Drug-resistant Triple-negative Breast Cancer Research Articles

Bufalin Induces Programmed Necroptosis in Triple-Negative Breast Cancer Drug-Resistant Cell Lines through RIP1/ROS-Mediated Pathway.

To explore the effect and mechanism of action of bufalin in triple-negative breast cancer (TNBC) drug-resistant cell lines. The normal human mammary epithelial cell line, TNBC cell line, TNBC adriamycin-resistant cell line, and TNBC docetaxel-resistant cell line were treated with different doses of bufalin (0-1,000 nmol/L) at different time points (0-72 h). Propidium iodide staining, AV-FITC/PI double staining, Hoechst 33342/PI double staining and transmission electron microscopy (TEM) were used to evaluate the death patterns of the cell lines. Bufalin killed the TNBC cell line and its drug-resistant cell lines in a dose/time-dependent manner (all P<0.01). After treatment with bufalin for 24 h, the adriamycin-resistant cell line showed a co-existing pattern of necroptosis and apoptosis. However, at 48 h, necroptosis was the main manifestation. After treatment with bufalin, the expressions of tumor necrosis factor α, phospho-tumor necrosis factor receptor 1, phospho-receptor interacting protein 1 and c-caspase 3 increased (all P<0.01), the killing effect of bufalin could be mostly inhibited by NEC-1, and by z-VAD-fmk (both P<0.01). Besides, the intracellular reactive oxygen species (ROS) levels increased considerably (P<0.01), the antioxidant N-acetyl cysteine or Nec-1 could inhibit the increase of ROS level and the killing effect of bufalin (all P<0.01). The adriamycin-resistant cell line exhibited necroptosis characteristic after 48 h of bufalin treatment under TEM. Bufalin could induce necroptosis through RIP1/ROS-mediated pathway to kill the drug-resistant TNBC cell lines. This finding provides critical experimental data and theoretical basis for the clinical application of bufalin to overcome the difficulties in the treatment of TNBC.

Evaluation of Extracellular Matrix Composition to Improve Breast Cancer Modeling.

The development of resistance to therapy is a significant obstacle to effective therapeutic regimens. Evaluating the effects of oncology drugs in the laboratory setting is limited by the lack of translational models that accurately recapitulate cell-microenvironment interactions present in tumors. Acquisition of resistance to therapy is facilitated, in part, by the composition of the tumor extracellular matrix (ECM), with the primary current in vitro model using collagen I (COL I). Here we seek to identify the prevalence of COL I-enhanced expression in the triple-negative breast cancer (TNBC) subtype. Furthermore, we identify if methods of response to therapy are altered depending on matrix composition. We demonstrated that collagen content varies in patient tumor samples across subtypes, with COL I expression dramatically increased in typically less aggressive estrogen receptor (ER)-positive(ER+)/progesterone receptor (PGR)-positive (PGR+) cancers irrespective of patient age or race. These findings are of significance considering how frequently COL I is implicated in tumor progression. In vitro analyses of ER+ and ER-negative (ER-) cell lines were used to determine the effects of ECM content (collagen I, collagen IV, fibronectin, and laminin) on proliferation, cellular phenotype, and survival. Neither ER+ nor ER- cells demonstrated significant increases in proliferation when cultured on these ECM substrates. ER- cells cultured on these substrates were sensitized to both chemotherapy and targeted therapy. In addition, MDA-MB-231 cells expressed different morphologies, binding affinities, and stiffness across these substrates. We also demonstrated that ECM composition significantly alters transcription of senescence-associated pathways across ER+ and ER- cell lines. Together, these results suggest that complex matrix composites should be incorporated into in vitro tumor models, especially for the drug-resistant TNBC subtype. Impact statement The importance of tumor extracellular matrix (ECM) in disease progression is often inadequately represented in models of breast cancer that rely heavily on collagen I and Matrigel. Through immunohistochemistry analysis of patient breast tumors, we show a wide variation in collagen content based on subtype, specifically a repression of fibril collagens in the receptor negative subtype, irrespective of age and race. We also demonstrated that tumor ECM composition alters cellular elasticity and oncogenic pathway activation demonstrating that physiologically relevant three-dimensional models of breast cancer should include an ECM that is subtype specific.

Thidiazuron suppresses breast cancer via targeting miR-132 and dysregulation of the PI3K-Akt signaling pathway mediated by the miR-202-5p-PTEN axis.

Chemo-resistance and metastasis are the most common causes of breast cancer recurrence and death. Thidiazuron (TDZ) is a plant growth regulator (phytohormone) whose biological effects on humans and animals has not yet been determined. In this study, we investigated the anticancer activity of this phytohormone on the drug resistant-triple negative breast cancer cell line MDA-MB-231. Treatment of the breast cancer cells with TDZ (1-50μmol/L) caused more stressful environment and induced a significant increase in active caspase-positive cells. In addition, TDZ treatment (5 and 10μmol/L) significantly attenuated the migration and the invasiveness of these highly metastatic cancer cells. Mechanistically, TDZ reduces cancer progression and invasiveness by targeting miR-202-5p, which stimulates the expression of phosphatase and tensin homolog (PTEN), the tumor suppressor that downregulates the PI3K-Akt signaling pathway. Treatment with TDZ significantly upregulates miRNA-132, the suppressor of breast cancer proliferation, which is also implicated in dysregulation of the TEN-Akt-NFκB signaling pathway. Interestingly, our molecular docking analysis revealed a potential non-covalent interaction between TDZ and Akt, PTEN, and PI3K. These findings suggest that TDZ suppresses breast cancer metastasis by targeting miRNA-132, the miR-202-5p-PTEN axis, and the PI3K-Akt signaling pathway downstream.

Gemcitabine resistance in triple‑negative breast cancer cells can be reverted by &lt;em&gt;Drosophila melanogaster&lt;/em&gt; deoxyribonucleoside kinase in the nucleus or cytosol

The development of drug resistance to chemotherapeutic agents has consistently presented a challenge in terms of the treatment of patients with triple-negative breast cancer (TNBC). In the present study, gemcitabine (dFdC)-resistant TNBC cells were established, and the effects of lentivirus-deoxyribonucleoside kinase (dNK) and a mutated form of dNK (lentivirus-dNKmut) on reversing the acquired drug resistance in dFdC-resistant TNBC cells were explored. Quantitative PCR and western blotting experiment results suggested that Drosophila melanogaster (Dm)-dNK was stably expressed in the lentivirus-infected MDA-MB-231 and MDA-MB-231R cells in the nucleus or cytosol, and autoradiography experiments revealed similar levels of enzymatic activity in the cells expressing dNK or dNKmut. In vitro cytotoxicity assay revealed that the IC50 values of dFdC were decreased 30~50-fold in the dFdC-resistant MDA-MB-231 cells following lentiviral transfection with dNK or dNKmut, and this effect was associated with a significantly increased rate of apoptosis compared with the cells transfected with the negative control lentivirus. In conclusion, Dm-dNK in the nucleus or cytosol may be a potential candidate for reversing acquired dFdC resistance in TNBC cells, which may form the basis of novel strategies for the treatment of patients with drug-resistant TNBC.

Aspirin attenuates YAP and \u03b2-catenin expression by promoting \u03b2-TrCP to overcome docetaxel and vinorelbine resistance in triple-negative breast cancer

The use of aspirin has been associated with reduced breast cancer risk, but it is litter known if aspirin overcomes chemoresistance in triple-negative breast cancer (TNBC). Herein, we demonstrated that changes in the expression of Yes-associated protein (YAP) and β-catenin might be a promising predictive biomarker for neoadjuvant chemotherapy sensitivity in TNBC patients. Inhibition of YAP or β-catenin enhanced the cytotoxicity of the anti-microtubule agents docetaxel and vinorelbine against drug-resistant TNBC cells as well as the sensitivity of these cells to the agents in vitro and in vivo. Interestingly, aspirin not only significantly inhibited the growth of TNBC cells, but also attenuated YAP and β-catenin expression by upregulating the E3 ubiquitin ligase β-TrCP to abolished docetaxel and vinorelbine resistance. The combination of aspirin and docetaxel or vinorelbine remarkably inhibited the growth of drug-resistant TNBC cells in vitro and in vivo. Moreover, TNBC patients with high YAP and/or β-catenin expression had a higher risk of relapse or mortality than patients with low YAP and/or β-catenin expression. Collectively, our study discovered a novel role of aspirin based on its anticancer effect, and put forward some possible mechanisms of chemoresistance in TNBC. The combined use of aspirin and anti-microtubule drugs presented several promising therapeutic approaches for TNBC treatment.

Aspirin attenuates YAP and β-catenin expression by promoting β-TrCP to overcome docetaxel and vinorelbine resistance in triple-negative breast cancer.

e13099 Background: YAP and β-catenin play critical roles in the carcinogenesis of several cancers, but their functions in chemoresistance remain unclear in triple-negative breast cancer (TNBC). Herein, we examined the expression and function of the two proteins in the chemoresistance of TNBC. Methods: We examined the expression levels of YAP and β-catenin in TNBC samples by immunohistochemistry (IHC) and their roles in prognosis. Dynamic changes in the two proteins in TNBC tissues were determined by RT-PCR. CCK-8 assays, colony formation assays, flow cytometry, migration assays and nude mouse models were used to investigate the functions of YAP and β-catenin in drug-resistant TNBC cells. The involved signalling pathways were detected by western blot assays. Results: TNBC patients with high YAP and/or β-catenin expression had a higher risk of relapse or mortality than patients with low YAP and/or β-catenin expression, and changes in the expression of the two proteins might be a promising predictive biomarker for neoadjuvant chemotherapy sensitivity. Inhibition of YAP or β-catenin enhanced the cytotoxicity of the anti-microtubule agents docetaxel and vinorelbine against TNBC cells in vitro and in vivo. Moreover, YAP and β-catenin were upregulated in docetaxel- or vinorelbine-resistant TNBC cells, while inhibition of YAP or β-catenin enhanced the sensitivity of drug-resistant cells. Interestingly, aspirin, a kind of acetylsalicylic acid, reversed the drug resistance of cells and in combination with aspirin and docetaxel or vinorelbine significantly inhibited the growth of drug-resistant TNBC cells. The involved mechanism is that aspirin impaired YAP or β-catenin expression by upregulating the E3 ubiquitin ligase β-TrCP. Conclusions: Both YAP and β-catenin act as key biomarkers of prognosis and anti-microtubule drug resistance in TNBC, and the combined use of aspirin and anti-microtubule drugs inhibits YAP and β-catenin, presenting several promising therapeutic approaches for TNBC treatment.

Abstract P3-06-14: Inhibition of PRKCQ enhances chemosensitivity of triple negative breast cancer by regulating EMT and Bim

Abstract Background/Rationale: While some patients with triple negative breast cancer (TNBC) achieve long-term remission with standard-of-care chemotherapy, many have cancers that are chemotherapy resistant, putting them at risk for metastatic recurrence. There is also a lack of targeted therapies for TNBC, highlighting the need for novel therapeutic strategies for this subtype. PRKCQ, a member of the novel protein kinase C family, is preferentially expressed in TNBC compared to other breast cancer subtypes and we previously reported that PRKCQ expression is sufficient to promote oncogenic activities (growth-factor independent growth, anoikis resistance and migration). In addition, PRKCQ suppression inhibits growth of TNBC tumor xenografts. We sought to determine if PRKCQ expression modulation impacts sensitivity of triple negative breast cancer cells to standard of care chemotherapy and whether PRKCQ inhibition could be a strategy to enhance chemosensitivity of drug-resistant TNBC. Methods: We determined the effects of modulating PRKCQ expression, using PRKCQ cDNA or shRNA vectors, on sensitivity of TNBC cells to the apoptotic effects of standard-of-care chemotherapy, including those cancers that are relatively chemotherapy resistant at baseline. We also determined the mechanisms by which PRKCQ regulates sensitivity to chemotherapy. Results: Increased PRKCQ expression in non-transformed MCF-10A breast epithelial cells promotes resistance to the apoptosis-inducing effects of Doxorubicin or Paclitaxel treatment. These effects are dependent on PRKCQ kinase activity as a catalytically inactive PRKCQ failed to promote resistance. Downregulation of PRKCQ or inhibition of its kinase activity enhanced sensitivity of TNBC cells to chemotherapy. PRKCQ impacts chemotherapy sensitivity of TNBC by regulating epithelial-mesenchymal transition (EMT) and expression of Bim, a pro-apoptotic Bcl2 family protein. Conclusions. PRKCQ promotes resistance of TNBC to standard of care chemotherapies by promoting EMT and suppressing pro-apoptotic Bim. Inhibiting PRKCQ expression or activity sensitizes TNBC cells to chemotherapy and could be an effective strategy to overcome chemotherapy resistance of a subset of triple negative breast cancers, thereby improving patient outcomes. Citation Format: Hanna Yoko Irie, Jessica H. Byerly, Elisa R. Port. Inhibition of PRKCQ enhances chemosensitivity of triple negative breast cancer by regulating EMT and Bim [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-14.

Abstract P6-03-16: Role of autophagy in CD24-mediated TNBC drug resistance

Abstract Background: Triple-negative breast cancer (TNBC) is defined by lack of estrogen and progesterone receptors and absence of HER2 amplification. Compared with other breast cancer subtypes, TNBC is more aggressive with more frequent metastasis especially involving visceral and central nervous system. Currently, the only available standard adjuvant treatment is chemotherapy. However, mortality rate remains high despite chemotherapy. To improve treatment, it is crucial to develop novel strategies to predict and overcome drug resistance. Our pervious study showed that CD44/CD24 expressions were associated with drug sensitivity and CD44+/CD24+ TNBC was shown to be resistant to docetaxel. In this study, we aimed to investigate the regulatory mechanism involved in CD24-mediated taxane resistance in TNBC. Methods: In this study, we used flow cytometry to determine the CD24 expressions in 7 TNBC cell lines. Western blot was used to demonstrate the levels of CD24 and LC3B-II (a marker for cellular autophagy activity) expressions in TNBC cell lines. CD24 expressions in TNBC cell lines were manipulated by target-specific shRNA and gene overexpression approaches. Cell apoptosis assay was used to determine docetaxel drug sensitivity in cells with manipulated CD24 expression. Using the same assay, autophagy inhibitors were used to further evaluate the involvement of autophagy in CD24-mediated drug resistance. To investigate the possible regulatory mechanism, autophagy related signaling pathway Akt/mTOR was studied in CD24 modified TNBC cells. Results: In this study, the relationship between CD24 expression and autophagy activities in TNBC was evaluated. LC3B-II protein expression was used to determinate autophagy activities. We have shown that TNBC cell lines with high CD24 expressions are associated with increased LC3B-II expression. Suppression of autophagy in CD24 high TNBC cells improves drug sensitivities toward docetaxel. We further demonstrated that knocking down CD24 in TNBC cells suppressed LC3B-II expression and improved docetaxel sensitivity. In addition, gene overexpression of CD24was found to enhance docetaxel resistance in TNBC cells. Our study has also shown that docetaxel increases the activation of Akt/mTOR signaling pathway in drug-resistant TNBC cells. Knocking down CD24 increased Akt/mTOR activation, and decreased the expression of PP2A and LC3B-II in TNBC. Conclusions: CD24 expression is associated with docetaxel sensitivity and autophagy activity in TNBC cells. Drug sensitivity of CD44+CD24+/high TNBC cells is regulated through autophagy related signaling pathway. Taken together, CD24 mediated TNBC drug resistance is at least partially through autophagy activity. Citation Format: Lihong Huo, Xinyu Deng, Xiang Huang, Helena R Chang. Role of autophagy in CD24-mediated TNBC drug resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-16.

Curcumin-Loaded Solid Lipid Nanoparticles Bypass P-Glycoprotein Mediated Doxorubicin Resistance in Triple Negative Breast Cancer Cells.

Multidrug resistance (MDR) is a critical hindrance to the success of cancer chemotherapy. The main thing responsible for MDR phenotypes are plasma-membranes associated with adenosine triphosphate (ATP) Binding Cassette (ABC) drug efflux transporters, such as the P-glycoprotein (Pgp) transporter that has the broadest spectrum of substrates. Curcumin (CURC) is a Pgp inhibitor, but it is poorly soluble and bioavailable. To overcome these limitations, we validated the efficacy and safety of CURC, loaded in biocompatible solid lipid nanoparticles (SLNs), with or without chitosan coating, with the goal of increasing the stability, homogeneous water dispersibility, and cellular uptake. Both CURC-loaded SLNs were 5–10-fold more effective than free CURC in increasing the intracellular retention and toxicity of doxorubicin in Pgp-expressing triple negative breast cancer (TNBC). The effect was due to the decrease of intracellular reactive oxygen species, consequent inhibition of the Akt/IKKα-β/NF-kB axis, and reduced transcriptional activation of the Pgp promoter by p65/p50 NF-kB. CURC-loaded SLNs also effectively rescued the sensitivity to doxorubicin against drug-resistant TNBC tumors, without signs of systemic toxicity. These results suggest that the combination therapy, based on CURC-loaded SLNs and doxorubicin, is an effective and safe approach to overcome the Pgp-mediated chemoresistance in TNBC.

Kinase Chemodiversity from the Arctic: The Breitfussins.

In this work, we demonstrate that the indole-oxazole-pyrrole framework of the breitfussin family of natural products is a promising scaffold for kinase inhibition. Six new halogenated natural products, breitfussin C-H (3 - 8) were isolated and characterized from the Arctic, marine hydrozoan Thuiaria breitfussi. The structures of two of the new natural products were also confirmed by total synthesis. Two of the breitfussins (3 and 4) were found to selectively inhibit the survival of several cancer cell lines, with the lowest IC50 value of 340 nM measured against the drug-resistant triple negative breast cancer cell line MDA-MB-468, while leaving the majority of the tested cell lines not or significantly less affected. When tested against panels of protein kinases, 3 gave IC50 and Kd values as low as 200 and 390 nM against the PIM1 and DRAK1 kinases, respectively. The activity was confirmed to be mediated through ATP competitive binding in the ATP binding pocket of the kinases. Furthermore, evaluation of potential off-target and toxicological effects, as well as relevant in vitro ADME parameters for 3 revealed that the breitfussin scaffold holds promise for the development of selective kinase inhibitors.

Abstract 5186: Discovery of a novel histone deacetylase 6 inhibitor that kills drug-resistant breast cancer

Abstract Cytotoxic chemotherapy is the standard of care for patients with triple negative breast cancer (TNBC). Most patients with advanced TNBC progress after chemotherapy and die from metastatic disease. Currently, no established targeted therapeutics or biomarkers of outcome/response have been clinically approved in the context of TNBC. The aggressive nature of TNBC is reflected by an increased likelihood of distant recurrence and death within five years following primary intervention and a shorter survival once diagnosed with metastatic disease. To identify novel therapeutics for the treatment of chemoresistant TNBC, we performed a high-throughput screen to identify small molecules that are cancer selective but can kill drug-resistant TNBC cells. We screened a total of 30,000 compounds in duplicate across the TNBC cell line MDA-MB-321 in which the pro-death proteins BAX/BAK were knocked down, and the non-transformed "normal" MCF10A cell line. There was a hit rate of 0.3% in the screen and 85 compounds were retested in the validation cherry pick screen. From this screen 18 compounds were further validated with low-throughput assessment for mitochondrial independent killing and selectivity for cancer cells. To identify the mechanism of action of the lead compound we used a genetic approach to generate an RNAi signature for the compound, which indicated the lead compound was most likely that of histone deacetylase inhibitor (HDAC). Using an in vitro HDAC inhibitor screen, we identified that the compound specifically inhibited HDAC6. Using a series of cell based assays, we determined that treatment of cells with the compound BAS-2 resulted in the same phenotypical response (inhibition of migration, invasion and 3D spheroid formation) as HDAC6 knockout. To identify substrates of HDAC6 in cells we analyzed the proteome for increased acetylation by mass spectrometry following HDAC6 knockout or inhibition with the specific inhibitor, BAS-2. Remarkably, we identified a series of new substrates for HDAC6 that are common in both the HDAC6 knockdown and BAS-2 treated cells. In conclusion, we have identified a novel HDAC6 specific inhibitor that selectively kills cancer cells independent of mitochondrial apoptosis, alters acetylation of over a 50 common proteins and inhibits spheroid formation of TNBC. Citation Format: Catríona Dowling, Eugene Dillion, Michael Hemann, Gerard Cagney, Anthony Letai, Triona Ni Chonghaile. Discovery of a novel histone deacetylase 6 inhibitor that kills drug-resistant breast cancer [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 5186.

Hsa-miRNA-143-3p Reverses Multidrug Resistance of Triple-Negative Breast Cancer by Inhibiting the Expression of Its Target Protein Cytokine-Induced Apoptosis Inhibitor 1 In Vivo.

PurposeMultidrug resistance (MDR) remains a major obstacle in the treatment of triple-negative breast cancer (TNBC) with conventional chemotherapeutic agents. A previous study demonstrated that hsa-miRNA-143-3p plays a vital role in drug resistance of TNBC. Downregulation of hsa-miRNA-143-3p upregulated the expression of its target protein cytokine-induced apoptosis inhibitor 1 (CIAPIN1) in order to activate MDR, while upregulation of hsa-miRNA-143-3p effectively enhances the sensitivity of drug-resistant TNBC cells to chemotherapeutics. The present study aimed to further verify these findings in vivo.MethodsWe established a hypodermic tumor nude mice model using paclitaxel-resistant TNBC cells. We expressed ectopic hsa-miRNA-143-3p under the control of a breast cancer-specific human mammaglobin promoter that guided the efficient expression of exogenous hsa-miRNA-143-3p only in breast cancer cells. Thereafter, we overexpressed hsa-miRNA-143-3p in xenografts using a recombinant virus system and quantified the expression of hsa-miRNA-143-3p, CIAPIN1 protein, and proteins encoded by related functional genes by western blot.ResultsWe successfully completed the prospective exploration of the intravenous virus injection pattern from extensive expression to targeted expression. The overexpression of hsa-miRNA-143-3p significantly alleviated chemoresistance of TNBC by inhibiting viability. In addition, we observed that the expression of CIAPIN1 as a hsa-miRNA-143-3p target protein was remarkably decreased.ConclusionWe partly illustrated the mechanism underlying the hsa-miRNA-143-3p/CIAPIN1 drug resistance pathway. HsamiRNA-143-3p as a tumor suppressive microRNA may be a novel target to effectively reverse MDR of TNBC in vivo.

MYC Inhibition Depletes Cancer Stem-like Cells in Triple-Negative Breast Cancer.

There is mounting evidence that cancer stem-like cells (CSC) are selectively enriched in residual tumors after anticancer therapies, which may account for tumor recurrence and metastasis by regenerating new tumors. Thus, there is a critical need to develop new therapeutic agents that can effectively eliminate drug-resistant CSCs and improve the efficacy of cancer therapy. Here, we report that Triptolide (C1572), a small-molecule natural product, selectively depletes CSCs in a dose-dependent fashion in human triple-negative breast cancer (TNBC) cell lines. Nanomolar concentrations of C1572 markedly reduced c-MYC (MYC) protein levels via a proteasome-dependent mechanism. Silencing MYC expression phenocopied the CSC depletion effects of C1572 and induced senescence in TNBC cells. Limited dilution assays revealed that ex vivo treatment of TNBC cells with C1572 reduced CSC levels by 28-fold. In mouse xenograft models of human TNBC, administration of C1572 suppressed tumor growth and depleted CSCs in a manner correlated with diminished MYC expression in residual tumor tissues. Together, these new findings provide a preclinical proof of concept defining C1572 as a promising therapeutic agent to eradicate CSCs for drug-resistant TNBC treatment. Cancer Res; 77(23); 6641-50. ©2017 AACR.

Reversal of drug-resistance by noscapine chemo-sensitization in docetaxel resistant triple negative breast cancer

Multidrug resistance (MDR) is a major impediment to cancer treatment. Here, for the first time, we investigated the chemo-sensitizing effect of Noscapine (Nos) at low concentrations in conjunction with docetaxel (DTX) to overcome drug resistance of triple negative breast cancer (TNBC). In vitro experiments showed that Nos significantly inhibited proliferation of TNBC wild type (p < 0.01) and drug resistant (p < 0.05) TNBC cells. Nos followed by DTX treatment notably increased the cell viability (~1.3 fold) markedly (p < 0.05) in 3D models compared to conventional 2D systems. In vivo oral administration of Nos (100 mg/kg) followed by intravenous DTX (5 mg/kg) liposome treatment revealed regression of xenograft tumors in both wild type (p < 0.001) and drug-resistant (p < 0.05) xenografts. In wild type xenografts, combination of Nos plus DTX group showed 5.49 and 3.25 fold reduction in tumor volume compared to Nos and DTX alone groups, respectively. In drug-resistant xenografts, tumor volume was decreased 2.33 and 1.41 fold in xenografts treated with Nos plus DTX significantly (p < 0.05) compared to Nos and DTX alone respectively and downregulated the expression of anti-apoptotic factors and multidrug resistance proteins. Collectively, chemo-sensitizing effect of Nos followed by DTX regime provide a promising chemotherapeutic strategy and its significant role for the treatment of drug-resistant TNBC.

MYC and MCL1 Cooperatively Promote Chemotherapy-Resistant Breast Cancer Stem Cells via Regulation of Mitochondrial Oxidative Phosphorylation

Most patients with advanced triple-negative breast cancer (TNBC) develop drug resistance. MYC and MCL1 are frequently co-amplified in drug-resistant TNBC after neoadjuvant chemotherapy. Herein, we demonstrate that MYC and MCL1 cooperate in the maintenance of chemotherapy-resistant cancer stem cells (CSCs) in TNBC. MYC and MCL1 increased mitochondrial oxidative phosphorylation (mtOXPHOS) and the generation of reactive oxygen species (ROS), processes involved in maintenance of CSCs. A mutant of MCL1 that cannot localize in mitochondria reduced mtOXPHOS, ROS levels, and drug-resistant CSCs without affecting the anti-apoptotic function of MCL1. Increased levels of ROS, a by-product of activated mtOXPHOS, led to the accumulation of HIF-1α. Pharmacological inhibition of HIF-1α attenuated CSC enrichment and tumor initiation invivo. These data suggest that (1) MYC and MCL1 confer resistance to chemotherapy by expanding CSCs via mtOXPHOS and (2) targeting mitochondrial respiration and HIF-1α may reverse chemotherapy resistance in TNBC.

Nanoparticle-Mediated Mitochondrial Damage Induces Apoptosis in Cancer.

Detouring of conventional DNA damaging anticancer drugs into mitochondria to damage mitochondrial DNA is evolving as a promising strategy in chemotherapy. Inhibiting single target in mitochondria would eventually lead to the emergence of drug resistance. Moreover, targeting mitochondria selectively in cancer cells, keeping them intact in healthy cells, remains a major challenge. Herein, triphenylphosphine (TPP)-coated positively charged 131.6 nm spherical nanoparticles (NPs) comprised of α-tocopheryl succinate (TOS, inhibitor of complex II in electron transport chain) and obatoclax (Obt, inhibitor of Bcl-2) were engineered. The TOS-TPP-Obt-NPs entered into acidic lysosomes via macropinocytosis, followed by lysosomal escape and finally homed into mitochondria over a period of 24 h. Subsequently, these TOS-TPP-Obt-NPs triggered mitochondrial outer membrane permeabilization (MOMP) by inhibiting antiapoptotic Bcl-2, leading to Cytochrome C release. These TOS-TPP-Obt-NPs mediated mitochondrial damage induced cellular apoptosis through caspase-9 and caspase-3 cleavage to show improved efficacy in HeLa cells. Moreover, TOS-TPP-Obt-NPs induced MOMP in drug-resistant triple negative breast cancer cells (MDA-MB-231), leading to remarkable efficacy, compared to the combination of free drugs in higher drug concentrations. Results presented here clearly stimulate the usage of multiple drugs to perturb simultaneously diverse targets, selectively in mitochondria, as next-generation cancer therapeutics.

MUC1-C Stabilizes MCL-1 in the Oxidative Stress Response of Triple-Negative Breast Cancer Cells to BCL-2 Inhibitors.

Aberrant expression of myeloid cell leukemia-1 (MCL-1) is a major cause of drug resistance in triple-negative breast cancer (TNBC) cells. Mucin 1 (MUC1) is a heterodimeric oncoprotein that is aberrantly overexpressed in most TNBC. The present studies show that targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in TNBC cells with silencing or pharmacologic inhibition with GO-203 is associated with downregulation of MCL-1 levels. Targeting MUC1-C suppresses the MEK → ERK and PI3K → AKT pathways, and in turn destabilizes MCL-1. The small molecules ABT-737 and ABT-263 target BCL-2, BCL-XL and BCL-w, but not MCL-1. We show that treatment with ABT-737 increases reactive oxygen species and thereby MUC1-C expression. In this way, MUC1-C is upregulated in TNBC cells resistant to ABT-737 or ABT-263. We also demonstrate that MUC1-C is necessary for the resistance-associated increases in MCL-1 levels. Significantly, combining GO-203 with ABT-737 is synergistic in inhibiting survival of parental and drug resistant TNBC cells. These findings indicate that targeting MUC1-C is a potential strategy for reversing MCL-1-mediated resistance in TNBC.

RIP1K and RIP3K provoked by shikonin induce cell cycle arrest in the triple negative breast cancer cell line, MDA-MB-468: necroptosis as a desperate programmed suicide pathway.

Resistance to cell death and reprogramming of metabolism are important in neoplastic cells. Increased resistance to apoptosis and recurrence of tumors are the major roadblocks to effective treatment of triple negative breast cancer. It has been thought that execution of necroptosis involves ROS generation and mitochondrial dysfunction in malignant cells. In this study, the effect of shikonin, an active substance from the dried root of Lithospermum erythrorhizon, on the induction of necroptosis or apoptosis, via RIP1K-RIP3K expressions has been examined in the triple negative breast cancer cell line. The expression levels of RIP1K and RIP3K, caspase-3 and caspase-8 activities, the levels of ROS, and mitochondrial membrane potential have been studied in the shikonin-treated MDA-MB-468 cell line. An increase in the ROS levels and a reduction in mitochondrial membrane potential have been observed in the shikonin-treated cells. Cell death has mainly occurred through necroptosis with a significant increase in the RIP1K and RIP3K expressions, and characteristic morphological changes have been observed. In the presence of Nec-1, caspase-3 mediating apoptosis has occurred in the shikonin-treated cells. The current findings have revealed that shikonin provoked mitochondrial ROS production in the triple negative breast cancer cell line, which works as a double-edged executioner's ax in the execution of necroptosis or apoptosis. The main route of cell death induced by shikonin is RIP1K-RIP3K-mediated necroptosis, but in the presence of Nec-1, apoptosis has prevailed. The present results shed a new light on the possible treatment of drug-resistant triple negative breast cancer.

Abstract 691: Down-regulation of Wnt10b following targeted theranostic nanoparticles in a patient derived xenograft model of triple negative breast cancer

Abstract BACKGROUND: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease with a poor clinical prognosis. While the primary therapeutic option for TNBC patients is pre-operative neo-adjuvant chemotherapy followed by surgical resection, about 60% of patients develop drug resistance and have a very high incidence of tumor recurrence and poorer prognosis. Given its poor survival and lack of available targeted therapeutics, the development of novel treatment options for chemo-resistant TNBC is critical. Cancer progenitor cells or stem-like cells (CSCs) are generally thought to contribute to the drug-resistant phenotype of TNBC which are typically identified as CD44+/CD24-. CSCs can also rely on activation of stem cell signaling through the Wnt/ β-catenin pathway. Components of the Wnt pathway, including Wnt ligands, have been shown to be over-expressed in human breast cancers. Our lab has developed urokinase plasminogen activator receptor (uPAR) targeted theranostic magnetic iron oxide nanoparticles (IONPs) carrying therapeutic agents that are clinically beneficial to TNBC patients such as doxorubicin (Dox). uPAR is an ideal target due to its high expression in malignant tumors including aggressive breast cancer tissues and tumor stromal cells that are enriched in TNBC tissues. METHODS: We generated patient derived xenografts of drug-resistant TNBC for testing the therapeutic efficacy of uPAR-targeted nanoparticle-drugs from tumor fragments from patients obtained from an ongoing Phase II clinical trial at Winship Cancer Institute. Histological analysis of post-chemotherapy TNBC with or without uPAR-targeted IONP-Dox was conducted using immunohistochemistry and immunofluorescence techniques. RESULTS: We have previously reported that treatment with uPAR-targeted IONP-Dox caused tumor growth inhibition following systemic delivery. We further investigated the effect of uPAR-targeted IONP on Wnt10b, a ligand of the Wnt signaling pathway. We observed that uPAR-targeted-IONP-Dox treatment decreases Wnt10b expression relative to the untreated and conventional Dox groups. We also investigated the level of CD44 expression, which is a target gene of the Wnt signaling pathway. We observed that Wnt10b and CD44 expression was co-localized and that treatment with uPAR-targeted IONP-Dox decreased the level of CD44 expression that is comparable to the level of decrease in Wnt10b expression. CONCLUSION: Our findings show that uPAR-targeted theranostic IONP can effectively target the Wnt activated CSC population. It is likely that uPAR expressing tumor cells are potential drug resistant cell populations with breast cancer stem-like cell properties. Our results support further investigation of theranostic nanoparticles as a viable therapeutic approach to overcome drug resistance in TNBC. Citation Format: Jasmine Miller-Kleinhenz, Weiping Qian, Ruth O'Regan, Toncred Styblo, Amelia Zelnak, Andrew Wang, Lily Yang. Down-regulation of Wnt10b following targeted theranostic nanoparticles in a patient derived xenograft model of triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 691. doi:10.1158/1538-7445.AM2014-691

Abstract B045: uPAR-targeted theranostic nanoparticles effectively decrease expression of IGF-1R and Ki67 in drug-resistant triple-negative breast cancer human xenograft

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype that affects about 20% of breast cancer patients and has a high incidence in young African American women. Currently the only therapeutic options for TNBC patients is chemotherapy, however, more than 60% of TNBC patients are highly resistant to chemotherapeutic treatment. The development of novel therapeutic approaches is essential to enable treatment of patients with chemo-resistant TNBC. Our lab focuses on the development of receptor-targeted theranostic nanoparticles. We have developed a class of urokinase plasminogen activator receptor (uPAR) targeted theranostic magnetic iron oxide nanoparticles (IONPs) carrying therapeutic agents that are clinically beneficial to TNBC patients, including DNA damaging agents, such as doxorubicin (Dox) or cisplatin (Cis). We are targeting uPAR because it is highly expressed in malignant cancers including aggressive breast cancer tissues and tumor stromal cells that are enriched in TNBC tissues. Methods: Tumor fragments from TNBC patients, obtained from a Phase II clinical trial that is ongoing at the Breast Cancer Clinic in the Winship Cancer Institute, were implanted in the mammary fat pad of SCID mice. The tumor xenografts grew to about 1 cm in diameter in 12 to 15 weeks. Tumor fragments were then implanted into the mammary fat pad and passaged through nude mice for testing the therapeutic efficacy of the uPAR-targeted nanoparticle-drugs. Histological analysis of post-chemotherapy TNBC and primary tumor xenograft tissues with or without uPAR-targeted IONP-Dox was conducted using immunohistochemistry and dual immunofluorescence techniques. Results: We have previously reported that the uPAR-targeted IONP-Dox causes tumor growth inhibition following systemic delivery. Using CD44 and uPAR as biomarkers we also reported histological analysis of post-chemotherapy TNBC and primary tumor xenograft tissues showed high levels of CD44+/CD24- tumor cells and up-regulation of uPAR. Primary TNBC xenografts treated with conventional Dox had increased levels of CD44+/CD24- cells and strong uPAR expression. Conversely, the tumor tissues from the uPAR-targeted IONP-Dox treated group showed decreased levels of CD44+/CD24- and uPAR positive cells. We have further investigated the biomarkers insulin-like growth factor 1 receptor (IGF-1R) and Ki67. IGF-1R has been shown to be overexpressed in more than 50% of TNBCs and may be associated with the drug-resistant TNBC phenotype, while Ki67 is a conventional biomarker for proliferation. We observed that uPAR-targeted IONP- Dox treatment decreased IGF-1R expression compared to the control group and the group treated with conventional Dox. We also observed that Ki67 expression was decreased in the uPAR-targeted IONP- Dox treatment group compared to control group and the group treated with conventional Dox. Conclusions: Results of our study demonstrated that uPAR-targeted theranostic IONPs selectively delivers therapeutic payloads of the chemotherapeutic drug Dox to inhibit the growth of uPAR positive tumor cells. It is likely that uPAR expressing tumor cells are an aggressive and potentially drug resistant cell population that overexpress breast cancer stem-like cell biomarker (CD44+/CD24-) as well as up-regulation of a growth factor receptor, IGF-1R. These results support further investigation of theranostic nanoparticles as an approach to overcome drug resistance in TNBC. Citation Format: Jasmine M. Miller-Kleinhenz, Hongyu Zhou, Weiping Qian, Ruth O'Regan, Amelia Zelnak, Toncred Styblo, Lily Yang. uPAR-targeted theranostic nanoparticles effectively decrease expression of IGF-1R and Ki67 in drug-resistant triple-negative breast cancer human xenograft. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B045.