Regimens For Triple-negative Breast Cancer Research Articles

Association between homologous recombination deficiency and outcomes with platinum and platinum-free chemotherapy in patients with triple-negative breast cancer.

The choice of chemotherapeutic regimen for triple-negative breast cancer (TNBC) remains controversial. Homologous recombination deficiency (HRD) has attracted increasing attention in informing chemotherapy treatment. This study was aimed at investigating the feasibility of HRD as a clinically actionable biomarker for platinum-containing and platinum-free therapy. Chinese patients with TNBC who received chemotherapy between May 1, 2008 and March 31, 2020 were retrospectively analyzed with a customized 3D-HRD panel. HRD positivity was defined by an HRD score ≥ 30 or deleterious BRCA1/2 mutation. A total of 386 chemotherapy-treated patients with TNBC were screened from a surgical cohort (NCT01150513) and a metastatic cohort, and 189 patients with available clinical and tumor sequencing data were included. In the entire cohort, 49.2% (93/189) of patients were identified as HRD positive (40 with deleterious BRCA1/2 mutations and 53 with BRCA1/2 intact with an HRD score of ≥ 30). In the first-line metastatic setting, platinum therapy was associated with longer median progression-free survival (mPFS) than platinum-free therapy [9.1 vs. 3.0 months; hazard ratio (HR), 0.43; 95% confidence interval 0.22-0.84; P = 0.01]. Among HRD-positive patients, the mPFS was significantly longer in those treated with platinum rather than platinum-free therapy (13.6 vs. 2.0 months; HR, 0.11; P = 0.001). Among patients administered a platinum-free regimen, HRD-negative patients showed a PFS significantly superior to that of HRD-positive patients (P = 0.02; treatment-biomarker P-interaction = 0.001). Similar results were observed in the BRCA1/2-intact subset. In the adjuvant setting, HRD-positive patients tended to benefit more from platinum chemotherapy than from platinum-free chemotherapy (P = 0.05, P-interaction = 0.02). HRD characterization may guide decision-making regarding the use of platinum treatment in patients with TNBC in both adjuvant and metastatic settings.

Treatment of Breast Cancer in Young Adults.

Although breast cancer is rare and understudied in adults age 40 and younger, recent epidemiologic data show an increasing incidence of breast cancer among young women in the United States and ongoing inferior long-term outcomes. Given breast cancers arising at a young age are more likely to present at advanced stages and to have aggressive biology, multimodal treatments are often indicated. Elevated local recurrence risks and greater propensity for germline cancer predisposition mutations can impact local therapy choices. Recently, escalated systemic therapy regimens for triple-negative breast cancer incorporating immunotherapy, de-escalated anti-HER2 therapy, and emerging targeted agents, including CDK4/6 inhibitors and PARP inhibitors, for early-stage disease may be employed in younger and older patients alike, with some special considerations. Prognostic genomic signatures can spare low-risk young women with hormone receptor-positive breast cancer adjuvant chemotherapy, but management of intermediate-risk patients remains controversial. Ovarian function suppression and extended endocrine therapy are improving outcomes in hormone receptor-positive breast cancer, but treatment adherence is a particular problem for young patients. Young women may also face greater challenges in long-term survivorship, including impaired fertility, difficulties in psychosocial adjustment, and other treatment-related comorbidities. Consideration of these age-specific issues through dedicated multidisciplinary strategies is necessary for optimal care of young women with breast cancer.

Abstract 2050: Treatment with anti-PD-1 monoclonal antibody increases levels of MIP-1α and other cytokines, and suppresses tumor growth in a mouse model of triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC), characterized by the lack of estrogen and progesterone receptors and HER2, is an aggressive form of breast cancer that accounts for 15-20% of all diagnosed cases. There are few targeted therapies available for TNBC, but immune checkpoint inhibitors (ICIs) such as Pembrolizumab have been shown to be effective in a subset of TNBC patients. However, because ICIs are rarely administered as a monotherapy, there remains little foundational understanding of how single-agent immunotherapies alter the immune landscape and which circulating immune profiles predict therapeutic response in TNBC. Therefore, further investigation into the distinct immune signatures of ICIs are necessary for developing a rational basis for selecting optimal treatment regimens for TNBC. Methods: E0771 cells, a C57Bl/6 mouse-derived TNBC cell line, were injected into the bilateral mammary pads of 8 week old female C57Bl/6J mice. Control mice were injected with saline into mammary pads. After tumors developed (14 days post-implantation), E0771 mice received 200 ug doses of immunotherapy (anti-PD-1, anti-LAG-3, or anti-TIM-3 antibodies) or IgG isotype control every four days. After 3 doses, treatments were stopped and serum was collected from “on-treatment” mice. Fourteen days after the third treatment, serum was collected from “post-treatment” mice. A panel of 31 cytokines, chemokines, and growth factors were analyzed in “pre-treatment”, “on-treatment”, “post-treatment”, and saline control mouse serum using a BioPlex 200 multiplex system (Eve Technologies). Results: Mice treated with anti-PD-1 antibody showed significantly higher levels of several cytokines and chemokines compared to isotype control mice. Among the most significantly increased cytokines in the post-treatment anti-PD-1 group was macrophage inflammatory protein-1α (MIP-1α), or CCL3. MIP-1α is a chemotactic cytokine secreted by several types of immune cells that is known to attract macrophages and CD8+ T cells. More subtle changes in levels of several cytokines, chemokines, and growth factors were observed in anti-LAG-3 and anti-TIM-3 treatment groups. At the post-treatment timepoint, mice treated with anti-PD-1 antibody had smaller tumor sizes than mice treated with anti-LAG-3 and anti-TIM-3 antibodies, indicating that PD-1 blockade slows tumor growth and disease progression. Conclusion: Anti-PD-1 treatment induced more distinct circulating immune profiles and was more effective at controlling tumor growth than anti-LAG-3 and anti-TIM-3 treatments. These results may also suggest MIP-1α-mediated tumor regression in response to anti-PD-1 treatment in TNBC. Citation Format: Payton De La Cruz, Kathryn Grive, Jennifer Ribeiro. Treatment with anti-PD-1 monoclonal antibody increases levels of MIP-1α and other cytokines, and suppresses tumor growth in a mouse model of 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 2050.

Quercetin attenuates the cardiotoxicity of doxorubicin-cyclophosphamide regimen and potentiates its chemotherapeutic effect against triple-negative breast cancer.

Doxorubicin combined with cyclophosphamide (AC) is the most commonly used regimen for triple-negative breast cancer (TNBC) chemotherapy; however, its clinical application is severely limited by its serious adverse effect on cardiomyocytes. The cardiotoxicity of AC is mainly the result of oxidative stress caused by the imbalance between reactive oxygen species (ROS) and antioxidants, and it also involves multiple signaling pathways. Quercetin (Que) has been proven to possess strong antioxidant activity, and therefore we investigated whether it had potential protective effect against AC-induced cardiotoxicity. Meanwhile, we also evaluated its effect on the antitumor activity of AC. Our in vitro studies showed that Que could attenuate AC-induced cardiotoxicity by inhibiting ROS accumulation and activating ERK1/2 pathway in cardiomyocytes, but interestingly, Que could enhance the antitumor activity of AC by inhibiting ROS accumulation and ERK1/2 pathway in TNBC cells. In addition, our in vivo studies further confirmed that Que could enhance the chemotherapeutic effect of AC against TNBC while it reduced the injury of cardiotoxicity induced by AC. Therefore, Que could be used as a novel agent for the treatment of cardiotoxicity induced by AC regimen in TNBC chemotherapy.

Immune Effect of Active Components of Traditional Chinese Medicine on Triple-Negative Breast Cancer.

Triple-negative breast cancers are heterogeneous, poorly prognostic, and metastatic malignancies that result in a high risk of death for patients. Targeted therapy for triple-negative breast cancer has been extremely challenging due to the lack of expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Clinical treatment regimens for triple-negative breast cancer are often based on paclitaxel and platinum drugs, but drug resistance and side effects from the drugs frequently lead to treatment failure, thus requiring the development of new therapeutic platforms. In recent years, research on traditional Chinese medicine in modulating the immune function of the body has shown that it has the potential to be an effective treatment option against triple-negative breast cancer. Active components of herbal medicines such as alkaloids, flavonoids, polyphenols, saponins, and polysaccharides have been shown to inhibit cancer cell proliferation and metastasis by activating inflammatory immune responses and can modulate tumor-related signaling pathways to further inhibit the invasion of triple-negative breast cancer. This paper reviews the immunomodulatory mechanisms of different herbal active ingredients against triple-negative breast cancer and provides an outlook on the challenges and directions of development for the treatment of triple-negative breast cancer with herbal active ingredients.

GABRP sustains the stemness of triple-negative breast cancer cells through EGFR signaling.

Effective treatment regimens for triple-negative breast cancer (TNBC) are relatively scarce due to a lack of specific therapeutic targets. Epidermal growth factor receptor (EGFR) signaling is highly active in TNBC and is associated with poor prognosis. Most EGFR antagonists, which significantly improve outcome in lung and colon cancer, have shown limited clinical effects in breast cancer. However, limiting EGFR expression in TNBC is a potential strategy for improving the clinical efficacy of EGFR antagonists. Here, we found that the gamma-aminobutyric acid type A receptor π subunit (GABRP), as a membrane protein enriched in TNBC stem cells, interacted with EGFR and significantly sustained its expression, resulting in stemness maintenance and chemotherapy resistance. Silencing GABRP induced down-regulation of EGFR signaling, which hindered cell stemness and enhanced sensitivity to chemotherapies, including paclitaxel, doxorubicin, and cisplatin. We also identified that retigabine, an FDA-approved drug for adjunctive treatment of seizures, increased the sensitivity of EGFR to gefitinib in gefitinib-resistant cells. Our findings show that GABRP can sustain the stemness of TNBC via modulating EGFR expression, suggesting that GABRP may be a potential therapeutic target that can address EGFR inhibitor resistance in TNBC.

An in vitro study on the reversal of epithelial to mesenchymal transition by brusatol and its synergistic properties in triple-negative breast cancer cells.

Taxane based conventional chemotherapy serves as the standard treatment regimen for triple-negative breast cancer (TNBC). However, the efficacy is plateaued due to toxicities, chemoresistance and metastasis. Hence, the development of new therapies that provide long-term cover is needed. Brusatol, a natural quassinoid, has been implicated to inhibit the migration and proliferation of metastatic cells in lung and liver carcinoma, but its efficacy in TNBC has not been explored. The growth inhibitory activity on TNBC cells was measured using MTT assay and flow cytometry. Epithelial to mesenchymal transition (EMT) and apoptotic markers were quantified using western blotting. The caspases using Calorimetric assay. Brusatol along with paclitaxel showed an enhanced growth inhibitory activity and a combined synergistic effect. In addition, brusatol was also observed to inhibit the invasion, migratory potential of TNBC cells. Mechanistically, brusatol and its combination were observed to decrease the matrix metalloproteinase (MMP) and a modest increase in the reactive oxygen species (ROS) production. Furthermore, brusatol treatment activated both intrinsic and extrinsic pathways with morphological changes of apoptosis in TNBC cells. This is the first in vitro report demonstrating antineoplastic, anti-EMT and synergistic activity of brusatol and in combination with paclitaxel in TNBC cell. Further in-vivo studies are needed to substantiate the above findings.

Molecular Targeting of RRM2, NF-κB, and Mutant TP53 for the Treatment of Triple-Negative Breast Cancer

Doxorubicin and other anthracycline derivatives are frequently used as part of the adjuvant chemotherapy regimen for triple-negative breast cancer (TNBC). While effective, doxorubicin is known for its off-target and toxic side effect profile, particularly with respect to the myocardium, often resulting in left ventricular (LV) dysfunction and congestive heart failure when used at cumulative doses exceeding 400 mg/m2. Previously, we have observed that the ribonucleotide reductase subunit M2 (RRM2) is significantly overexpressed in estrogen receptor (ER) negative cells as compared with ER-positive breast cancer cells. Here we inhibited RRM2 in ER-negative breast cancer cells as a target for therapy in this difficult to treat population. We observed that through the use of Didox, a ribonucleotide reductase inhibitor, the reduction in RRM2 was accompanied by reduced NF-κB activity in vitro. When Didox was used in combination with doxorubicin, we observed significant downregulation of NF-κB proteins accompanied by reduced TNBC cell proliferation. As well, we observed that protein levels of mutant p53 were significantly reduced by Didox or combination therapy in vitro. Xenograft studies showed that combination therapy was found to be synergistic in vivo, resulting in a significantly reduced tumor volume as compared to doxorubicin monotherapy. Additionally, the use of Didox was also found to ameliorate the toxic myocardial effects of doxorubicin in vivo as measured by heart mass, left ventricular diameter, and serum troponin T levels. The data present a novel and promising approach for the treatment of TNBC that merits further clinical evaluation in humans.

Abstract 6430: Redefining therapy regimens for triple negative breast cancer - Exploiting the epigenetic effects of eribulin action

Abstract While the advent of targeted therapy has led to vast improvements in outcomes for certain types of breast cancer, the standard of care for triple negative breast cancer (TNBC) still remains chemotherapy. A major clinical hurdle in the successful management of this disease is the eventual development of therapeutic resistance and disease relapse in more aggressive forms. Our research centers on the improving therapeutic outcomes for TNBC through the induction of tumor differentiation. Current therapy regimens for TNBC involve the administration of anthracyclines such as doxorubicin, and taxanes such as paclitaxel, typically in combination. When patients present with advanced or metastatic disease, or upon relapse following treatment with an anthracycline or a taxane, eribulin is administered as third-line therapy. We observe that treatment of TNBC cell lines such as SUM159, BT549 or Hs578T, or mouse models such as MMTV-PyMT or C3(1)-Tag with either doxorubicin, paclitaxel or eribulin results in the emergence of resistant subpopulations of cells. In the case of doxorubicin and paclitaxel, the resistant clones exhibit mesenchymal properties due to therapy-induced epithelial-to-mesenchymal transition (EMT). In the case of eribulin, however, these resistant subpopulations are epithelial in nature, having undergone mesenchymal-to-epithelial transition (MET). Our work sheds like on the epigenetic mechanisms that drive eribulin-induced changes to cell state, providing insights into its non-antimitotic actions. Assaying the chromatin landscape of eribulin-resistant vs. paclitaxel-resistant cells by ATAC-Seq reveals genome-wide repression of transcription upon treatment with eribulin. This is accompanied by a severe loss in H3K4me3 that suppresses the transcription of thousands of promoter regions. By carrying out thermal proteome profiling, we identify targets of eribulin that could be responsible for its effects in epigenomic reprogramming and induction of differentiation. Surprisingly, doxorubicin- or paclitaxel-resistant cells are no longer susceptible to subsequent treatment with eribulin, do not undergo MET, and remain difficult to eliminate. Eribulin-resistant cells, however, owing to their epithelial phenotype, are eliminated by subsequent treatment with other drugs including doxorubicin and paclitaxel. Current treatment regimens in the clinic administer eribulin only to patients that have previously received anthracyclines and/or taxanes, never in the treatment-naive or neoadjuvant setting. Our findings highlight the importance of designing treatment regimens based on a clear understanding of the effects of individual drugs on cellular phenotype, including altered responsiveness induced by prior lines of therapy. Our work has major implications for the clinical use and the sequence of administration of chemotherapy with the potential to rapidly alter the course of tumor progression in TNBC by altering treatment regimens. Citation Format: Meisam Bagheri, Aisha Mohamed, Nevena B. Ognjenovic, Diwakar R. Pattabiraman. Redefining therapy regimens for triple negative breast cancer - Exploiting the epigenetic effects of eribulin action [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6430.

Neoadjuvant chemotherapy regimens for triple-negative breast cancer: Insights from network meta-analysis.

e12635 Background: The best regimen for neoadjuvant chemotherapy for triple-negative breast cancer (TNBC) is unknown. Recent studies have shown promising data that adding carboplatin improves the rate of pathological complete response (pCR) in TNBC). Therefore, we performed a network meta-analysis to define the overall most effective neoadjuvant systemic therapy for TNBC. Methods: We searched MEDLINE, Cochrane Library, and EMBASE from inception to December 2019 for studies comparing the efficacy of different neoadjuvant regimens in patients with TNBC. We performed a network meta-analysis comparing the regimens in the selected studies using the random-effects model. We focused on anthracycline (A), bevacizumab (B), everolimus (E), and platinum salts (Pl) because these are the medications commonly added to taxane based regimens. All studies contained a taxane regimen. We analyzed the rate of pCR (ypT0/is, N0) and the incidence of grade 3-4 neutropenia, thrombocytopenia, nausea/vomiting, and diarrhea. Results: We identified a total of 13 randomized control trials for this analysis. We compared eight different classes of regimens. We found that regimens containing Pl were significantly superior to Pl non-containing regimens for the rate of pCR. (risk ratio (RR) for pCR [95% confidence interval (CI)]: 1.66 [1.19-2.31], 1.37 [1.14-1.64], and 1.36 [1.14-1.62] for no medications of special interest vs Pl, A vs A+Pl, and A+B vs A+B+Pl, respectively). Regimens containing B were associated with a significantly better rate of pCR. (RR for pCR [95% CI]: 1.24 [1.06-1.46] and 1.23 [1.01-1.51] for A vs A+B and A+Pl vs A+B+Pl, respectively) In contrast, adding A into the regimen did not improve the rate of pCR. In the safety analysis, regimens containing Pl were associated with a significantly higher incidence of grade 3-4 neutropenia and thrombocytopenia. Regimens containing B showed a higher incidence of grade 3-4 nausea/vomiting, and diarrhea. Conclusions: For TNBC, adding Pl or B into the neoadjuvant therapy regimen brings about a higher pCR rate, though they are associated with an increase in hematologic or gastrointestinal complications. The benefit of adding A to the neoadjuvant chemotherapy regimen for TNBC is not apparent.

Efficacy and safety of neoadjuvant chemotherapy regimens for triple-negative breast cancer: a network meta-analysis.

Different neoadjuvant chemotherapies are available for triple-negative breast cancer (TNBC). Here, we performed a network meta-analysis to evaluate the pathological complete response (pCR) benefit and safety of treatment regimens. Pairwise and Bayesian network meta-analyses were performed to compare direct and indirect evidence, respectively. Twenty-three studies involving 12 regimens namely standard chemotherapeutic agents, bevacizumab (B)-, platinum salts (P)-, B plus P (BP)-, poly(ADP-ribose) polymerase inhibitors (Pi)-, P plus Pi (PPi)-, capecitabine (Ca)-, gemcitabine (Ge)-, zoledronic acid (Za)-, everolimus (E)-, P plus E (PE)-, and gefitinib (G)-containing regimens. The results showed that P-, B-, PPi-, and Za-containing regimens achieved higher pCR than standard chemotherapeutic agents. BP-containing regimens had a better pCR than B-containing regimens. In indirect comparisons, Za-, BP-, P-, and B-containing regimens were the top four strategies with the highest probability for pCR. Benefit-risk analysis showed that B-containing regimens had the highest acceptability of being the best treatment for better pCR achievement with fewer SAEs. The addition of P, B, BP, PPi, and Za to standard chemotherapeutic agents enhanced the pCR, but a balance between efficacy and safety should be carefully considered. B-containing regimens might be the best choice for neoadjuvant chemotherapy due to its better efficacy and tolerability.

Pathological complete response rate (pCR) in thirty-three women with triple-negative breast cancer (TNBC) treated with a neoadjuvant carboplatin-based regimen.

e12099 Background: The purpose of this retrospective case series was to assess pCR rate, progression-free survival and prognostic factors in TNBC. Methods: We reviewed medical records for 33 consecutive female patients with TNBC (41% node+) treated between July 2015 and April 2018 with neoadjuvant paclitaxel 80 mg/m2 IV weekly plus concurrent carboplatin (AUC 4) every 3 weeks for a total of 12 weeks followed by dose-dense doxorubicin 60 mg/m2 plus cyclophosphamide 600 mg/m2 every 2 weeks for a total of 4 cycles. Surgical pathology was studied to determine the presence or absence of residual disease at surgery. Age at treatment, tumor stage, subsequent hospitalizations, and genetic testing were recorded. Patients with residual disease were treated with adjuvant capecitabine 1500 mg PO BID, one week on, one week off, for 6 monthly cycles ± radiation; some patients with a complete pathological response also received postoperative radiation. Results: Among 33 patients, 17 patients had pCR (52%, median age 58 yrs), 10 had a partial response and 6 had no response or progression (median age 63.5). After surgery 24 patients received radiation therapy (XRT). There were 6 hospitalizations, 3 that were treatment related, 2 for neutropenic fever and one for renal failure induced by carboplatin; all 3 resulted in chemotherapy dose reductions; all 3 had pCR. 3 progressions/recurrences were recorded: 2 after treatment and 1 progression during treatment. Two deaths occurred, 1 secondary to progressive disease. Median progression free survival time was 8.5 months (range 0.1 to 24.0 mos). Median time since diagnosis is 16.7 months (range 8.1 to 37.6 mos). There was no significant difference in the median age of patients who had a pCR compared with patients with residual disease. Conclusions: We observed pCR in patients with TNBC treated with a pre-operative carboplatin-containing regimen (superior to historical pCR rates in patients receiving taxanes and anthracyclines only). Although there are insufficient data to demonstrate increased overall survival, we show an improvement in prognosis with a carboplatin-containing regimen for TNBC.

Abstract 3836: Imbalanced nucleotide metabolism sensitizes breast cancer cells to anthracyclines

Abstract Introduction. Triple negative breast cancer (TNBC) makes up 15% of breast cancers and is associated with poor prognosis. TNBC treatment remains hampered by early visceral metastasis and lymph node involvement at the time of diagnosis and limited effective therapeutic options. Advances in treatment that translate to significant improvements in outcome have been painstakingly incremental, despite advances in research technologies and TNBC subtyping. We have identified deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumor DNA from the genotoxic misincorporation of uracil during treatment with anthracyclines that are commonly used in the FEC chemotherapy regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP). This reaction has the dual function of providing dUMP for thymidylate synthase (TS) as part of the thymidylate biosynthesis pathway and of maintaining low intracellular dUTP pools. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporated into DNA. dUTPase is being evaluated as a target in cancer therapy alongside TS-targeted therapies and uracil misincorporation is believed to be a potential mechanism of cytotoxicity. Hypothesis: Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by anthracyclines represents a novel strategy to induce TNBC cell lethality. Methods. Inhibition of dUTPase (DUTi) was carried out using SMARTpool siRNA or small molecule inhibition. The effect of DUTi in combination with anthracyclines was determined by growth inhibition and clonogenics assays. DNA damage was assessed by Western blot, immune-fluorescent foci detection, and flow cytometry. Nucleotide metabolites were quantified by LC/MS. Results. DUTi significantly sensitised TNBC cell lines to doxorubicin and epirubicin. In the MDA-MB-231 cancer cells, loss of dUTPase expression resulted in a synergistic 80% reduction in survival compared to 0.075µM doxorubicin alone at 40%. This decrease in survival correlated with increased activation of proteins involved in DNA damage response, including γ-H2AX(Ser139), a marker for dsDNA breaks. We anticipate that quantification of nucleotides will show an imbalance of dUTP:dTTP, subsequently leading to uracil misincorporation and resulting cell death. Discussion. These results suggest that repair of anthracycline-induced DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a potential novel strategy to enhance the efficacy of anthracyclines. This shows the potential advantage of a dUTPase inhibitor being added to current chemotherapy regimens for TNBC. Future work will elucidate the mechanism of sensitisation and in vivo translation of combination therapy for TNBC. Citation Format: Craig Davison, Olivier P. Chevallier, Catherine Knowlson, Melanie McKechnie, Robbie Carson, Jaime Esteve, Richard Wilson, Robert D. Ladner, Melissa J. LaBonte (Wilson). Imbalanced nucleotide metabolism sensitizes breast cancer cells to anthracyclines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3836.

Abstract 5470: IKK-epsilon supports anchorage independent growth via alternative NF-kB signaling in triple-negative breast cancer

Abstract Metastatic breast cancer carries a poor prognosis despite the success of newly targeted therapies and treatment options remain especially limited for the subtype of triple negative breast cancer (TNBC). Several signaling pathways, including NF-κB, are altered in TNBC. Given that ΙΚΚε behaves as an oncogene in breast cancer we hypothesized that IKKε regulates classical or alternative NF-κB signaling to control diverse oncogenic functions in TNBC. Vector expression and RNA interference were used to investigate the functional role of IKKε in triple-negative breast cancer cells. Viability, protein expression, NF-κB binding activity, invasion, anoikis, and spheroid formation were examined in cells expressing high or low levels of IKKε in conjunction with alternative NF-κB p52 RNA interference or MEK inhibition. Our data show that p52 protein levels and binding activity are inversely proportional to ΙΚΚε. Moreover, knockdown of IKKε leads to increased expression of p52 and the p52-regulated gene CXCL1. This interaction was confirmed by CHiP-PCR experiments showing increased binding of p52 in the CXCL1 promoter of cells with IKKε knockdown relative to control. We further found that IKKε and MEK were required for growth in anchorage supportive conditions whereas p52 was required in anchorage resistant conditions. Western blots confirm diminished IKKε expression and enhanced p52 processing in anchorage resistant conditions relative to anchorage supportive conditions. In this model, IKKε and MEK cooperate to support growth of cancer cells in the attached, anchored environment, whereas p52 is required for growth in unattached 3-D environment. These studies reveal novel information about the role of IKKε in TNBC and highlight the adaptability of NF-κB signaling in maintaining cancer cell survival under different growth conditions. Current studies are underway to clarify the mechanism of IKKε regulation of p52. A better understanding of the diversity of NF-κB signaling may ultimately improve the development of novel therapeutic regimens for TNBC. Citation Format: Carrie D. House, Valentina Grajales, Michelle Ozaki, Elizabeth Jordan, Helmae Wubneh, Danielle Kimble, Marianne Kim, Christina M. Annunziata. IKK-epsilon supports anchorage independent growth via alternative NF-kB signaling in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5470.

I\u039a\u039a\u03b5 cooperates with either MEK or non-canonical NF-kB driving growth of triple-negative breast cancer cells in different contexts

BackgroundMetastatic breast cancer carries a poor prognosis despite the success of newly targeted therapies. Treatment options remain especially limited for the subtype of triple negative breast cancer (TNBC). Several signaling pathways, including NF-κB, are altered in TNBC, and the complexity of this disease implies multi-faceted pathway interactions. Given that IKKε behaves as an oncogene in breast cancer, we hypothesized that IKKε regulates NF-κB signaling to control diverse oncogenic functions in TNBC.MethodsVector expression and RNA interference were used to investigate the functional role of IKKε in triple-negative breast cancer cells. Viability, protein expression, NF-κB binding activity, invasion, anoikis, and spheroid formation were examined in cells expressing high or low levels of IKKε, in conjunction with p52 RNA interference or MEK inhibition.ResultsThis study found that non-canonical NF-κB p52 levels are inversely proportional to ΙΚΚε, and growth of TNBC cells in anchorage supportive, high-attachment conditions requires IKKε and activated MEK. Growth of these cells in anchorage resistant conditions requires IKKε and activated MEK or p52. In this model, IKKε and MEK cooperate to support overall viability whereas the p52 transcription factor is only required for viability in low attachment conditions, underscoring the contrasting roles of these proteins.ConclusionsThis study illustrates the diverse functions of IKKε in TNBC and highlights the adaptability of NF-κB signaling in maintaining cancer cell survival under different growth conditions. A better understanding of the diversity of NF-κB signaling may ultimately improve the development of novel therapeutic regimens for TNBC.

Abstract 4506: UPR biomarker expression correlates with inhibition of migration of TNBC cells treated with low doses of chemotherapy

Abstract Much attention has been directed to the study of triple-negative breast cancer (TNBC) because of its short disease-free interval from diagnosis and more aggressive course in the metastatic setting. TNBC also lacks specific target receptors for treatment. Third-generation metronomic chemotherapy using available agents that target DNA repair complexes (i.e., taxanes), and cell proliferation (i.e., anthracycline) have shown promise results. However, specific regimens for TNBC at the different disease stages have not been clearly defined. Therefore, researchers are seeking molecular biomarkers to predict response to current chemotherapeutic agents. In a previous study of TNBC, we demonstrated that low doses of anthracyclines and taxanes, which are known to increase endoplasmic reticulum stress, increased tumor cell expression of glucose-regulated protein 78 (GRP78), a key regulator of the unfolding protein response (UPR), resulting in tumor cell apoptosis. GRP78 gene expression is upregulated via CREB2L1, an endoplasmic reticulum transmembrane transcription factor and member of the CREB/ATF family of transcription factors. CREB3L1 is a metastatic suppressor and functions as a transducer of UPR. The aim of the present study was to investigate the effect of low doses of doxorubicin and paclitaxel on UPR activation in metastatic TNBC cells by determining CREB3L1 protein expression in correlation to cell-surface GRP78 expression. Furthermore, we related CREB3L1 and cell-surface GRP78 expression with the migration potential of the cells in response to treatment. We found that metronomic doses of doxorubicin significantly induced CREB3L1 and cell surface GRP78 expression in TNBC cells. CREB3L1 increased by 2.5-fold in MDAMB231 and by 3-fold in MDAMB468 (p<0.04); GRP78 increased by 3.7-fold and 6.1-fold, respectively (p<0.01). Similar results were obtained with low doses of paclitaxel. A strong correlation was observed between CREB3L1 and cell-surface GRP78 protein expression. The increased expression of both proteins in MDAMB231 (highly metastatic) and MDAMB468 (moderated metastatic) was associated with significant inhibition of the migration capacity of the treated TNBC cells. In contrast, the migration capacity of treated estrogen-positive MCF7 cells (non-metastatic) and Her2- positive BT474 cells (highly metastatic) was poorly inhibited, coinciding with a non-significant increase in CREB3L1 and GRP78 expression. The results demonstrated that the drug effect via the UPR was specific for TNBC and unrelated to the metastatic profile of the cells. This study is the first stage in the identification of UPR-related biomarkers of beneficial outcome of metronomic chemotherapy for TNBC. Future mechanistic studies should focus on the possible use of cell surface GRP78 and CREB3L1 as targets for combination therapies. Citation Format: Annat Raiter, Julia Lipovetsky, Britta Hardy, Rinat Yerushalmi. UPR biomarker expression correlates with inhibition of migration of TNBC cells treated with low doses of chemotherapy [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 4506. doi:10.1158/1538-7445.AM2017-4506

Enhancing accrual to chemotherapy trials for patients with early stage triple-negative breast cancer: a survey of physicians and patients.

The optimal chemotherapy regimen for patients with early stage triple-negative breast cancer (TNBC) remains unknown. The purpose of the study is to survey physicians and breast cancer patients about preferred chemotherapy regimens for early stage TNBC and clinical trial strategies. A standardised online questionnaire was developed and circulated to medical oncologists known to treat breast cancer. A separate questionnaire was given to patients who had received chemotherapy for breast cancer. The questionnaire was completed by 41/84 medical oncologists (48.8% response rate) and 74 patients. The most commonly used neoadjuvant and adjuvant chemotherapy regimens for TNBC were dose-dense doxorubicin and cyclophosphamide (AC)-paclitaxel (P), dose-dense AC followed by weekly P and fluorouracil, epirubicin, cyclophosphamide-docetaxel (FEC-D). The majority of medical oncologists (80%) would be willing to enrol patients in trials evaluating the most effective chemotherapy regimen for TNBC. Oncologists favoured a three arm trial design comparing currently available standard of care treatments (36%) and trials of novel or non-standard of care agents 22% (9/41). Sixty percent (41/74) of patients indicated that they would be willing to be enrolled in trials evaluating various adjuvant regimens for TNBC. Both oncologists and patients were interested in novel consent approaches such as using the integrated consent model. Optimisation of chemotherapy for TNBC is an important and unmet clinical need. It is apparent that various chemotherapy regimens are used for patients with early stage TNBC. The majority of medical oncologists and patients are interested in entering trials to optimise chemotherapy choices.

Integrated in vivo genetic and pharmacologic screening identifies co-inhibition of EGRF and ROCK as a potential treatment regimen for triple-negative breast cancer.

Breast cancer is the second most common cause of cancer-related deaths worldwide among women. Despite several therapeutic options, 15% of breast cancer patients succumb to the disease owing to tumor relapse and acquired therapy resistance. Particularly in triple-negative breast cancer (TNBC), developing effective treatments remains challenging owing to the lack of a common vulnerability that can be exploited by targeted approaches. We have previously shown that tumor cells have different requirements for growth in vivo than in vitro. Therefore, to discover novel drug targets for TNBC, we performed parallel in vivo and in vitro genetic shRNA dropout screens. We identified several potential drug targets that were required for tumor growth in vivo to a greater extent than in vitro. By combining pharmacologic inhibitors acting on a subset of these candidates, we identified a synergistic interaction between EGFR and ROCK inhibitors. This combination effectively reduced TNBC cell growth by inducing cell cycle arrest. These results illustrate the power of in vivo genetic screens and warrant further validation of EGFR and ROCK as combined pharmacologic targets for breast cancer.

Cbcsg-10: Adjuvant capecitabine in combination with docetaxel and cyclophosphamide plus epirubicin for triple negative breast cancer.

1012Background: Standard adjuvant chemotherapy regimens for triple-negative breast cancer (TNBC) contain taxane and anthracycline. We aimed to investigate the efficacy and safety of integrate capec...

Abstract 1955: IKKϵ maintains MEK activation and suppresses non-canonical NF-κB signaling in triple-negative breast cancer

Abstract Metastatic breast cancer carries a poor prognosis despite the success of newly targeted therapies. Treatment options remain especially limited for the subtype of triple negative breast cancer (TNBC). Several signaling pathways, including NF-κB, are altered in TNBC, and the complexity of this disease implies multifaceted pathway interactions and compensatory signaling. We hypothesized that a subset of TNBCs expresses IKKϵ to promote a specific NF-κB signaling program that can enhance metastatic potential. Our data suggest that IKKϵ supports MEK activation and suppresses non-canonical signaling by the p52 NF-κB transcription factor. We found that NF-κB p52 expression is inversely proportional to activated MEK. We further demonstrate that IKKϵ and MEK cooperate to support viability and invasion potential under adherent conditions, whereas the p52 transcription factor supports viability under non-adherent conditions, underscoring the contrasting roles of these proteins. This study illustrates the diverse roles of NF-κB signaling components in TNBC, and highlights the adaptability of NF-κB in maintaining cancer cell survival under different microenvironments. A better understanding of the diversity of NF-κB signaling may ultimately improve the development of novel therapeutic regimens for TNBC. Citation Format: Carrie House, Valentina Grajales, Helmae Wubneh, Danielle Kimble, Marianne Kim, Christina Annunziata. IKKϵ maintains MEK activation and suppresses non-canonical NF-κB signaling in triple-negative breast cancer. [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 1955. doi:10.1158/1538-7445.AM2015-1955