BackgroundFat grafting is used to restore breast defects after surgical resection of breast tumors. Supplementing fat grafts with adipose tissue-derived stromal/stem cells (ASCs) is proposed to improve the regenerative/restorative ability of the graft and retention. However, long term safety for ASC grafting in proximity of residual breast cancer cells is unknown. The objective of this study was to determine the impact of human ASCs derived from abdominal lipoaspirates of three donors, on a human breast cancer model that exhibits early metastasis.Methodology/Principal FindingsHuman MDA-MB-231 breast cancer cells represents “triple negative” breast cancer that exhibits early micrometastasis to multiple mouse organs [1]. Human ASCs were derived from abdominal adipose tissue from three healthy female donors. Indirect co-culture of MDA-MB-231 cells with ASCs, as well as direct co-culture demonstrated that ASCs had no effect on MDA-MB-231 growth. Indirect co-culture, and ASC conditioned medium (CM) stimulated migration of MDA-MB-231 cells. ASC/RFP cells from two donors co-injected with MDA-MB-231/GFP cells exhibited a donor effect for stimulation of primary tumor xenografts. Both ASC donors stimulated metastasis. ASC/RFP cells were viable, and integrated with MDA-MB-231/GFP cells in the tumor. Tumors from the co-injection group of one ASC donor exhibited elevated vimentin, matrix metalloproteinase-9 (MMP-9), IL-8, VEGF and microvessel density. The co-injection group exhibited visible metastases to the lung/liver and enlarged spleen not evident in mice injected with MDA-MB-231/GFP alone. Quantitation of the total area of GFP fluorescence and human chromosome 17 DNA in mouse organs, H&E stained paraffin sections and fluorescent microscopy confirmed multi-focal metastases to lung/liver/spleen in the co-injection group without evidence of ASC/RFP cells.ConclusionsHuman ASCs derived from abdominal lipoaspirates of two donors stimulated metastasis of MDA-MB-231 breast tumor xenografts to multiple mouse organs. MDA-MB-231 tumors co-injected with ASCs from one donor exhibited partial EMT, expression of MMP-9, and increased angiogenesis.

Src kinase is elevated in breast tumors that are ER/PR negative and do not overexpress HER2 but clinical trials with Src inhibitors have demonstrated little activity. The present study evaluated preclinical efficacy of a novel peptidomimetic compound, KX-01 (KX2-391), that exhibits dual action as a Src and pretubulin inhibitor. KX-01 was evaluated as a single agent and in combination with paclitaxel in MDA-MB-231, MDA-MB-157, and MDA-MB-468 human ER/PR/HER2-negative breast cancer cells. Treatments were evaluated by growth/apoptosis, isobologram analysis, migration/invasion assays, tumor xenograft volume, metastasis, and measurement of Src, FAK, microtubules, Ki67, and microvessel density. KX-01 inhibited cell growth in vitro and in combination with paclitaxel resulted in synergistic growth inhibition. KX-01 resulted in a dose dependent inhibition of MDA-MB-231 and MDA-MB-157 tumor xenografts (1 and 5 mg/kg, BID). KX-01 inhibited activity of Src and downstream mediator FAK in tumors that was coincident with reduced proliferation and angiogenesis, and increased apoptosis. KX01 also resulted in microtubule disruption in tumors. Combination of KX-01 with paclitaxel resulted in significant regression of MDA-MB-231 tumors and reduced metastasis to mouse lung and liver. KX-01 is a potently active Src/pretubulin inhibitor that inhibits breast tumor growth and metastasis. As ER/PR/HER2-negative patients are candidates for paclitaxel therapy, combination with KX-01 may potentiate antitumor efficacy in management of this aggressive breast cancer subtype.

Human ASCs did not alter growth of human head/neck cancer cells or tumor xenografts, but stimulated migration and early micrometastasis to mouse brain.

Unlike breast cancer that is positive for estrogen receptor-α (ERα), there are no targeted therapies for triple negative breast cancer (TNBC). ERα is silenced in TNBC through epigenetic changes including DNA methylation and histone acetylation. Restoring ERα expression in TNBC may sensitize patients to endocrine therapy. Expression of c-Src and ERα are inversely correlated in breast cancer suggesting that c-Src inhibition may lead to re-expression of ERα in TNBC. KX-01 is a peptide substrate-targeted Src/pretubulin inhibitor in clinical trials for solid tumors. KX-01 (1 mg/kg body weight-BID) inhibited growth of tamoxifen-resistant MDA-MB-231 and MDA-MB-157 TNBC xenografts in NUDE mice that was correlated with Src kinase inhibition. KX-01 also increased ERα mRNA and protein, as well as increased the ERα targets progesterone receptor (PR), pS2 (TFF1), cyclin D1 (CCND1) and c-myc (MYC) in MDA-MB-231 and MDA-MB-468 but not MDA-MB-157 xenografts. MDA-MB-231 and MDA-MB-468 tumors exhibited reduction in mesenchymal markers (vimentin, β-catenin) and increase in epithelial marker (E-cadherin) suggesting mesenchymal-to-epithelial transition (MET). KX-01 sensitized MDA-MB-231 and MDA-MB-468 tumors to tamoxifen growth inhibition and tamoxifen repression of the ERα targets pS2, cyclin D1 and c-myc. Chromatin immunoprecipitation (ChIP) of the ERα promoter in KX-01 treated tumors demonstrated enrichment of active transcription marks (acetyl-H3, acetyl-H3Lys9), dissociation of HDAC1, and recruitment of RNA polymerase II. Methylation-specific PCR and bisulfite sequencing demonstrated no alteration in ERα promoter methylation by KX-01. These data demonstrate that in addition to Src kinase inhibition, peptidomimetic KX-01 restores ERα expression in TNBC through changes in histone acetylation that sensitize tumors to tamoxifen. Implications: Src kinase/pretubulin inhibitor KX-01 restores functional ERα expression in ERα- breast tumors, a novel treatment strategy to treat triple-negative breast cancer.

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PURPOSE:Stromal vascular fraction (SVF) is used in breast reconstruction in FDA clinically approved studies. In SVF, adipose derived stem cells (ASC) may play a role in wound healing. In patients following chemotherapy for breast cancer, poor soft tissue wound healing is a major problem. Previous work in our laboratory demonstrated that direct exposure to Paclitaxel (PTX) has cytotoxic effects on ASCs by decreasing proliferation and multipotency. This study examined human ASCs pretreated with PTX and then exposed to a washout period (no PTX) of 8 days to evaluate 1) the influence on ASC function after chemotherapy cessation and 2) influence on tumor cell growth in ASC co-culture. METHODS:IRB-approved human ASCs were isolated and MDA-MB-231 cells (breast cancer cell line) were treated with PTX at different concentrations. Cell proliferation, viability, and migration were measured by growth curves, MTT assays, and scratch assays. ASCs were cultured in derivative-specific differentiation media with or without PTX (1uM) for 3 weeks. Adipogenic and endothelial differentiation were measured by quantitative RT-PCR, Oil-Red-O staining and cord formation on Matrigel, respectively. MDA-MB-231 cells and ASCs were co-cultured at equal cell numbers. ASC conditional media were collected every 3 days from ASCs, both no-PTX treatment control and PTX treated -washout groups.

Unlike breast cancer that is positive for estrogen receptor α (ERα) or amplified/overexpressed HER2/neu, there are no targeted therapies for triple negative breast cancer (TNBC). ERα is silenced in TNBC through epigenetic changes including DNA methylation and histone acetylation. Restoring ERα expression in TNBC tumors may sensitize patients to endocrine therapy. Expression of c-Src kinase and ERα are inversely correlated in breast cancer suggesting that c-Src inhibition may lead to re-expression of ERα in TNBC. KX-01 is a peptide substrate targeted Src kinase/pretubulin inhibitor in clinical trials for solid tumors. At low dose, KX-01 is an effective Src kinase inhibitor in breast tumor xenografts in mice, but does not affect microtubules. MDA-MB-231 and MDA-MB-468 TNBC xenografts in NUDE mice were treated with low dose (1 mg/kg b.wt. BID) KX-01 for 40 days. KX-01 treatment decreased Src kinase activity in tumors and increased ERα mRNA and protein. Immunohistochemistry and Western blot demonstrated that the epithelial markers progesterone receptor and E-cadherin were increased, whereas the mesenchymal markers vimentin and nuclear β-catenin were decreased, suggesting a mesenchymal to epithelial transition (MET) in the tumors. At the ERα promoter in MDA-MB-231 tumors, KX-01 treatment led to enrichment of transcriptionally active (acetyl-H3, acetyl-H3Lys9) chromatin marks. There was no change in ERα promoter methylation as assessed by methylation PCR analysis and bisulfite sequencing. KX-01 treatment did not alter histone deacetylase 1 (HDAC1) levels or activity in tumors, but did result in HDAC1 dissociation from the ERα promoter, and a concomitant recruitment of RNA Polymerase II as assessed by chromatin immunoprecipitation (ChIP). Tamoxifen-resistant MDA-MB-231 xenografts in NUDE mice were treated with vehicle, tamoxifen alone (5 mg pellet; 60 day release), KX-01 alone (1 mg/kg b.wt. BID), or KX-01 + tamoxifen for 40 days. KX-01 alone and KX-01 + tamoxifen reduced tumor volume by 59% and 70%, respectively, compared to vehicle. Tumor volume for the KX-01 + tamoxifen group was significantly reduced compared to the KX-01 alone group (P = .023), and tumor weight of the KX-01 + tamoxifen group was 32% lower compared to the KX-01 alone group (P< 0.01). Only the KX-01 + tamoxifen group exhibited reduced levels of ERα targets pS2, c-Myc and cyclin D1 indicating that estradiol signaling was attenuated by tamoxifen only in tumors treated with KX-01. In MDA-MB-468 tumors, tamoxifen alone (10 mg pellet; 60 day release) and KX-01 alone (1 mg/kg b.wt. BID) had no effect on tumor volume compared to vehicle, but KX-01 + tamoxifen reduced tumor volume 67% compared to vehicle (P = 0.0025). Collectively, these data demonstrate that the peptidomimetic Src inhibitor KX-01 can restore ERα expression in TNBC through changes in histone acetylation that sensitize TNBC tumors to endocrine therapy (tamoxifen). Citation Format: Murali Anbalagan, Mei Sheng, Brian Fleischer, David Hangauer, Brian G. Rowan. Peptidomimetic Src kinase inhibitor KX-01 sensitizes estrogen receptor α-negative breast tumor xenografts to tamoxifen by inducing ERα re-expression. [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 3859.