Orthotopic Triple-negative Breast Cancer Model Research Articles

Abstract 3908: The combination of NVP-BEZ235 & endostatin exerts synergistic anticancer activity against triple-negative breast cancer in vitro and in vivo

Abstract Background: Triple-negative breast cancer(TNBC) take up 15% of invasive breast cancer. TNBC has no effective molecular targets & unfavourable prognosis. We investigated the effects of NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, a combine with or not an angiogenesis inhibitor Endostatin in the orthotopic TNBC model & explored its anti-tumor & anti-angiogenesis mechanisms. Material & Methods: Assess sensitivity of the cells to the drug by MTT assays.Orthotopic xenografts were performed for tumor growth and survival studies. Additionally, Immunohistochemistry for VEGF and MVD(CD34) was performed using the EnVision/HRP technique,Serum VEGF was detected by ELISA method. Results: BEZ235 effectively inhibited cell proliferation in vitro & provided additive effects when combine with Endostatin.Treatment with BEZ235 & Endostatin resulted in inhibition of tumor growth & prolongation of survival time of tumor-bearing mice in vivo.Immunohistochemical analysis revealed that intratumoral proliferation & angiogenesis was significantly suppressed. Furthermore, Changing of the number of Serum VEGF support the finding of angiogenesis inhibition too. Conclusion: Our findings suggest that BEZ235 exerts antitumor effects against TNBC & enhances effects of Endostatin through inhibition of cell proliferation & tumor angiogenesis. This approach may represent a promising combination targeted therapy for TNBC treatment. Note: This abstract was not presented at the meeting. Citation Format: Xiaoshan Wang, Xiao Zhang, Fanghua Li. The combination of NVP-BEZ235 & endostatin exerts synergistic anticancer activity against triple-negative breast cancer in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3908.

Pharmacologic inhibition of liver X receptors enhances T cell anti-tumor function in triple negative breast cancer

Abstract While immunotherapies have demonstrated success in solid malignancies, they have limited efficacy in triple negative breast cancer (TNBC). The liver-X-receptors (LXR) are modulators of metabolism and are known to inhibit pro-inflammatory (Th17 and Th1) and increase anti-inflammatory (Tregs) T-cell differentiation. Colorectal and hematologic cancer cells have also been demonstrated to suppress immune function through production of endogenous LXR agonists. Here we show that targeted suppression of LXR activity using the inverse agonist, SR9243, induces immune-mediated tumor destruction in an orthotopic TNBC model. FACs analysis on tumors revealed a significant increase in Th1 and cytotoxic CD8+ T-cells and a decrease in tumor promoting myeloid derived suppressor cells (MDSCs) in response to SR9243. Intratumor immune cell activation coincided with enhanced dendritic cell (DC) lymph node homing and activity. SR9243 also enhanced Th1 CD4+ and CD8+ cell proliferation in the presence of immune-suppressive tumor conditioned media and cancer cell lysates an in vitro model of the tumor-microenvironment. Metabolic analysis of immune cells exposed to LXR-ligands revealed that LXR activation by tumor-produced ligands or synthetic agonists blocks T-cell glycolytic activity essential to immune cell proliferation and activation in response to tumor antigens. Similarly, we observed that LXR activation disrupts de novo cholesterol synthesis which has been shown to be vital to immune synapse formation between T-cells and DCs. Our results suggest that LXR inverse agonists can be used as immunotherapy drugs to treat TNBC by blocking tumor-induced dysregulation of T-cell metabolism.

Breast cancer cell-derived exosomes and macrophage polarization are associated with lymph node metastasis.

Crosstalk between breast cancer and macrophages has potential implications for tumor metastasis. This study investigates macrophage polarization induced by triple-negative breast cancer (TNBC) cell-derived exosomes that promote lymph node (LN) metastasis in orthotopic TNBC models. The MDA-MB-231 cancer cell line expressing the exosomal CD63-red fluorescence (RFP) fusion protein was generated to noninvasively visualize exosome transfer into cancer cells and macrophages. Administration of RFP-tagged exosomes enhanced migration of macrophages and induced macrophage polarization in vitro. In orthotopic TNBC models, noninvasive bioluminescent imaging, ultrasound-guided photoacoustic imaging, and histological analysis revealed that intravenous injection of RFP-tagged exosomes promoted primary tumor growth and axillary LN metastasis in which expression of CD206, a marker or alternatively activated type 2 (M2) macrophages, was significantly higher than expression of NOS2, a marker of classically activated type 1 (M1) macrophages. These results suggest breast cancer cell-derived exosomes stimulate macrophage polarization that creates favorable conditions for LN metastatic processes in TNBC.

Abstract 3849: The antidiabetic drug metformin increases pro-apoptotic effect of TRAIL therapy by reducing X-linked inhibitor of apoptosis protein (XIAP) levels in triple-negative breast cancer cells

Abstract Most metastatic tumors, such as triple negative breast cancer (TNBC) respond poorly to conventional chemotherapy. We have previously demonstrated that targeting TNF-related apoptosis-inducing ligand receptor-2 (TRAIL-R2) might be an effective pathway against metastatic TNBC. However, many TNBCs are resistant to TRAIL receptor agonists. Here we demonstrate that diabetes drug metformin, previously linked to preventing and treating several types of cancer, enhances sensitivity of TNBC cells to recombinant TRAIL and TRAIL-R2 agonistic humanized monoclonal antibody (lexatumumab)-induced apoptosis. We have demonstrated that combination of metformin with lexatumumab or TRAIL augments response to TRAIL therapy and reduce cancer cell viability via induction of apoptosis and inhibiting long-term survival of three different metastatic TNBC cell lines. Intriguingly, metformin treatment also sensitized transformed MCF10A-RasV12 cells, but not untransformed MCF10A-Vector cells, to TRAIL agonist therapy. This effect was associated with reduction of X-linked Inhibitor of Apoptosis Protein (XIAP) levels after metformin treatment. Similar to metformin treatment, inhibition of XIAP by small interfering RNA sensitized breast cancer cells to TRAIL-induced apoptosis. Moreover, metformin treatment in combination with TRAIL more efficiently inhibited primary tumor growth and lung metastases in orthotopic model of TNBC. Overall, our results demonstrate that metformin treatment in combination with targeting TRAIL receptors may be a good strategy to augment the antitumor effects of TRAIL receptors agonistic therapy and provide the foundation for a clinical trial combining dietary metfomin and TRAIL agonists. Citation Format: Elena Strekalova, Dmitry Malin, Vincent Cryns. The antidiabetic drug metformin increases pro-apoptotic effect of TRAIL therapy by reducing X-linked inhibitor of apoptosis protein (XIAP) levels in triple-negative breast cancer cells. [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 3849.

Abstract 1384: CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, reverses the HIF-1α-mediated increase in cancer stem cells caused by bevacizumab in a preclinical model of triple-negative breast cancer

Abstract Antiangiogenic agents are designed to impede the development of new tumor blood vessels, thereby starving tumors of oxygen and nutrients to ultimately block tumor growth. Despite advances made in developing antiangiogenic therapies, clinical and preclinical data suggest that these drugs have limited efficacy in breast cancer patients. Tumors inevitably develop resistance to antiangiogenic agents and patients frequently fail to demonstrate significantly better overall survival. Acquired resistance to antiangiogenic agents has been attributed in part to the induction of intra-tumoral hypoxia and the concomitant stabilization of hypoxia-inducible factor 1α (HIF-1α), a transcription factor that promotes tumor angiogenesis, invasion, metastasis, and cancer stem cell (CSC) self-renewal. In this preclinical model, we demonstrate that CRLX101, an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin (CPT), can reverse the stimulatory effects of hypoxia on the CSC population in an orthotopic triple-negative xenograft model. The payload, camptothecin, a topoisomerase 1 inhibitor, inhibits HIF-1α protein accumulation at concentrations below those that were cytotoxic to tumor cells, and inhibits hypoxia-mediated CSC induction at these low concentrations in vitro. In an orthotopic triple-negative breast cancer model, CRLX101 is synergistic with bevacizumab. Tumor reimplantation experiments confirm that the combination therapy effectively targets both the bulk tumor cell and CSC populations. Results generated from these preclinical studies support the clinical research of combining antiangiogenic agents with CRLX101 to increase patient survival. CRLX101 is currently in phase 2 clinical trials in combination with bevacizumab in both recurrent ovarian cancer and metastatic renal cell carcinoma. Citation Format: Sarah J. Conley, Trenton L. Baker, Joseph P. Burnet, Rebecca L. Thiesen, Douglas Lazarus, Christian G. Peters, Shawn G. Clouthier, Scott Eliasof, Max S. Wicha. CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, reverses the HIF-1α-mediated increase in cancer stem cells caused by bevacizumab in a preclinical model of 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 1384. doi:10.1158/1538-7445.AM2015-1384

αB-Crystallin: A Novel Regulator of Breast Cancer Metastasis to the Brain

Basal-like breast tumors are typically (ER/PR/HER2) triple-negative and are associated with a high incidence of brain metastases and poor clinical outcomes. The molecular chaperone αB-crystallin is predominantly expressed in triple-negative breast cancer (TNBC) and contributes to an aggressive tumor phenotype in preclinical models. We investigated the potential role of αB-crystallin in brain metastasis in TNBCs. αB-crystallin expression in primary breast carcinomas and brain metastases was analyzed by immunohistochemistry among patients with breast cancer with brain metastases. αB-crystallin was overexpressed or silenced in two different TNBC cell lines. The effects on cell adhesion to human brain microvascular endothelial cells (HBMEC) or extracellular matrix proteins, transendothelial migration, and transmigration across a HBMEC/astrocyte coculture blood-brain barrier (BBB) model were examined. In addition, the effects of overexpressing or silencing αB-crystallin on brain metastasis in vivo were investigated using orthotopic TNBC models. In a cohort of women with breast cancer brain metastasis, αB-crystallin expression in primary breast carcinomas was associated with poor overall survival and poor survival after brain metastasis, even among patients with TNBC. Stable overexpression of αB-crystallin in TNBC cells enhanced adhesion to HBMECs, transendothelial migration, and BBB transmigration in vitro, whereas silencing αB-crystallin inhibited these events. αB-crystallin promoted adhesion of TNBC cells to HBMECs, at least in part, through an α3β1 integrin-dependent mechanism. αB-crystallin overexpression promoted brain metastasis, whereas silencing αB-crystallin inhibited brain metastasis in orthotopic TNBC models. αB-crystallin is a novel regulator of brain metastasis in TNBC and represents a potential biomarker and drug target for this aggressive disease.

Abstract 1788: The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition.

Abstract Background: Triple Negative Breast Cancer (TNBC) comprises 15% of all breast cancers, and has a poor prognosis relative to other breast cancer subtypes. Inhibition of poly (ADP-ribose) polymerase (PARP) causes the degeneration of single-strand DNA breaks to more lethal double-strand breaks (DSBs), and also traps PARP-DNA complexes, which must be repaired or bypassed by homologous recombination (HR). BRCA1 plays a critical role in HR and its activity is in part regulated by cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation. PARP inhibition results in synthetic lethality in cells that have impaired HR, such as BRCA-deficient cells. Other CDK family members activate additional components of the HR pathway. Here, we show that HR-proficient TNBC cells can be sensitized to PARP inhibition through use of CDK inhibition to impair BRCA1 function and disrupt HR. Methods: We examined the effects of dinaciclib (CDK1, 2, 5 and 9 inhibitor), γ-irradiation and veliparib (PARP inhibitor) on a panel of BRCA1 wild type TNBC cell lines (MDA-MB-231, MDA-MB-468, and BT549). Levels of phospho-S1497 BRCA1 (pBRCA1; CDK phosphorylation site), γ-H2AX, and RAD51 were measured by western blot to assess effects on HR proteins. A direct assessment of DSB repair was measured using the U2OS-DR-GFP reporter system. Cell viability was measured using Cell-Titer Glo assays. Patient-derived TNBC xenograft models were established in immunocompromised NOD-SCID-IL2γ-/- mice, and implanted orthotopically into cohorts of mice for in vivo efficacy studies. Results: There was a significant reduction in total and pBRCA1 and RAD51 protein levels with increasing concentrations of dinaciclib in all TNBC cell lines. In response to 10 Gy γ-irradiation treatment, pretreatment with dinaciclib (20 nM) reduced the percentage of cells with greater than five BRCA1 foci from 54% to 5% (P < 0.001); and the percentage of cells with greater than 5 RAD51 foci from 26% to 4% (P = 0.001). In U2OS-DR-GFP assays, expression of GFP was detected in 3.4% of the vehicle group and in 1.9% of dinaciclib treated cells. Cell viability assays following five days of dinaciclib, veliparib or the combination revealed in vitro cytotoxic synergy in all three TNBC cell lines. In an orthotopic TNBC model derived from a primary patient sample, the relative tumor volumes over a 30-day treatment period for vehicle, dinaciclib, veliparib and combination- treated mice were 5.6-fold, 3.3-fold, 3.2-fold and 0.66-fold (P < 0.001) that of the initial tumor volume at the start of treatment. Tumor volume decreased only in the combination arm. Conclusion: CDK inhibition effectively inhibits HR, and renders BRCA-proficient TNBC cells sensitive to PARP inhibition. This combination represents an effective strategy for the treatment of TNBC. Citation Format: Shawn F. Johnson, Neil Johnson, David Chi, Benjamin Primack, Alan D. D'Andrea, Elgene Lim, Geoffrey I. Shapiro. The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition. [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 1788. doi:10.1158/1538-7445.AM2013-1788