Triple-negative Breast Cancer Xenograft Mouse Research Articles

Radiosensitizing effect of a novel CTSS inhibitor by enhancing BRCA1 protein stability in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) patients harboring wild-type breast cancer susceptibility gene 1 (BRCA1) account for most TNBC patients but lack adequate targeted therapeutic options. Although radiotherapy (RT) is the primary treatment modality for TNBC patients, radioresistance is one of the major challenges. RT-induced increase in cathepsin S (CTSS) causes radioresistance through suppressing BRCA1-mediated apoptosis of tumor cells, which was induced by CTSS-mediated degradation of BRCA1. Targeting CTSS may provide a novel therapeutic opportunity for TNBC patients. Publicly available data and human tissue microarray slides were analyzed to investigate the relationship between CTSS and BRCA1 in breast cancer patients. A CTSS enzyme assay and in silico docking analysis were conducted to identify a novel CTSS inhibitor. RO5461111 was used first to confirm the concept of targeting CTSS for radiosensitizing effects. The MDA-MB-231 TNBC cell line was used for invitro and invivo assays. Western blotting, promoter assay, cell death assay, clonogenic survival assay, and immunohistochemistry staining were conducted to evaluate novel CTSS inhibitors. CTSS inhibitors were further evaluated for their additional benefit of inhibiting cell migration. A novel CTSS inhibitor, TS-24, increased BRCA1 protein levels and showed radiosensitization in TNBC cells with wild-type BRCA1 and invivo in a TNBC xenograft mouse model. These effects were attributed by BRCA1-mediated apoptosis facilitated by TS-24. Furthermore, TS-24 demonstrated the additional effect of inhibiting cell migration. Our study suggests that employing CTSS inhibitors for the functional restoration of BRCA1 to enhance RT-induced apoptosis may provide a novel therapeutic opportunity for TNBC patients harboring wild-type BRCA1.

LncRNA MCM3AP-AS1 promotes chemoresistance in triple-negative breast cancer through the miR-524-5p/RBM39 axis.

Triple-negative breast cancer (TNBC) is the most lethal subtype of BC, with unfavorable treatment outcomes. Evidence suggests the engagement of lncRNA MCM3AP-AS1 in BC development. This study investigated the action of MCM3AP-AS1 in chemoresistance of TNBC cells. Drug-resistant TNBC cell lines SUM159PTR and MDA-MB-231R were constructed by exposure to increasing concentrations of doxorubicin/docetaxel (DOX/DXL). MCM3AP-AS1 and miR-524-5p expression levels were determined by RT-qPCR. RNA binding motif 39 (RBM39) level was measured using Western blot. Cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry. The targeted binding of miR-524-5p with MCM3AP-AS1 or RBM39 was predicted by ECORI database and validated by dual-luciferase assays. The gain-and-loss of function assays were conducted in cells to investigate the interactions among MCM3AP-AS1, miR-524-5p, and RBM39. TNBC xenograft mouse models were established through subcutaneous injection of MCM3AP-AS1-silencing MDA-MB-231R cells and intraperitoneally administrated with DOX/DXL to verify the role of MCM3AP-AS1 in vivo. MCM3AP-AS1 was upregulated in drug-resistant TNBC cells, and MCM3AP-AS1 silencing could sensitize drug-resistant TNBC cells to chemotherapeutic drugs by promoting apoptosis. MCM3AP-AS1 targeted miR-524-5p. After DOX/DXL treatment, miR-524-5p inhibition partially reversed the effect of MCM3AP-AS1 silencing on inhibiting chemoresistance and promoting apoptosis of drug-resistant TNBC cells. miR-524-5p targeted RBM39. Silencing MCM3AP-AS1 promoted apoptosis via the miR-524-5p/RBM39 axis, thereby enhancing chemosensitivity of drug-resistant TNBC cells. MCM3AP-AS1 knockdown upregulated miR-524-5p, downregulated RBM39, and restrained tumor development in vivo. MCM3AP-AS1 silencing potentiates apoptosis of drug-resistant TNBC cells by upregulating miR-524-5p and downregulating RBM39, thereby suppressing chemoresistance in TNBC.

ANXA2 (annexin A2) is crucial to ATG7-mediated autophagy, leading to tumor aggressiveness in triple-negative breast cancer cells

ABSTRACT Triple-negative breast cancer (TNBC) is associated with a poor prognosis and metastatic growth. TNBC cells frequently undergo macroautophagy/autophagy, contributing to tumor progression and chemotherapeutic resistance. ANXA2 (annexin A2), a potential therapeutic target for TNBC, has been reported to stimulate autophagy. In this study, we investigated the role of ANXA2 in autophagic processes in TNBC cells. TNBC patients exhibited high levels of ANXA2, which correlated with poor outcomes. ANXA2 increased LC3B-II levels following bafilomycin A1 treatment and enhanced autophagic flux in TNBC cells. Notably, ANXA2 upregulated the phosphorylation of HSF1 (heat shock transcription factor 1), resulting in the transcriptional activation of ATG7 (autophagy related 7). The mechanistic target of rapamycin kinase complex 2 (MTORC2) played an important role in ANXA2-mediated ATG7 transcription by HSF1. MTORC2 did not affect the mRNA level of ANXA2, but it was involved in the protein stability of ANXA2. HSPA (heat shock protein family A (Hsp70)) was a potential interacting protein with ANXA2, which may protect ANXA2 from lysosomal proteolysis. ANXA2 knockdown significantly increased sensitivity to doxorubicin, the first-line chemotherapeutic regimen for TNBC treatment, suggesting that the inhibition of autophagy by ANXA2 knockdown may overcome doxorubicin resistance. In a TNBC xenograft mouse model, we demonstrated that ANXA2 knockdown combined with doxorubicin administration significantly inhibited tumor growth compared to doxorubicin treatment alone, offering a promising avenue to enhance the effectiveness of chemotherapy. In summary, our study elucidated the molecular mechanism by which ANXA2 modulates autophagy, suggesting a potential therapeutic approach for TNBC treatment. Abbreviation: ATG: autophagy related; ChIP: chromatin-immunoprecipitation; HBSS: Hanks’ balanced salt solution; HSF1: heat shock transcription factor 1; MTOR: mechanistic target of rapamycin kinase; TNBC: triple-negative breast cancer; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3

Transcriptome reprogramming through alternative splicing triggered by apigenin drives cell death in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is characterized by its aggressiveness and resistance to cancer-specific transcriptome alterations. Alternative splicing (AS) is a major contributor to the diversification of cancer-specific transcriptomes. The TNBC transcriptome landscape is characterized by aberrantly spliced isoforms that promote tumor growth and resistance, underscoring the need to identify approaches that reprogram AS circuitry towards transcriptomes, favoring a delay in tumorigenesis or responsiveness to therapy. We have previously shown that flavonoid apigenin is associated with splicing factors, including heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2). Here, we showed that apigenin reprograms TNBC-associated AS transcriptome-wide. The AS events affected by apigenin were statistically enriched in hnRNPA2 substrates. Comparative transcriptomic analyses of human TNBC tumors and non-tumor tissues showed that apigenin can switch cancer-associated alternative spliced isoforms (ASI) to those found in non-tumor tissues. Apigenin preferentially affects the splicing of anti-apoptotic and proliferation factors, which are uniquely observed in cancer cells, but not in non-tumor cells. Apigenin switches cancer-associated aberrant ASI in vivo in TNBC xenograft mice by diminishing proliferation and increasing pro-apoptotic ASI. In accordance with these findings, apigenin increased apoptosis and reduced tumor proliferation, thereby halting TNBC growth in vivo. Our results revealed that apigenin reprograms transcriptome-wide TNBC-specific AS, thereby inducing apoptosis and hindering tumor growth. These findings underscore the impactful effects of nutraceuticals in altering cancer transcriptomes, offering new options to influence outcomes in TNBC treatments.

A specific RAGE-binding peptide inhibits triple negative breast cancer growth through blocking of Erk1/2/NF-κB pathway

Triple-negative breast cancer (TNBC) is an aggressive cancer that poses a significant threat to women's health. Unfortunately, the lack of clinical targets leads the poor clinical outcomes in TNBC. Many cancers demonstrate overexpression of receptor for advanced glycation end products (RAGE), which can contribute to cancer progression. Despite the potential therapeutic value of blocking RAGE for TNBC treatment, effective peptide drugs have yet to be developed. In our study, we observed that RAGE was highly expressed in TNBC and was associated with poor disease progression. We subsequently investigated the antitumor effects and underlying mechanisms of the RAGE antagonist peptide RP7 in both in vitro and in vivo models of TNBC. Our study revealed that RP7 selectively binds to RAGE-overexpressing TNBC cell lines, including MDA-MB-231 and BT549, and significantly inhibits cell viability, migration, and invasion in both cell lines. Furthermore, RP7-treatment suppressed tumor growth in TNBC xenograft mouse models without inducing detectable toxicity in normal tissues. Mechanistically, RP7 was found to inhibit the phosphorylation of ERK1/2, IKKα/β, IKBα, and p65 to block the NF-κB pathway, prevent the entry of p65 into the nucleus, decrease the protein expression of Bcl-2 and HMGB1, and promote the release of cytochrome C from the mitochondria into the cytoplasm. These effects were observed to activate apoptosis and inhibit epithelial-mesenchymal transition (EMT) in TNBC cells. This study highlights RAGE as a candidate therapeutic target for TNBC treatment and suggests that the RAGE antagonist peptide RP7 is a promising anticancer drug for TNBC.

Combating breast cancer progression through combination therapy with hypomethylating agent and glucocorticoid

Breast cancer is the leading cause of cancer-related death in women. Among breast cancer types, triple-negative breast cancer (TNBC) accounts for 15% of all breast cancers with aggressive tumor behavior. By using bioinformatic approaches, we observed that the microRNA-708 promoter is highly methylated in breast carcinomas, and this methylation is linked to a poor prognosis. Moreover, microRNA-708 expression correlates with better clinical outcomes in TNBC patients. Combination treatment with the hypomethylating agent decitabine and synthetic glucocorticoid significantly increased the expression of microRNA-708, reactivated DNMT-suppressed pathways, and decreased the expression of multiple metastasis-promoting genes such as matrix metalloproteinases (MMPs) and IL-1β, leading to the suppression of breast cancer cell proliferation, migration, and invasion, as well as reduced tumor growth and distant metastasis in the TNBC xenograft mouse model. Overall, our study reveals a therapeutic opportunity in which a combined regimen of decitabine with glucocorticoid may have therapeutic potential in treating TNBC patients.

PLAG co-treatment increases the anticancer effect of Adriamycin and cyclophosphamide in a triple-negative breast cancer xenograft mouse model

Chemotherapy induces tumor cell death and inhibits tumor progression, but the accompanying immune responses in the surrounding dying tissue cause significant inflammation. These responses, such as excessive neutrophil infiltration into tumor tissue, are the main causes of resistance to anticancer treatment. The development of drugs that reduce neutrophil infiltration into tumors is necessary to increase the anticancer effect of chemotherapy.Here, we show that the antitumor effect of the chemotherapy AC regimen (Adriamycin and cyclophosphamide) was increased by 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) cotreatment in the MDA-MB-231 triple-negative breast cancer xenograft mouse model. Tumor growth was inhibited up to 56% in mice treated with AC and inhibited up to 94% in mice cotreated with AC and PLAG. Side effects of chemotherapy, such as a reduction in body weight, were alleviated in mice cotreated with AC and PLAG. Excessive neutrophil infiltration caused by the AC regimen was successfully cleared in mice cotreated with AC and PLAG.We conclude that PLAG inhibits excessive neutrophil infiltration that aids tumor growth. Reduced neutrophils and increased lymphocytes in PLAG-treated mice can maximize the antitumor effect of the AC regimen and inhibit tumor growth.

Abstract 5310: Physiochemical and biological characterization of anti-EGFR based bispecific antibodies in triple negative breast cancer (TNBC) models

Abstract Triple-negative breast cancer (TNBC) accounts for approximately 10-20% of all diagnosed breast cancer. Both epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) frequently overexpress in TNBC and cooperate with each other in autocrine and paracrine manner to enhance tumor growth and angiogenesis. Therapeutic mAbs targeting EGFR (cetuximab) and VEGFR2 (ramucirumab) are approved by FDA for numerous cancer indications, but none of them are approved to treat breast cancers. TNBC cells secrete VEGF-A, which mediate angiogenesis on endothelial cells in a paracrine fashion and promote cancer cell growth in autocrine manner. To disrupt autocrine/paracrine loop in TNBC models in addition to mediating anti-EGFR tumor growth signaling and anti-VEGFR2 angiogenic pathway, we generated a bispecific antibody co-targeting EGFR and VEGFR2 (designated as anti-EGFR/VEGFR2 BsAb), in which cetuximab IgG backbone is connected to the single chain variable fragment (scFv) of ramucirumab via a glycine linker. Physiochemical characterization data shows that anti-EGFR/VEGFR2 BsAb binds to both EGFR and VEGFR2 in a similar binding affinity comparable to parental antibodies. Anti-EGFR/VEGFR2 BsAb demonstrates potent in vitro and in vivo anti-tumor activity in TNBC models. Mechanistically, anti-EGFR/VEGFR2 BsAb not only directly inhibits both EGFR and VEGFR2 in TNBC cells but also disrupts autocrine mechanism in TNBC xenograft mouse model. Furthermore, anti-EGFR/VEGFR2 BsAb blocks paracrine pathway mediated by VEGF/VEGFR2 in endothelial cells. Collectively, our novel findings demonstrate that anti-EGFR/VEGFR2 BsAb inhibits tumor growth via multiple mechanisms of action and has potential to be developed as an attractive targeted therapy for TNBC. Citation Format: Nishant Mohan, Xiao Luo, Yi Shen, Zachary Olson, Atul Agrawal, Yukinori Endo, David Rotstein, Lorraine Pelosof, Wen Jin Wu. Physiochemical and biological characterization of anti-EGFR based bispecific antibodies in triple negative breast cancer (TNBC) models [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 5310.

Abstract P5-08-14: Maternal embryonic leucine zipper kinase is associated with metastasis in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) has high rates of relapse and metastasis. It has a high proportion of cancer stem-like cells (CSCs), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, plays a critical role in promoting CSC maintenance, cell proliferation, and malignant transformation. Here, we assessed the role of MELK in TNBC aggressiveness. Methods: The clinical relevance of MELK in TNBC was analyzed using the World IBC Consortium dataset (n = 314). The effects of MELK knockdown (siRNA), knockout (CRISPR-Cas9), and MELK inhibition with a novel selective small molecule inhibitor MELK-In-7 on migration and invasion were determined using the migration and invasion assays, respectively. MELK’s role in regulating CSC phenotypes was assessed using the mammosphere assay and flow cytometry. The impact of MELK knockdown on epithelial-to-mesenchymal transition (EMT) was determined using Western blotting. MELK’s protumorigenic role was determined in vitro using soft agar assay and in vivo using TNBC xenograft mouse models. Results: MELK mRNA expression level was higher in TNBC tumors than in tumors expressing hormone receptor, HER2, or both (P < 0.0001). In univariate analysis, breast cancer patients with high-MELK-expressing tumors had worse overall survival (OS, P < 0.001) and distant metastasis-free survival (DMFS, P < 0.01) than patients with low-MELK-expressing tumors, suggesting a prognostic impact of MELK in breast cancer. MELK knockdown using siRNA or MELK inhibition using MELK-In-7 significantly reduced migration, invasion, mammosphere formation, CD24-/CD44+ subpopulations, and ALDH activity, and reversed EMT in TNBC cells. This result suggests a role for MELK in regulating CSCs and EMT, which play crucial roles in tumor invasion and metastasis. Nude mice injected with MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and significantly improved OS compared with the mice injected with parental MDA-MB-231 cells (P < 0.05). Further, MELK-selective inhibitor (MELK-In-7) suppressed the growth of 4T1 mouse TNBC tumors in a dose-dependent manner in syngeneic BALB/c mice (P < 0.001). These results indicate an essential role for MELK in promoting TNBC tumor growth and metastasis. To uncover the underlying molecular mechanism of MELK’s regulation of TNBC metastasis, we performed microarray analysis and identified SERPINE1, a serine protease inhibitor known to regulate metastasis, as a downstream target of MELK. We validated that mRNA levels of SERPINE1 were reduced in MELK-knockout MDA-MB-231 cells and MELK-In-7-treated MDA-MB-231 cells. Moreover, SERPINE1 knockdown using siRNA reduced migration and invasion of TNBC cells. These results suggest that MELK regulates TNBC cell motility through SERPINE1. Conclusion: This work confirms MELK as a driver of aggressiveness and metastasis in TNBC. The mechanisms underlying MELK’s regulation of TNBC metastasis via SERPINE1 require further studies. Citation Format: Xuemei Xie, Gaurav B. Chauhan, Ramakrishna Edupuganti, Takahiro Kogawa, Jihyun Park, Moises T Tacam, Fnu Vidhu, Diane D. Liu, Juliana M. Taliaferro, Mary Kathryn Pitner, Yu Shen, Naoto T. Ueno, Savitri Krishnamurthy, Gabriel N. Hortobagyi, Debu Tripathy, Steven J. Van Laere, Geoffrey Bartholomeusz, Kevin Dalby, Chandra Bartholomeusz. Maternal embryonic leucine zipper kinase is associated with metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-14.

TOP1 inhibition induces bifurcated JNK/MYC signaling that dictates cancer cell sensitivity.

Rationale: Triple-negative breast cancer (TNBC) does not respond to anti-estrogen and anti-HER2 therapies and is commonly treated by chemotherapy. TNBC has a high recurrence rate, particularly within the first 3 years. Thus, there is an urgent clinical need to develop more effective therapies for TNBC. Topoisomerase I (TOP1) inhibitors cause DNA damage, making these drugs desirable for TNBC treatment since DNA repair machinery is defective in this subtype of breast cancer. Among the main molecular subtypes of breast cancer, the TNBC cell lines exhibited the highest TOP1 inhibition sensitivity. However, clinically used TOP1 inhibitors, such as topotecan and irinotecan, have shown limited clinical applications and the reasons remain unclear. Understanding the mechanism of differential responses to TOP1 blockade and identifying the predictive markers for cancer cell sensitivity will help further TOP1-targeted therapy for TNBC treatment and improve the clinical use of TOP1 inhibitors.Methods: Viability assays were used to evaluate breast cancer cell sensitivity to topotecan and other TOP1 inhibitors as well as TOP2 inhibitors. An in vitro-derived topotecan-resistant TNBC cell model and TNBC xenograft models were employed to confirm cancer cell response to TOP1 blockade. RNA-seq was used to identify potential predictive markers for TNBC cell response to TOP1 blockade. Western blotting and qRT-PCR were performed to measure the protein levels and RNA expression. ATAC-seq and luciferase reporter assays were used to examine MYC transcriptional regulations. The effects of MYC and JNK in cancer cell response to TOP1 inhibition were validated via loss-of-function and gain-of-function experiments.Results: We observed two distinct and diverging cancer cell responses - sensitive versus resistant to TOP1 inhibition, which was confirmed by TNBC xenograft mouse models treated by topotecan. TNBC cells exhibited bifurcated temporal patterns of ATR pathway activation upon TOP1 inhibitor treatment. The sensitive TNBC cells showed an “up then down” dynamic pattern of ATR/Chk1 signaling, while the resistant TNBC cells exhibited a “persistently up” profile. On the contrary, opposite temporal patterns of induced expression of MYC, a key regulator and effector of DNA damage, were found in TNBC cells treated by TOP1 inhibitors. Mechanistically, we showed that TOP1-induced JNK signaling upregulated MYC expression. Furthermore, pharmacological inhibition of ATR reversed TNBC cell resistance to topotecan, whereas MYC knockdown and JNK inhibition reduced cancer cell sensitivity.Conclusions: Dynamic temporal profiles of induced ATR/Chk1 and JNK activation as well as MYC expression, may predict cancer cell response to TOP1 inhibitors. JNK activation-mediated constitutive elevation of MYC expression may represent a novel mechanism governing cancer cell sensitivity to TOP1-targeting therapy. Our results may provide implications for identifying TNBC patients who might benefit from the treatment with TOP1 inhibitors.

Abstract 14414: Embryonic Stem Cell Derived Exosomes Enhance Anti-Tumor Efficacy of Doxorubicin and Improve Cardiac Function in Triple-Negative Breast Cancer Xenograft Mice

Background: Doxorubicin (Dox) is a potent chemotherapeutic drug widely used for the treatment of triple negative breast cancer (TNBC). However, Dox treatment results in inadvertent cardiotoxic effects which often require supplemental therapy. We previously showed that treatment with embryonic stem cells derived exosomes (ES-Exos) enhanced chemosensitivity of Dox in breast cancer cells. Here we tested the hypothesis whether adjuvant therapy with ES-Exos in mice with orthotopic TNBC xenograft can enhance the anti-tumor effect of Dox and improve cardiac function. Methods and Results: Athymic female nude mice (6-wk old) were injected with human MDA-MB-231-Luc cell line in mammary intraductal T4 region to generate orthotopic xenografts of TNBC. After 2 weeks of tumor growth, mice (n=10/group) were treated for 4 weeks with: Saline (Con); Dox (5 mg/kg/week); ES-Exos (50 μg, twice a week); Dox+ES-Exos; and MEF (50 μg/twice a week; non-stem cell exosomes derived from mouse fibroblasts). ES-Exos were labelled with ExoGlow-Vivo near-IR dye to track the localization of exosomes in the tumor. Treatment with Dox+ES-Exos and Es-Exos showed improved survival as compared to Dox (2 deaths) and Con (3 deaths) (Fig.A) . Tumor size was significantly reduced with Dox+ES-Exos and ES-Exos treatments as compared to Con and Dox treatment (Fig.B) . Cardiac function measured by echocardiography showed improved ejection fraction in Dox+ES-Exos and ES-Exos as compared to Con group (Fig.C) . Cell proliferation rate assessed by Ki-67 score with immunofluorescence showed significantly higher rate in Con as compared to Dox and Dox+ES-Exos groups. RNA seq analysis identified upregulation of unique antitumorigenic regulators such as Semaphorin 5 (Sema5) and FOXO15 in ES-Exos compared to MEF, which may contribute to the therapeutic effect. Conclusion: Treatment with ES-Exos may be a promising novel strategy to improve chemosensitivity with attenuation of its cardiotoxic effect in TNBC.

Co-loading of an anthracycline analogue Pirarubicin and Salinomycin in a 1:3 ratio into Quatramerbiodegradable polymeric nanoparticles synergistically inhibit growth of triple-negative breast cancer.

e15047 Background: Triple negative breast cancer (TNBC) accounts for about 10-15% of all breast cancers and differ from other types of invasive breast cancers in that they grow and spread faster. TNBCs have limited treatment options and a worse prognosis. Therapy with anthracyclines considered to be one of the most effective agents in the treatment. Unfortunately, resistance to anthracycline therapy is very common due to drug efflux mediated by overexpression of ABC transporter. Pirarubicin (PIRA), an analogue of doxorubicin (DOX), is approved in Japan, Korea and China and is shown to be less cardiotoxic than DOX. Recent studies suggest that cancer stem cells (CSCs) play an important role in tumorigenesis and biology of TNBC. Targeting CSCs may be a promising, novel strategy for the treatment of this aggressive disease. Recent studies have shown that salinomycin (SAL) preferentially targets the viability of CSCs. Methods: SAL and PIRA were co-encapsulated in polylactic acid (PLA)-based block copolymeric nanoparticles (NPs) to efficiently co-deliver these agents to treat TNBC cells. Results: Generated SAL-PIRA co-encapsulated dual drug-loaded NPs showed an average diameter of 110 ± 7 nm, zeta potential of -12.5 mV and PDI of less than 0.25. Both of these anti-cancer agents showed slow and sustained release profile in non-physiological buffer (PBS, pH 7.4) from these dual drug-encapsulated NPs. Additionally, multiple ratios (PIRA:SAL = 3:1, 1:1, 1:3) were encapsulated to generate diverse dual drug-loaded NPs. The results demonstrate that, in contrast to 1:1 and 3:1, treatment of TNBC cells with 1:3 ratio of PIRA:SAL dual drug-loaded NPs, was associated with significant inhibition of growth in vitro in multiple TNBC cell lines. Interestingly, PIRA:SAL (1:3) was synergistic as compared to either SAL- or PIRA single drug-loaded NPs. The IC50 of PIRA and SAL in single drug-encapsulated NPs is 150 nM and 700 nM respectively in MDA-MB-468. Importantly, the IC50 of PIRA in dual drug-encapsulated NPs dropped down to 30 nM (5-fold). Similar results were obtained in SUM-149 TNBC cell line. Studies are underway to evaluate in vivo biological activity of PIRA:SAL (1:3) on tumor growth in a TNBC xenograft mice model. Conclusions: These results demonstrate that a novel dual drug-loaded NP formulation of PIRA and SAL in a unique ratio of 1:3 represents an approach for successful targeting of CSCs and bulk tumor cells in TNBC and potentially other cancer types.

Tea polyphenol epigallocatechin-3-gallate inhibits cell proliferation in a patient-derived triple-negative breast cancer xenograft mouse model via inhibition of proline-dehydrogenase-induced effects.

Triple-negative breast cancers (TNBCs) lack specific targeted therapy options and have evolved into highly chemo-resistant tumors that metastasize to multiple organs. The present study demonstrated that the proline dehydrogenase (PRODH) mRNA level in paired (tumor vs. normal) human breast tissue samples (n=234) was 6.6-fold greater than normal cells (*p=0.021). We established stable PRODH-overexpressing TNBC (HS578T) cells, and the malignant phenotypes were evaluated using soft agar colony formation and Transwell migration assays. The results demonstrated that PRODH induced epithelial-mesenchymal transition in cancer cells and increased cell proliferation. The present study found that the tea polyphenol epigallocatechin-3-gallate (EGCG) significantly inhibited PRODH and its regulated proteins, such as alpha-smooth muscle actin (alpha-SMA) expression in TNBC cells. These findings support the targeting of the PRODH signaling pathway as a potential therapeutic strategy in preventing cancer cell metastasis. The patient-derived xenograft (PDX) mouse model is highly relevant to real human tumor growth. We established a TNBC-PDX (F4, n=4 in each group)mouse model. The PDX mice were treated with EGCG (50 mg/kg), and the results indicated that EGCG significantly inhibited PDX tumor growth (*p = 0.013). These experiments provide additional evidence to evaluate the antitumor effects of EGCG-induced PRODH inhibition for clinical therapeutic application, especially in TNBC patients.

Highly Efficient Photothermal Therapy with Cell-Penetrating Peptide-Modified Bumpy Au Triangular Nanoprisms using Low Laser Power and Low Probe Dose.

Photothermal therapy (PTT) exploits nanomaterials with optimal heat conversion and cellular penetration using near-infrared (NIR) laser irradiation. However, current PTT agents suffer from inefficient heat conversion, poor intracellular delivery, and a high dose of probes along with excessive laser irradiation, causing limited therapeutic outcomes. Here, bumpy Au triangular nanoprisms (BATrisms) are developed for increasing the surface area, improving cell penetration, shifting the absorption peak to the NIR region, and enhancing the photothermal conversion efficiency (∼86%). Further, leucine (L)- and lysine (K)-rich cell-penetrating peptides (LK peptides) were employed to largely improve their cellular uptake efficiency. Importantly, a significant in vivo therapeutic efficacy with LK-BATrisms was demonstrated in a triple-negative breast cancer xenograft mice model. A very small dose of LK-BATrism (2.5 μg Au) was enough to exert antitumor efficacy under very low laser power (808 nm, 0.25 W/cm2), causing minimal tissue damages while very efficiently killing cancer cells.

Abstract 548: Birinapant enhances gemcitabine's anti-tumor efficacy in triple-negative breast cancer by inducing intrinsic pathway-dependent apoptosis

Abstract Background: Apoptosis resistance is a hallmark of cancer, and apoptosis regulators have been targeted for cancer treatment. A class of such regulators is inhibitor of apoptosis proteins (IAPs), which suppress caspase activity by inducing degradation of active caspases or blocking interaction of caspases with their substrates. Dysregulated IAP expression has been reported in many cancers, including breast cancer, and is involved in chemoresistance. IAPs have therefore become attractive targets for cancer treatment. Birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspase (SMAC, an endogenous antagonist of IAPs), has been shown to effectively activate apoptotic signaling by targeting IAPs and has potential application for treatment of multiple cancers. In addition, birinapant showed synergistic anti-tumor effects with several widely used chemotherapeutic agents in various cancers. Here, we assessed whether birinapant synergizes with commonly used anti-cancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), everolimus (mTOR inhibitor), and gemcitabine, in triple-negative breast cancer (TNBC). Among these drugs, gemcitabine strongly synergized with birinapant (combination index [CI] <1). We hypothesize that birinapant enhances anti-tumor efficacy of gemcitabine in TNBC by inducing intrinsic pathway-dependent apoptosis. Methods: The in vitro anti-tumor efficacy of birinapant, gemcitabine, and their combination was determined using CellTiter-Blue viability and soft agar colony formation assays. The CI (CalcuSyn software) was used to quantify the synergy. The effects of drug treatment on cell cycle distribution were analyzed by flow cytometry; the effects on apoptosis were assessed by annexin V-PE and 7-AAD staining, and induction of apoptosis was further confirmed by pan-caspase inhibitor treatment; and the effects on IAP degradation were examined by Western blotting. The in vivo antitumor efficacy was determined using TNBC xenograft mouse models. Results: Birinapant as a single agent had varying anti-proliferation effects in TNBC cells (IC50 = 0.21 to >20 µM). It had no synergistic effects with entinostat, cisplatin, paclitaxel, voxtalisib, dasatinib, or everolimus but strongly synergized with gemcitabine (CI <1 µM) in tested TNBC cells. Birinapant and gemcitabine synergistically inhibited the growth of TNBC cells by activating the intrinsic apoptosis pathway, accomplished by inducing degradation of cIAP2 and XIAP, which led to apoptotic cell death. Furthermore, birinapant significantly enhanced anti-tumor effectiveness of gemcitabine in TNBC xenograft mouse models (n = 10; P < 0.05) by inducing apoptosis. Conclusion: Our findings demonstrate the therapeutic potential of birinapant for enhancing the anti-tumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins. Citation Format: Xuemei Xie, Jangsoon Lee, Troy Pearson, Alexander Y. Lu, Debu Tripathy, Gayathri R. Devi, Chandra Bartholomeusz, Naoto T. Ueno. Birinapant enhances gemcitabine's anti-tumor efficacy in triple-negative breast cancer by inducing intrinsic pathway-dependent apoptosis [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 548.

Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate.

Sphingosine kinase 2 (SphK2) is known to phosphorylate the nuclear sphingolipid metabolite to generate sphingosine-1-phosphate (S1P). Nuclear S1P is involved in epigenetic regulation of gene expression; however, the underlying mechanisms are not well understood. In this work, we have identified the role of nuclear S1P and SphK2 in regulating hypoxia-responsive master transcription factors hypoxia-inducible factor (HIF)-1α/2α, and their functions in breast cancer, with a focus on triple-negative breast cancer (TNBC). We have shown SphK2 is associated with HIF-1α in protein complexes, and is enriched at the promoters of HIF target genes, including vascular endothelial growth factor (VEGF), where it enhances local histone H3 acetylation and transcription. S1P specifically binds to the PAS domains of HIF-1α. SphK2, and HIF-1α expression levels are elevated in metastatic estrogen receptor-positive (ER+) and TNBC clinical tissue specimens compared to healthy breast tissue samples. To determine if S1P formation in the nucleus by SphK2 is a key regulator of HIF functions, we found using a preclinical TNBC xenograft mouse model, and an existing selective SphK2 inhibitor K-145, that nuclear S1P, histone acetylation, HIF-1α expression, and TNBC tumor growth were all reduced in vivo. Our results suggest that S1P and SphK2 in the nucleus are linked to the regulation of HIF-1α/2α functions associated with breast cancer progression, and may provide potential therapeutic targets.

Anti-tumor activity of BET inhibitors in androgen-receptor-expressing triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a heterogeneous disease comprising several subtypes. Androgen-receptor (AR) signaling has been targeted by several investigational agents in luminal AR subtype TNBCs. Bromodomain (BRD) and extra-terminal motif (BET) protein inhibitors have been shown to attenuate AR signaling in metastatic castration-resistant prostate cancer and to overcome enzalutamide resistance. We demonstrated potent anti-tumor effects of the BET inhibitor JQ1 against AR-positive TNBC cell lines using cell viability and cell cycle analysis. To reveal the mechanisms of JQ1 effects, multiplex gene expression analysis and immunoblotting assays were used. We examined in vivo effects of JQ1 in a xenograft model of AR expressing TNBC. JQ1 exhibited its anti-proliferative activity by inducing apoptosis and cell cycle arrest. JQ1 activity was not mediated by MYC downregulation. Instead, JQ1 blocked the interactions among the ATPase-family AAA-domain-containing 2 protein (ATAD2), BRD2, BRD4, and AR; effectively suppressing the expression of AR associated targets. In addition, JQ1 showed significant anti-tumor activity in vivo in TNBC xenograft mouse models as a monotherapy and in combination with anti-AR therapy. Taken together, our results showed that the BET inhibitor JQ1 is a promising therapeutic agent for the treatment of AR-positive TNBC.

Abstract 2697: YPN005, an oral CDK7 inhibitor, exhibits a significant antitumor activity in Myc-driven cancers

Abstract Background: CDK7 plays an important role in regulating cell cycle progression and gene via activation of cell cycle kinases (CDK1, CDK2, CDK4 and CDK6) and RNA polymerase II (PolII). Recent studies indicate that the inhibition of CDK7 is an attractive strategy for the treatment of cancer by down-regulation of c-Myc expression. (Wang et al 2018) We have investigated the therapeutic efficacy of YPN005, a novel oral CDK7 inhibitor, in triple negative breast cancer (TNBC) and hepatocellular carcinoma (HCC). Methods: We evaluated antiproliferative activity of YPN005 on TNBC and HCC cells and the expression of RNA PolII and c-Myc was studied by Western Blot analyses to investigate the mechanism of action. To identify a biomarker, we examined the correlation between the level of c-Myc expression and anticancer activity of YPN005 in vitro using HCC cells. The therapeutic efficacy of YPN005 was evaluated in TNBC xenograft mouse model. Results: YPN005 significantly inhibited the proliferation of TNBC and HCC cells and IC50s was determined as 10-20 nM and 5-30 nM range, respectively. Inhibition of cell proliferation was accompanied by a decrease in the levels of RNA PolII phosphorylation and c-Myc expression. Western Blot analyses revealed that the sensitivity of HCC cells to YPN005 was correlated with the level of c-Myc expression. In vivo xenograft model of TNBC showed that oral daily administration of YPN005 for 21 days (once, and 10mg/kg) efficiently inhibited the growth of tumor. All mice survived during the dosing period without significant changes of the hematologic profiles. Conclusion: We propose that oral administration of YPN005, an orally available CDK7 inhibitor, could be a potent and attractive approach to treat the Myc overexpressing cancers. Citation Format: Kwang-Ok Lee, Jakyung Yoo, Mi Jung Lee, Kang Woo Lee, Ji Eun Min, Jinhwan Kim, Ki-Nam Min, Tae Chul Roh, Kang-Sik Seo, Hae In Rhee, Jun Hee Lee, Da-Hye Jeon, Dae Seong Lim. YPN005, an oral CDK7 inhibitor, exhibits a significant antitumor activity in Myc-driven cancers [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 2697.

CAPE-pNO2 Inhibited the Growth and Metastasis of Triple-Negative Breast Cancer via the EGFR/STAT3/Akt/E-Cadherin Signaling Pathway.

Overexpressed epidermal growth factor receptor (EGFR) and overactivated epithelial-mesenchymal transition (EMT) in triple-negative breast cancer (TNBC) can enhance tumorigenesis and tumor recurrence and metastasis. Caffeic acid p-nitro-phenethyl ester (CAPE-pNO2) has various pharmacological activities in our previous research, but its effect on metastasis and growth of TNBC has not been studied. In this study, Caffeic acid phenethyl ester (CAPE) was as a positive control. in vitro, MTT, Transwell, wound healing, colony formation and cell adhesion assays were performed to examine the effect on viability, invasion, migration, colony formation and adhesion of MDA-MB-231 cells by CAPE-pNO2, the results indicated that CAPE-pNO2 significantly dose-dependently inhibited metastasis of MDA-MB-231 cells (p < 0.05). in vivo, TNBC xenograft mice were established by subcutaneously injected with MDA-MB-231 cells, and they were used to estimate the effect on metastasis and growth of CAPE-pNO2 after 38 days of treatment. HE staining and TUNEL staining were carried out in tumor tissues, results showed that CAPE-pNO2 obviously suppressed the tumor growth, induced cells apoptosis (p < 0.01) and decreased pulmonary and splenic metastatic tumor cells. The results of IHC demonstrated that the VEGFA and Ki-67 proteins expression were downregulated (p < 0.01) in tumor tissues. Furthermore, western blot analysis was used to quantify key metastasis- and growth-associated proteins expression in vitro and in vivo, the results suggested that CAPE-pNO2 downregulated the proteins expression of p-EGFR, p-STAT3, p-Akt, MMP-2, MMP-9, Survivin, and key EMT-related proteins (Vimentin and N-cadherin) (p < 0.01), and increased the expression of E-cadherin (p < 0.01) in vivo and in vitro. Besides, CAPE-pNO2 had a similar effect as erlotinib in regulating the EGFR downstream proteins in EGF-induced MDA-MB-231cells. Collectively, these results indicated that CAPE-pNO2 possessed inhibitory effect on the growth and metastasis of TNBC may via the EGFR/STAT3/Akt/E-cadherin signaling pathway, and CAPE-pNO2 is better than CAPE in inhibiting growth and metastasis.

GDF10 inhibits proliferation and epithelial-mesenchymal transition in triple-negative breast cancer via upregulation of Smad7.

Triple-negative breast cancer (TNBC) cannot be treated with current hormonal therapies and has a higher risk of relapse than other breast cancers. To identify potential therapeutic targets for TNBC, we conducted microRNA sequencing (RNA-Seq) in human TNBC specimens and tumor-matched controls. We found that growth differentiation factor-10 (GDF10), a member of the TGF-β superfamily, was downregulated in tumor samples. Further analysis of GDF10 expression in a larger set of clinical TNBC samples using qPCR confirmed its downregulation and association with parameters of disease severity. Using human-derived TNBC cell lines, we carried out GDF10 under- and overexpression experiments, which showed that GDF10 loss promoted cell proliferation and invasion. By contrast, overexpression of GDF10 inhibited proliferation, invasion, and epithelial mesenchymal transition (EMT) via upregulation of Smad7 and E-Cadherin, downregulation of p-Smad2 and N-Cadherin, and reduction of nuclear Smad4 expression. In addition, overexpression of GDF10 reduced tumor burden and induced apoptosis in a TNBC xenograft mouse model. These findings indicate that GDF10 acts as a tumor suppressor in mammary epithelial cells that limits proliferation and suppresses EMT. Efforts aimed at restoring GDF10 expression may thus bring a long-sought therapeutic alternative in the treatment of patients with TNBC.