly, TNBC cells were 5-20 times more sensitive than the non-TNBC cells in their migration and cell adhesion responses to SLDM. The protein array platform further revealed that, of the wide range of protein kinases, IR and IGRR1 were the most affected in that SLDM resulted in 25-50% reduction in the phosphorylation of IR and IR substrate in TNBC cells. SLDM also caused a contrasting response in IGFR1 phosphorylation in TNBC and non-TNBC cells. Metabolically, TNBC and non-TNBC cells responded to SLDM in very different fashions. For example, in TNBC cells SLDM resulted in a significant decrease in glycolytic activities, in particular that driven by insulin (30.2±12.2 pmol/min in control, 47.7±10 pmol/min with insulin and 35.4±4.2 pmol/min with insulin/SLDM, p=0.01). There is evidence that the mitochondria oxygen consumption (OCR) was also affected by SLDM in TNBC cells (p=0.01). These changes induced by SLDM were in clear contrast to non-TNBC cells which did not respond with significant reduction. Both TNBC and non-TNBC breast cancer tissues have higher IR staining than normal mammary tissues (p<0.001). TNBC tumours also demonstrated significantly more positive IR staining than the non-TNBC tumours (p=0.04). Conclusion. ShenLingLan has a profound inhibitory effect on the migration and cell-matrix adhesion of TNBC cells, with marked effect on the metabolics of these cells. This effect connects with reduction of the IR and IGFR activation, mainly through the reduction in glycolysis. Together with the clinical implication of IR and IGFR in breast cancer, ShenLingLan has an important role in the treatment of breast cancer with an emphasis in TNBCs. Citation Format: Jiang WG, Owen S, Ruge F, Gao Y, Wang H, Wei C, Wu Y, Davies E. The impact of the Chinese medicine ShenLingLan on triple negative breast cancer, the metabolic and signalling pathways and clinical implications [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-10.

Approximately 10-20% of breast cancers lack estrogen, progesterone or HER2 receptors, the so-called triple negative breast cancer cells (TNBCs). These women have a poor prognosis and treatment options are limited. Therefore, the development of novel therapies are urgently required. It is well established that repair of DNA double-strand breaks (DSBs), one of the most lethal forms of DNA damage, is abnormal in breast cancers and can lead to genomic instability. We have recently reported that an alternative and highly error-prone form of NHEJ (ALT NHEJ) is found to be upregulated in TNBCs, as evidenced by increased protein levels of ALT NHEJ components, DNA ligase III (LIG3) and poly ADP ribose polymerase 1 (PARP1) and increased ALT NHEJ activity. Notably, these cells are highly sensitive to a combination of DNA ligase and PARP inhibitors in colony survival assays. This suggests that ALT NHEJ may not only contribute to the genomic instability, but it is also important for survival. The aim of this study is to determine the mechanisms underlying ALT-NHEJ upregulation in TNBCs that will likely identify potential novel targets for therapy. Herein, we demonstrate that c-MYC plays a novel role in driving error-prone repair by ALT NHEJ. A significant decrease in PARP1 and LIG3 is observed with siRNA-mediated knockdown (KD) of c-MYC in several TNBC cell lines (N=4). Furthermore, overexpression of c-MYC in wild-type breast epithelial cells (WT) with c-MYC-pcDNA3 construct leads to >2-fold increase in steady-state levels of PARP1 and LIG3. To determine whether c-MYC expression affects the functional outcome of DSB repair via ALT NHEJ, we performed in vivo and in vitro plasmid-based end-joining assays following knockdown or overexpression of c-MYC. Notably, c-MYC KD led to an increase in fidelity of NHEJ repair in TNBCs. In contrast, c-MYC overexpression leads to an increase in DSB repair errors, characterized by larger DNA deletions. Given the pro-proliferative role of c-MYC we determined whether error-prone ALT NHEJ was S-phase specific. In vivo NHEJ assays on cells released from synchronization at S- and G2/M phases of cell cycle reveal that DSB repair errors in the plasmid are introduced mainly in the S-phase. Putative c-MYC binding sites are present on the promoters of both PARP1 and LIG3. To investigate whether c-MYC is involved in transcriptional regulation of ALT NHEJ proteins, we cloned the PARP1 promoter into a luciferase vector and transfected this construct into WT and TNBCs (N=4). PARP1 promoter activity decreases upon c-MYC knockdown in TNBCs and increases upon c-MYC overexpression in WT cells, suggesting that c-MYC is likely involved in direct regulation of PARP1. Our findings demonstrate a novel role of c-MYC in driving error-prone DSB repair by ALT NHEJ pathway, which could have important implications in genomic instability, disease progression and the development of novel therapies in TNBCs. Citation Format: Pratik K. Nagaria, Khadiza Chowdhury, Angela Brodie, Feyruz Rassool. C-MYC plays a novel role in driving the error-prone double-strand break repair in triple negative breast cancers. [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 1778. doi:10.1158/1538-7445.AM2013-1778

The Polycomb Group Protein L3MBTL2 Modulates DNA Double Strand Break Repair and Predicts Response of Triple Negative Breast Cancer Cells to Poly (ADP-ribose) Polymerase Inhibition

Background: Epithelial to Mesenchymal transition (EMT) is an important and coordinated series of events associated with tumor metastasis and invasion. Recent studies had shown the importance of CCN5 (also known as WISP-2,Wnt-1-induced signaling protein-2) in the regulation of various carcinomas including the breast cancer. Recent studies had showed that ectopic expression of CCN5 can reverse Epithelial-Mesenchymal transition (EMT) and inhibit cancer metastasis. Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been extensively studied as a bioactive dietary component against various types of carcinomas through multiple mechanisms such as anti-oxidation, induction of apoptosis, inhibition of angiogenesis and metastasis. However, the mechanism of action of EGCG in breast carcinoma is uncertain. Objective: The objective of the present study is to determine whether CCN5 plays any significant role in EGCG-mediated cytotoxicity in triple negative breast cancer cells. Results: Exposure of triple negative human breast cancer (TNBC) cells, MDA-MB-231 and HCC70 to EGCG resulted in a dose-dependent inhibition of proliferation after 72h and the IC50 was observed around 75μM for MDA-MB-231 cells and 50μM for HCC70 respectively. We found EGCG-treatment effectively induces MET and inhibits the in vitro migration parallel with the induction of CCN5 expression in TNBC cells in a dose-dependent fashion. Furthermore, consistent with in vitro findings, tumor progression was drastically inhibited in EGCG-treated MDA-MB-231-tumor xenograft in nude mouse model. Conclusion: EGCG imparts its anti-cancer activity in both TNBC cells as well as MB-231-tumor xenografts via induction of CCN5. Citation Format: Amlan Das, Snigdha Banerjee, Archana De, Inamul Haque, Gargi Maity, Matt McEwen, Sushanta K. Banerjee. The green tea polyphenol EGCG induces mesenchymal to epithelial transition (MET) and tumor regression in triple negative breast cancer (TNBC) cells and mouse xenograft model: involvement of CCN5. [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 4385. doi:10.1158/1538-7445.AM2013-4385

Triple-negative breast cancers (TNBC) are often resistant to treatment with ionizing radiation (IR). We sought to investigate whether pharmacologic inhibition of Chk1 kinase, which is commonly overexpressed in TNBC, preferentially sensitizes TNBC cells to IR. Ten breast cancer cell lines were screened with small molecule inhibitors against Chk1 and other kinases. Chk1 inhibition was also tested in isogenic KRAS mutant or wild-type cancer cells. Cellular radiosensitization was measured by short-term and clonogenic survival assays and by staining for the DNA double-strand break (DSB) marker γ-H2AX. Radiosensitization was also assessed in breast cancer biopsies using an ex vivo assay. Aurora B kinase-dependent mitosis-like chromatin condensation, a marker of radioresistance, was detected using a specific antibody against co-localized phosphorylation of serine 10 and trimethylation of lysine 9 on histone 3 (H3K9me3/S10p). Expression of CHEK1 and associated genes was evaluated in TNBC and lung adenocarcinoma. Inhibition of Chk1 kinase preferentially radiosensitized TNBC cells in vitro and in patient biopsies. Interestingly, TNBC cells displayed lower numbers of IR-induced DSBs than non-TNBC cells, correlating with their observed radioresistance. We found that Chk1 suppressed IR-induced DSBs in these cells, which was dependent on H3K9me3/S10p-a chromatin mark previously found to indicate radioresistance in KRAS mutant cancers. Accordingly, the effects of Chk1 inhibition in TNBC were reproduced in KRAS mutant but not wild-type cells. We also observed co-expression of genes in this Chk1 chromatin pathway in TNBC and KRAS mutant lung cancers. Chk1 promotes an unexpected, common phenotype of chromatin-dependent DSB suppression in radioresistant TNBC and KRAS mutant cancer cells, providing a direction for future investigations into overcoming the treatment resistance of TNBC.

Aberrant activation of the phosphatidylinositol 3'-kinase (PI3K)-Akt signaling pathway is observed in many types of human cancer including triple negative breast cancer (TNBC). Additionally, dysregulation in the homologous recombination (HR)-dependent DNA-repair is associated with TNBC phenotype due to BRCA1/2 mutations or HR deficiency. Therefore, the hypothesis of this study was to evaluate the association of PI3K inhibition with HR pathway in TNBC in terms of BRCA1 mutation status. To examine the potential therapeutic effect of LY294002, an inhibitor of PI3K, on TNBC cell lines with known BRCA1 status, WST-1, annexin V, cell cycle analysis and AO/EB staining were performed. Additionally, RT-PCR and immunofluorescence analysis was used to explore the interaction between the inhibition of PI3K and HR functionality. The findings showed that LY294002 could significantly inhibited the proliferation of TNBC cells. Furthermore, the suppression of PI3K resulted in HR impairment by BRCA1 and RAD51 downregulation and apoptotic cell death by the induction of DNA damage and BAX overexpression. Therefore, LY294002 was more effective in BRCA1-deficient TNBC cells. Consequently, targeted therapies based on the interaction of PI3K inhibition with BRCA1 mutations or HR deficiency in TNBC may be a promising strategy for the treatment of patients with TNBC.

Breast cancer (BC) is the most frequent tumor in women worldwide. Although its mortality has significantly decreased owing to advanced therapies, triple negative breast cancer (TNBC) is still difficult to treat because of lack of estrogen receptor, progesterone receptor and HER-2. TNBC accounts for 12-24% of total BC and contributes to the aggressiveness and poorer outcomes. The risk of BC increases significantly in obese women. Obesity is also associated with the worse clinical prognosis of BC. But the underlying mechanisms between obesity and BC, particularly TNBC, remain unclear. Therefore, we compare the molecular mechanisms by which human adipocyte conditioned media (CM) affect TNBC cells (BT549) and non-TNBC cells (MCF7A). Adipocyte CM had no effect on the proliferation of both TNBC and non-TNBC cells. However, adipocyte CM enhanced significantly migration in TNBC cells compared to non-TNBC cells. Next, we examined the signaling pathway by which adipocyte CM promote migration of TNBC cell. AKT and ERK were activated in both TNBC cells and non-TNBC cells. However, phospho-STAT3 was significantly increased in TNBC cells. Also, N-cadherin, a marker for epithelial to mesenchymal transition (EMT), was increased at the late time point in TNBC cells. Furthermore, CXCR7 was specifically increased in TNBC cells after treatment with adipocyte CM using a PCR array. CXCR7 is chemoattractive to CXCL12 which is highly expressed in the lung, the bone marrow, and the liver, common sites of BC metastasis. Taken together, the results indicate that adipocyte CM may promote metastasis of TNBC cells through the CXCL12-CXCR7 axis by activating STAT3. Citation Format: Hyeongjwa Choi, Rosa Mistica C. Ignacio, Carla R. Gibbs, Eunsook Lee, Samuel E. Adunyah, Deok-Soo Son. Involvement of CXCL12-CXCR7 axis in adipocyte-induced TNBC metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2933. doi:10.1158/1538-7445.AM2017-2933

Bisnaphthalimidopropyl diaminodicyclohexylmethane induces DNA damage and repair instability in triple negative breast cancer cells via p21 expression

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.

Breast cancer (BC) is the most frequent female cancer and a leading cause of female deaths worldwide. BC-related mortality rates are high among African American (AA) women despite the low incidence rates of breast cancer observed in this population compared with Caucasian Americans (CA). The triple negative breast cancer (TNBC) subtype lacks expression of three biomarkers used to clinically classify BC, and thus TNBCs cannot be treated with traditional receptor therapies. Moreover, as TNBC is biologically aggressive and women diagnosed with TNBC have poor outcomes. Interestingly, TNBC is most prevalent in young women of African Ancestry (WAA) compared to women of other ethnicities, but the cause of this racial disparity remains unknown. Recent studies in our lab revealed that the transcription factor Kaiso is highly expressed in TNBC tissues of WAA patients compared with those from Caucasian patients, suggesting a role for Kaiso in TNBC racial disparity. Intriguingly, our lab and others have also reported a correlation between high Kaiso expression, poor overall survival of AA BC patients compared with Caucasian patients, and increased TNBC aggressiveness/metastasis that is in part mediated via the TGFβ signaling pathway. Notably, Kaiso has also been implicated in tumor cell migration via its regulation of the tumor-suppressing microRNA-31 (miR-31) in prostate cancer cells. Remarkably, the pleiotropic miR-31 functions to suppress metastasis and its expression has been shown to be inversely correlated with aggressive breast tumor metastasis. Although Kaiso has been implicated in epithelial-to-mesenchymal transition (EMT) and TNBC metastasis, Kaiso's exact roles in the regulation of miRNAs in the context of TNBC remains to be elucidated. Using chromatin immunoprecipitation (CHIP) analysis, we found that Kaiso binds to the miR-31 and miR-200 promoters, and we detected increased expression of these microRNAs in Kaiso-depleted TNBC cells using qRT-PCR analysis. Furthermore, using immunoblot analysis, we found that Kaiso depletion resulted in reduced expression of the actin remodelling protein WAVE3, which is a downstream target of both miR-31 and miR-200. Consistent with these molecular changes, transfection of TNBC cells with miR-31 and miR-200 mimics resulted in reduced migration of these cells compared to control TNBC cells as assessed via migration assays. These data suggest that Kaiso regulates miR-31 and miR-200 in TNBC cells, and promotes TNBC cell migration via downregulation of these miRNAs. Ongoing studies seek to assess and correlate miR-31 and miR-200 expression with Kaiso expression in TNBC tissues of WAA. Together, our findings raise the exciting possibility that Kaiso may be developed as a potential target for the treatment of TNBC patients. Citation Format: Rayner LGA, Bassey-Archibong BI, Jaber S, Daniel JM. Kaiso regulates miRNA-31 and miRNA-200 expression in triple negative breast cancer (TNBC) cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-07-06.