Breast Cancer Cell Line Xenografts Research Articles

Abstract P4-08-20: Inhibition of TACC3 blocks the growth of highly aggressive breast cancers with centrosome amplification

Abstract Centrosome amplification (CA) is a hallmark of cancer that is strongly associated with highly aggressive disease and worse clinical outcome. Enhanced mitotic progression via clustering of extra centrosomes is a major coping mechanism utilized by cancer cells with CA that would otherwise undergo mitotic cell death due to formation of multipolar spindles. However, the underlying molecular mechanisms have largely been unexplored. Furthermore, mitosis-targeting inhibitors have mostly been unsuccessful in clinical settings with poor efficacy and severe side effects. Therefore, there is a dire need to uncover novel molecular mechanisms of CA-driven tumor growth and identify therapeutic targets playing key roles not only in mitosis, but also in interphase of cancer cells with CA to achieve durable anti-tumor effect with minimal toxicity. Here, we identified Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) as a novel CA-directed dependency, driving highly aggressive cell growth by forming distinct functional interactomes during cell cycle progression. We demonstrated, for the first time, that TACC3 interacts with the Kinesin Family Member C1 (KIFC1) via its TACC domain in mitotic cells with CA to promote centrosome clustering (CC) and facilitate mitotic progression. On the other hand, TACC3 interacts with the members of the nucleosome remodeling and deacetylase (NuRD) complex (HDAC2 and MBD2) in the nucleus of interphase cells with CA, thereby suppressing the transcription of key tumor suppressors to facilitate G1/S progression and cell survival. Inhibiting TACC3 in mitotic cells blocks the formation of TACC3/KIFC1 complex, leading to formation of multipolar spindles and activation of spindle assembly checkpoint (SAC)/CDK1/p-Bcl2 axis that ultimately results in mitotic cell death; whereas TACC3 inhibition in interphase cells blocks TACC3/HDAC2/MBD2 complex, leading to enhanced transcription of cyclin-dependent kinase inhibitors (e.g., p21 and p16) and apoptosis regulators (e.g., APAF1), ultimately causing p53-independent G1 arrest and strong apoptosis. Notably, inducing CA by chemical (cytochalasin D) or genomic (PLK4 overexpression or p53 loss) modulations renders cancer cells highly sensitive to TACC3 inhibition, showing the dependency of cells with CA to TACC3. Targeting TACC3 by small molecule inhibitors or CrispR-CAS9-mediated knock-out significantly reduces colony formation ability, inhibits the growth of organoids of patient-derived xenografts (PDXs) with CA, and strongly inhibits tumor growth in breast cancer cell line xenografts and PDXs with CA. Notably, we demonstrated that high CA tumors express much higher levels of TACC3, and high TACC3 expression, in association with its downstream effectors, KIFC1, HDAC2 and MBD2, leads to drastically worse clinical outcome in cancer patients with CA. Altogether, our results show, for the first time, that TACC3 is a multifunctional driver of the growth of the highly aggressive breast tumors with CA and that targeting TACC3 is a promising approach to tackle this aggressive disease. Citation Format: Ozge Saatci, Ozge Akbulut, Metin Cetin, Vitali Sikirzhytski, Ozgur Sahin. Inhibition of TACC3 blocks the growth of highly aggressive breast cancers with centrosome amplification [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-20.

Cromolyn chitosan nanoparticles reverse the DNA methylation of RASSF1A and p16 genes and mitigate DNMT1 and METTL3 expression in breast cancer cell line and tumor xenograft model in mice

BackgroundDeveloping epigenetic drugs for breast cancer (BC) remains a novel therapeutic approach. Cromolyn is a mast cell stabilizer emerging as an anticancer drug; its encapsulation in chitosan nanoparticles (CSNPs) improves its effect and bioavailability. However, its effect on DNA and RNA methylation machineries has not been previously tackled. MethodsThe possible anticancer effect of cromolyn CSNPs and its potential as an epigenetic drug was investigated in vitro using MCF-7 human BC cell line and in vivo using Ehrlich ascites carcinoma-xenograft model in mice symbolizing murine mammary adenocarcinoma. Mice were injected with a single dose of Ehrlich ascites carcinoma cells subcutaneously for the induction of tumor mass, and then randomized into three groups: control, cromolyn CSNPs (equivalent to 5 mg cromolyn/kg, i.p.) and plain CSNPs twice/week for 2 weeks. ResultsCromolyn CSNPs showed prominent anticancer effect in MCF-7 cells by reducing the cell viability percent and enhancing DNA damage in the comet assay demonstrating its apoptotic actions. Mechanistically, cromolyn CSNPs influenced potential epigenetic processes through mitigating DNA methyltransferase 1 (DNMT1) expression, reversing the hypermethylation pattern of the tumor suppressor RASSF1A and p16 genes and attenuating the expression of the RNA N6-methyladenosine writer, methyltransferase-like 3 (METTL3). Cromolyn CSNPs diminished ERK1/2 phosphorylation, a possible arm influencing DNMT1 expression. In vivo, cromolyn CSNPs lessened the tumor volume and halted DNMT1 and METTL3 expression in Ehrlich carcinoma mice. ConclusionsCromolyn CSNPs have the premise as an epigenetic drug through inhibiting ERK1/2 phosphorylation/DNMT1/DNA methylation and possibly impacting the RNA methylation machinery via mitigating METTL3 expression.

Abstract 1777: Androgen receptor (AR) agonists inhibit AR- and estrogen-receptor-positive breast cancer

Abstract Introduction: Breast cancer is the most common cancer diagnosed in women. About 13% of women will develop invasive breast carcinoma in their lifetime and it is estimated that about 280,000 new cases will be diagnosed in 2021. Seventy-percent of diagnosed breast cancers are estrogen receptor (ER)-positive and about 90% of the ER-positive breast cancers are androgen receptor (AR)-positive. We have previously shown that AR agonists inhibit ER-positive breast cancer growth by sequestering the pioneer-transcription factor FOXA1 from ER-cistrome. Tissue-selective AR modulators (SARMs) such as enobosarm are in clinical trials to treat ER-positive breast cancers. Since resistance is a major impediment to sutained treatment, it is important to identify new therapeutic strategies and their mechanisms of resistance. Here, we evaluated the possible mechanisms of resistance to AR agonists in ER-positive breast cancer. Description: ER-positive breast cancer cell line xenografts and patient-derived xenografts (PDX) were used as experimental models. Once tumors grew to ~100-300 mm3, mice were randomized and treated orally with vehicle or enobosarm (30 mg/kg/day). Animals were sacrificed after 4 weeks when the tumors responded to treatment and after 10 weeks once resistance developed to treatment. Tumor volumes were measured twice weekly and tumors were collected at sacrifice for further analyses. RNA-seq and ChIP-seq were performed on tumor tissues. Summary: AR agonists, enobosarm and dihydrotestosterone (DHT), inhibited the proliferation of ER-positive breast cancer cells ZR-75-1 and T47D. Transcriptome analysis revealed that AR agonists activate AR in ER-positive breast cancer cells and inhibit ER-target gene signature. AR agonists inhibited the growth of breast cancer cell line xenograft T47D and breast cancer PDXs HCI-7 that express wildtype ER, and HCI-13 and WHIM-23, two models that express Y537S mutant ER. Treatment of WHIM-23 and T47D xenograft for over ten weeks resulted in resistance and regrowth. AR agonist-sensitive and -resistant tumors were analyzed using RNA-seq, ChIP-seq, and ATAC-seq to understand the mechanism of resistance development. The results indicate that the prolonged activation of AR will result in resistance. These mechanisms of resistance can be utilized as possible therapeutic targets. Conclusion: These results suggest that the AR is a promising therapeutic target in the treatment of ER-positive breast cancer. However, resistance development is possible with AR agonists and the mechanism provides suggestions for future combination therapies. Disclosure: This work was supported by an NCI grant (CA229164 and CA229164S1 to RN) and by a DOD grant (W81XWH2110055 to RN). Citation Format: Sarah Asemota, Suriyan Ponnusamy, Thirumagal Thiyagarajan, Ramesh Narayanan. Androgen receptor (AR) agonists inhibit AR- and estrogen-receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1777.

Modulated TRPC1 Expression Predicts Sensitivity of Breast Cancer to Doxorubicin and Magnetic Field Therapy: Segue Towards a Precision Medicine Approach.

Chemotherapy is the mainstream treatment modality for invasive breast cancer. Unfortunately, chemotherapy-associated adverse events can result in early termination of treatment. Paradoxical effects of chemotherapy are also sometimes observed, whereby prolonged exposure to high doses of chemotherapeutic agents results in malignant states resistant to chemotherapy. In this study, potential synergism between doxorubicin (DOX) and pulsed electromagnetic field (PEMF) therapy was investigated in: 1) MCF-7 and MDA-MB-231 cells in vitro; 2) MCF-7 tumors implanted onto a chicken chorioallantoic membrane (CAM) and; 3) human patient-derived and MCF-7 and MDA-MB-231 breast cancer xenografts implanted into NOD-SCID gamma (NSG) mice. In vivo, synergism was observed in patient-derived and breast cancer cell line xenograft mouse models, wherein PEMF exposure and DOX administration individually reduced tumor size and increased apoptosis and could be augmented by combined treatments. In the CAM xenograft model, DOX and PEMF exposure also synergistically reduced tumor size as well as reduced Transient Receptor Potential Canonical 1 (TRPC1) channel expression. In vitro, PEMF exposure alone impaired the survival of MCF-7 and MDA-MB-231 cells, but not that of non-malignant MCF10A breast cells; the selective vulnerability of breast cancer cells to PEMF exposure was corroborated in human tumor biopsy samples. Stable overexpression of TRPC1 enhanced the vulnerability of MCF-7 cells to both DOX and PEMF exposure and promoted proliferation, whereas TRPC1 genetic silencing reduced sensitivity to both DOX and PEMF treatments and mitigated proliferation. Chronic exposure to DOX depressed TRPC1 expression, proliferation, and responses to both PEMF exposure and DOX in a manner that was reversible upon removal of DOX. TRPC1 channel overexpression and silencing positively correlated with markers of epithelial-mesenchymal transition (EMT), including SLUG, SNAIL, VIMENTIN, and E-CADHERIN, indicating increased and decreased EMT, respectively. Finally, PEMF exposure was shown to attenuate the invasiveness of MCF-7 cells in correlation with TRPC1 expression. We thus demonstrate that the expression levels of TRPC1 consistently predicted breast cancer sensitivity to DOX and PEMF interventions and positively correlated to EMT status, providing an initial rationale for the use of PEMF-based therapies as an adjuvant to DOX chemotherapy for the treatment of breast cancers characterized by elevated TRPC1 expression levels.

Abstract 738: Targeting metabolic vulnerabilities of endocrine resistant breast cancers using XPO1 and NAMPT inhibition

Abstract Introduction: Approximately 70% of human breast cancers express estrogen receptor alpha (ERα), providing the potential for targeted endocrine therapy for patients. Unfortunately, 30-40% of ER+ patients still experience recurrence and metastasis with a 5-year relative overall survival rate of just 24%. Previously, we described metabolic changes in metastatic breast cancer cells that are driven by single therapies and the metastatic organ microenvironment. Combining selinexor (SEL), a small molecule inhibitor of exportin 1 (XPO1) approved by the FDA for treatment of some hematologic malignancies, with the current therapies used for advanced breast cancers (e.g. tamoxifen) decreased metabolic adaptations in response to individual drug treatments and prevented colony formation in hydrogels and tumor growth in xenograft models. Since we observed a reliance on NAD+ metabolism in cancer cells that are treated with single therapies, in the current study we investigated co-targeting metabolic alterations using SEL and the NAMPT inhibitor KPT-9274. Methods: We established 3D cell culture systems in novel hydrogels derived from decellularized bone, lung, and liver tissues - the major sites of metastasis of ER+ breast cancers, and compared with standard Matrigel culture. Endocrine responsive (MCF7 and T47D) and resistant (MCF7 and T47D cells with ESR1 Y537S or D538G mutations) breast cancer cell lines were treated with SEL or KPT-9274. Tumorigenicity was assessed using in vitro cell viability and colony formation assays and in vivo breast cancer cell line xenografts in athymic mice. Metabolic changes were assessed using whole metabolite profiling by GC-MS and a Seahorse XF analyzer. Results: Combining an FDA-approved XPO1 small molecule inhibitor, Selinexor (SEL) with the NAMPT inhibitors decreased metabolic adaptations in response to individual drug treatments, and prevented colony formation in hydrogels and tumor growth in xenograft models. Conclusions: Targeting drug and metastatic-site induced adaptations to regenerate new vulnerabilities in endocrine-resistant breast tumors are novel. Given the need for better strategies for improving therapy response of metastatic ER+ tumors, our findings show uncovering the role XPO1-NAMPT crosstalk plays in metastatic breast cancer could lead to new combined therapies that reduce mortality. Citation Format: Qianying Zuo, Ayca Mogol, Ozan Imir, Yosef Landesman, Zeynep Madak Erdogan. Targeting metabolic vulnerabilities of endocrine resistant breast cancers using XPO1 and NAMPT inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 738.

Abstract 1130: First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation

Abstract KAT6A is a lysine histone acetyltransferase (HAT) of the MYST family of HATs. KAT6A, and its paralog KAT6B, have been shown to acetylate histone H3K23Ac and regulate diverse biological processes, including transcription, cell-cycle progression, stem cell maintenance and development. Molecular dysregulation of KAT6A has been observed in several cancers, including amplifications in breast, lung, ovarian cancer along with oncogenic fusions in AML. In breast cancer, KAT6A is amplified as part of the 8p11 amplicon in 10-15% of the patient population, which correlates with a worse clinical outcome in the estrogen receptor+ (ER+) subtype. Here we present identification of a first-in-class potent KAT6A/KAT6B tool inhibitor CTx-648 (PF-9363), that possesses high selectivity versus other MYST family members (KAT7, KAT5, KAT8) and other KATs, demonstrating anti-tumor activity in breast cancer. Using genetic and pharmacological approaches, we have demonstrated several ER+ breast cancer cell lines including KAT6A amplified and over-expressing models, are dependent on KAT6A enzymatic function. Epigenomic profiling studies using bulk and nascent RNA-seq combined with ATAC-seq revealed CTx-648 leads to downregulation of a specific set of genes involved in ESR1 pathway, cell cycle and stem cell pathways. In vivo target validation studies showed strong anti-tumor activity of CTx-648 in several ER+ breast cancer cell line and patient-derived xenograft models, including models harboring endocrine therapy resistance ESR1 mutations, highlighting promise for this novel therapy in ER+ breast cancer population. Based on the strength of the pre-clinical data, a selective KAT6 inhibitor (PF-07248144) is now commencing a Phase 1 clinical study in Advanced or Metastatic Solid Tumors. Citation Format: Shikhar Sharma, Jay Chung, Sean Uryu, Amanda Rickard, Natalie Nady, Showkhin Khan, Zhenxiong Wang, Yong Zhang, Haikuo Zhang, Pei-Pei Kung, Eric Greenwald, Karen Maegley, Patrick Bingham, Hieu Lam, Ylva E. Bozikis, Hendrik Falk, Elizabeth Allan, Vicky M. Avery, Miriam S. Butler, Michelle A. Camerino, Catalina Carrasco-Pozo, Susan A. Charman, Melissa J. Davis, Mark A. Dawson, Dawson Sarah-Jane, Melanie de Silva, Matthew L. Dennis, Olan Dolezal, Rachel Lagiakos, Geoffrey J. Lindeman, Laura MacPherson, Stewart Nuttall, Thomas S. Peat, Bin Ren, Alexandra E. Stupple, Elliot Surgenor, Chin Wee Tan, Tim Thomas, Jane E. Visvader, Anne K. Voss, Francois Vaillant, Karen L. White, James Whittle, Yuqing Yang, Soroor Hediyeh-Zadeh, Paul A. Stupple, Ian P. Street, Brendon J. Monahan, Thomas Paul. First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1130.

19P Deep eutectic solvent mixture formed from 2-deoxy-D-glucose and metformin targets cancer cell metabolism and induces apoptosis in breast cancer cell line xenografts

19P Deep eutectic solvent mixture formed from 2-deoxy-D-glucose and metformin targets cancer cell metabolism and induces apoptosis in breast cancer cell line xenografts

Abstract 5675: Metastatic organ-specific changes in ERα and XPO1 signaling and metabolic adaptations dictate the therapy responses in endocrine resistant breast cancers

Abstract Introduction: Approximately 70% of human breast cancers express estrogen receptor alpha (ERα), providing a potential for targeted endocrine therapy for patients. Unfortunately, 30-40% of ER+ patients still experience recurrence and metastasis with a 5-year relative overall survival rate of just 24%. The tumor microenvironment (TME) plays a key role in the behavior of cancer cells and their response to endocrine therapies. However, there is a critical need for relevant 3D models of different metastatic sites to use for studying the molecular mechanisms driving resistance in these tumor phenotypes. Methods: We established 3D cell culture systems in novel hydrogels derived from decellularized bone, lung and liver tissues - the major sites of metastasis of ER+ tumors, and compared with standard Matrigel culture. Endocrine responsive (MCF7 and T47D) and resistant (MCF7 and T47D cells with ESR1 Y537S or D538G mutations) breast cancer cell lines were treated with fulvestrant (FUL), tamoxifen (TAM) and palbociclib (PAL). Tumorigenicity was assessed using in vitro cell viability and colony formation assays and in vivo breast cancer cell line xenografts in athymic mice. ERα cistromes and transcriptomes were assessed using ChIP-Seq and RNA-Seq analysis. Metabolic changes were assessed using whole metabolite profiling by GC-MS, a Seahorse XF analyzer, and flux analysis with labeled glucose and glutamine. Results: We observed metastatic site-specific responses to each drug. For example, TAM blocked colony formation in Matrigel and lung hydrogel but increased colony formation in bone and liver hydrogels. FUL acted as an agonist and increased colony number and size in all three-tissue matrices, but not in Matrigel. PAL increased colony size and number in Matrigel and liver hydrogel but not in bone or lung hydrogel. Genome-wide ERα recruitment to chromatin was reduced in liver hydrogels, consistent with decreased clinical response to endocrine agents in patients with liver metastasis. We found that treatment with individual drugs activated novel metabolic pathways to increase survival in different metastatic sites. Using RNA-Seq analysis, we identified increased expression of nuclear export pathway components (XPO1, KPNA2, etc.) in various metastatic sites. Combining an FDA-approved XPO1 small molecule inhibitor, Selinexor (SEL) with the current therapies used for advanced breast cancers (FUL, TAM or PAL) decreased metabolic adaptations in response to individual drug treatments and prevented colony formation in hydrogels and tumor growth in xenograft models. Conclusions: Further developing and characterizing 3D metastatic site models will enable us to elucidate the underlying mechanisms of metastasis and resistance, and target these mechanisms with novel drug combinations in already metastasized patients. Targeting metastatic-site specific adaptations to regenerate new vulnerabilities in endocrine-resistant breast tumors is novel. Given the need for better strategies for improving therapy response of metastatic ER+ tumors, our findings show uncovering the role ERα-XPO1 crosstalk plays in metastatic breast cancer could lead to new combined therapies that reduce mortality. Citation Format: Qianying Zuo, Eylem Kulkoyluoglu Cotul, Ashlie Santaliz-Casiano, Ozan Berk Imir, Elif Tunc, Chengjian Mao, David J. Shapiro, Ben Ho Park, Yosef Landesman, Zeynep Madak-Erdogan. Metastatic organ-specific changes in ERα and XPO1 signaling and metabolic adaptations dictate the therapy responses in endocrine resistant breast cancers [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 5675.

Potent Anticancer Effect of the Natural Steroidal Saponin Gracillin Is Produced by Inhibiting Glycolysis and Oxidative Phosphorylation-Mediated Bioenergetics.

Metabolic rewiring to utilize aerobic glycolysis is a hallmark of cancer. However, recent findings suggest the role of mitochondria in energy generation in cancer cells and the metabolic switch to oxidative phosphorylation (OXPHOS) in response to the blockade of glycolysis. We previously demonstrated that the antitumor effect of gracillin occurs through the inhibition of mitochondrial complex II-mediated energy production. Here, we investigated the potential of gracillin as an anticancer agent targeting both glycolysis and OXPHOS in breast and lung cancer cells. Along with the reduction in adenosine triphosphate (ATP) production, gracillin markedly suppresses the production of several glycolysis-associated metabolites. A docking analysis and enzyme assay suggested phosphoglycerate kinase 1 (PGK1) is a potential target for the antiglycolytic effect of gracillin. Gracillin reduced the viability and colony formation ability of breast cancer cells by inducing apoptosis. Gracillin displayed efficacious antitumor effects in mice bearing breast cancer cell line or breast cancer patient-derived tumor xenografts with no overt changes in body weight. An analysis of publicly available datasets further suggested that PGK1 expression is associated with metastasis status and poor prognosis in patients with breast cancer. These results suggest that gracillin is a natural anticancer agent that inhibits both glycolysis and mitochondria-mediated bioenergetics.

Abstract P5-04-14: Recombinant Slit2 suppresses breast cancer metastasis by activating anti-tumor macrophages

Abstract Metastasis is a major cause of mortality in breast cancer patients in part due to the lack of clinically established targeted therapies. Approximately 5%-20% of patients with Stage II, and ~50% of patients with stage III will recur distally and are likely to die from their disease. Metastatic, or stage IV breast cancers, have a 5-year relative survival rate of about 22%. The expression of a tumor suppressor protein, Slit2 has been shown to be downregulated in various types of tumors including breast cancer. The Slit2 acts through Roundabout Homolog1 (Robo1) receptor. Previously it has been shown that ectopic expression of Slit2 inhibits human MCF-7 breast cancer cell line xenograft tumor growth in mice. However, its role in breast cancer metastasis, tumor microenvironment (TME) and anti-tumor immunity has not been studied before. By using genetically engineered human breast cancer cells, spontaneous mammary tumor and pre-clinical mouse models, we evaluated the role of Slit2 in inhibiting breast cancer growth, metastasis by activating anti-tumor immune response. To study the role of Slit2 in breast cancer, we implanted Slit2 overexpressing human breast cancer cell line MDA-MB-231 (231-Slit2) or vector control cells (231-Vec) to the mammary fat-pads of NOD/SCID/gamma (NSG) mice and observed that 231-Slit2 had significantly reduced tumor growth and metastasis to the lungs compared to 231-Vec. To further confirm the anti-metastatic role of Slit2, we treated mouse mammary tumor virus- Polyoma Middle T antigen (MMTV-PyMT) mammary tumor model and MVT-1 orthotopic tumor bearing FVB/J wildtype mice with recombinant Slit2 (rSlit2) that resulted in significantly reduced tumor growth and metastasis to the lungs in both the models compared to PBS treated mice. The ex-vivo immunofluorescence and flow cytometry studies revealed that Slit2 treated tumors possess a very high number of tumor phagocytic macrophages compared to PBS. In-vitro analysis also showed that rSlit2 treated mouse macrophages (RAW264.7) has higher bacterial particle phagocytic ability. Further analysis of tumors elucidated that Slit2 treated tumors recruited higher number of CD4+ and CD8+ T-cells. In addition, The CD8+ cells were also positive for Granzyme-b showing higher number of effector T-cells in the Slit2 tumors compared to PBS. By using human breast cancer tissue microarray (TMA), we have found that Slit2 expression significantly correlates with better overall survival. These observations highlight the ability of Slit2 to enhance tumor phagocytic macrophages and anti-tumor CD8+/Granzyme-b+ T-cells, thereby restricting tumor growth and lung metastasis. These studies suggest that Slit2 could be used as a novel immunomodulatory therapeutic agent metastatic breast cancer. Citation Format: Dinesh Kumar Ahirwar, Ramesh Kumar Ganju, Sanjay Mishra, Konstantin Shilo, Kirti Kaul. Recombinant Slit2 suppresses breast cancer metastasis by activating anti-tumor macrophages [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 P5-04-14.

Specific, reversible G1 arrest by UCN-01 in vivo provides cytostatic protection of normal cells against cytotoxic chemotherapy in breast cancer

BackgroundLow-dose UCN-01 mediates G1 arrest in normal proliferating cell lines with an intact G1 to S transition but not tumour cells with a deregulated G1 to S checkpoint. Here we hypothesised that UCN-01 is effective in mediating a selective, reversible G1 arrest of normal proliferating cells, resulting in decreased chemotoxicity, improved tolerance and enhanced chemotherapeutic efficacy in vivo in both non-tumour-bearing mice and in breast cancer cell line xenograft models.MethodsMurine small bowel epithelium was used to examine the kinetics and mechanism of low-dose UCN-01-mediated arrest of normal proliferating cells and if it can protect tumour-bearing mice (MDA-MB-468 xenografts) against the toxic effects of chemotherapy (5-fluorouricil (5-FU)) allowing for its full therapeutic activity.ResultsUCN-01 causes significant, reversible arrest of normal gut epithelial cells at 24 h; this arrest persists for up to 7 days. Normal cellular proliferation returns by 2 weeks. Pre-treatment of both non-tumour-bearing and MDA-MB-468 tumour-bearing mice with UCN-01 prior to bolus 5-FU (450 mg/kg) yielded enhanced therapeutic efficacy with significantly decreased tumour volumes and increased survival.ConclusionsUCN-01 mediates a specific, reversible G1 arrest of normal cells in vivo and provides a cytoprotective strategy that decreases toxicity of cytotoxic chemotherapy without compromising efficacy.

Amphiregulin deletion strongly attenuates the development of estrogen receptor-positive tumors in p53 mutant mice.

The epidermal growth factor receptor ligand, Amphiregulin, is a transcriptional target of estrogen receptor alpha and is required for pubertal mammary gland development. Previous studies using immortalized human breast cancer cell line xenografts have suggested that Amphiregulin may be an important effector of estrogen receptor alpha during breast cancer development, at least in immune-compromised animals. Here, we evaluate the requirement for Amphiregulin in an immune-competent mouse model which is prone to developing estrogen receptor-positive tumors. We have intercrossed mice with mammary-specific mutation of p53 with mice deficient in Amphiregulin in order to assess the requirement for Amphiregulin in the initiation and progression of both estrogen receptor-positive and estrogen receptor-negative mammary tumors. Deletion of Amphiregulin significantly delayed the onset of palpable mammary tumors and also strongly reduced the proportion of estrogen receptor alpha-positive tumors formed. Upon necropsy, no substantial differences in the prevalence of non-palpable lesions were observed between cohorts, suggesting that the importance of Amphiregulin in mammary tumorigenesis is limited to the post-initiation phase. This study underlines the importance of the EGFR ligand, Amphiregulin, as a key mediator of estrogen receptor action in breast cancer.

Abstract 4561: Slit2 inhibits breast cancer growth and metastasis by activating anti-tumor immune response

Abstract Metastasis is a major cause of mortality in breast cancer patients in part due to the lack of clinically established targeted therapies. Approximately 5%-20% of patients with Stage II, and ~50% of patients with stage III will recur distally and are likely to die from their disease. Metastatic, or stage IV breast cancers, have a 5-year relative survival rate of about 22%. The expression of a tumor suppressor protein, Slit2 has been shown to be downregulated in various types of tumors including breast cancer. The Slit2 acts through Roundabout Homolog1 (Robo1) receptor. Previously it has been shown that ectopic expression of Slit2 inhibits human MCF-7 breast cancer cell line xenograft tumor growth in mice. However, its role in breast cancer metastasis, tumor microenvironment (TME) and anti-tumor immunity has not been studied before. By using genetically engineered human breast cancer cells, spontaneous mammary tumor and pre-clinical mouse models, we evaluated the role of Slit2 in inhibiting breast cancer growth, metastasis by activating anti-tumor immune response. To study the role of Slit2 in breast cancer, we implanted Slit2 overexpressing human breast cancer cell line MDA-MB-231 (231-Sli2) or vector control cells (231-Vec) to the mammary fat-pads of NOD/SCID/gamma (NSG) mice and observed that 231-Slit2 had significantly reduced tumor growth and metastasis to the lungs compared to 231-Vec. To further confirm the anti-metastatic role of Slit2, we treated mouse mammary tumor virus- Polyoma Middle T antigen (MMTV-PyMT) mammary tumor model and MVT-1 orthotopic tumor bearing FVB/J wildtype mice with recombinant Slit2 (rSlit2) that resulted in significantly reduced tumor growth and metastasis to the lungs in both the models compared to PBS treated mice. The ex-vivo immunofluorescence and flow cytometry studies revealed that Slit2 treated tumors possess a very high number of tumor phagocytic macrophages compared to PBS. In-vitro analysis also showed that rSlit2 treated mouse macrophages (RAW264.7) has higher bacterial particle phagocytic ability. Further analysis of tumors elucidated that Slit2 treated tumors recruited higher number of CD4+ and CD8+ T-cells. In addition, The CD8+ cells were also positive for Granzyme-b showing higher number of effector T-cells in the Slit2 tumors compared to PBS. By using human breast cancer tissue microarray (TMA), we have found that Slit2 expression significantly correlates with better overall survival. These observations highlight the ability of Slit2 to enhance tumor phagocytic macrophages and anti-tumor CD8+/Granzyme-b+ T-cells, thereby restricting tumor growth and lung metastasis. These studies suggest that Slit2 could be used as a novel immunomodulatory therapeutic agent. Citation Format: Dinesh K. Ahirwar, Nabanita Chatterjee, Sanjay Mishra, Kontestine Shilo, Ramesh Ganju. Slit2 inhibits breast cancer growth and metastasis by activating anti-tumor immune response [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 4561.

Characterization of immune responses to anti-PD-1 mono and combination immunotherapy in hematopoietic humanized mice implanted with tumor xenografts

BackgroundThe success of agents that reverse T-cell inhibitory signals, such as anti-PD-1/PD-L1 therapies, has reinvigorated cancer immunotherapy research. However, since only a minority of patients respond to single-agent therapies, methods to test the potential anti-tumor activity of rational combination therapies are still needed. Conventional murine xenograft models have been hampered by their immune-compromised status; thus, we developed a hematopoietic humanized mouse model, hu-CB-BRGS, and used it to study anti-tumor human immune responses to triple-negative breast cancer (TNBC) cell line and patient-derived colorectal cancer (CRC) xenografts (PDX).MethodsBALB/c-Rag2nullIl2rγnullSIRPαNOD (BRGS) pups were humanized through transplantation of cord blood (CB)-derived CD34+ cells. Mice were evaluated for human chimerism in the blood and assigned into experimental untreated or nivolumab groups based on chimerism. TNBC cell lines or tumor tissue from established CRC PDX models were implanted into both flanks of humanized mice and treatments ensued once tumors reached a volume of ~150mm3. Tumors were measured twice weekly. At end of study, immune organs and tumors were collected for immunological assessment.ResultsHumanized PDX models were successfully established with a high frequency of tumor engraftment. Humanized mice treated with anti-PD-1 exhibited increased anti-tumor human T-cell responses coupled with decreased Treg and myeloid populations that correlated with tumor growth inhibition. Combination therapies with anti-PD-1 treatment in TNBC-bearing mice reduced tumor growth in multi-drug cohorts. Finally, as observed in human colorectal patients, anti-PD-1 therapy had a strong response to a microsatellite-high CRC PDX that correlated with a higher number of human CD8+ IFNγ+ T cells in the tumor.ConclusionHu-CB-BRGS mice represent an in vivo model to study immune checkpoint blockade to human tumors. The human immune system in the mice is inherently suppressed, similar to a tumor microenvironment, and thus allows growth of human tumors. However, the suppression can be released by anti-PD-1 therapies and inhibit tumor growth of some tumors. The model offers ample access to lymph and tumor cells for in-depth immunological analysis. The tumor growth inhibition correlates with increased CD8 IFNγ+ tumor infiltrating T cells. These hu-CB-BRGS mice provide a relevant preclinical animal model to facilitate prioritization of hypothesis-driven combination immunotherapies.

The novel function of tumor protein D54 in regulating pyruvate dehydrogenase and metformin cytotoxicity in breast cancer

BackgroundThe role of tumor protein D54 in breast cancer has not been studied and its function in breast cancer remains unclear. In our previous pharmacogenomic studies using lymphoblastoid cell line (LCL), this protein has been identified to affect metformin response. Although metformin has been widely studied as a prophylactic and chemotherapeutic drug, there is still a lack of biomarkers predicting the response to metformin in breast cancer. In this study, we revealed the novel function of TPD54 in breast cancer through understanding how TPD54 altered the cancer cell sensitivity to metformin.MethodsThe role of TPD54 in altering cellular sensitivity to metformin treatment was carried out by either knockdown or overexpression of TPD54, followed by measuring cell viability and reactive oxygen species (ROS) production in MCF7 breast cancer cell line and breast cancer patient-derived xenografts. Functional analysis of TPD54 in breast cancer cells was demonstrated by studying TPD54 protein localization and identification of potential binding partners of TPD54 through immunoprecipitation followed by mass spectrometry. The effect of TPD54 on pyruvate dehydrogenase (PDH) protein regulation was demonstrated by western blot, immunoprecipitation, and site-directed mutagenesis.ResultsTPD54 inhibited colony formation and enhanced cellular sensitivity to metformin treatment in MCF7 cells and breast cancer patient-derived xenografts. Mechanistic study indicated that TPD54 had mitochondrial localization, bound to and stabilized pyruvate dehydrogenase E1α by blocking pyruvate dehydrogenase kinase 1 (PDK1)-mediated serine 232 phosphorylation. TPD54 knockdown increased PDH E1α protein degradation and led to decreased PDH enzyme activity, which reduced mitochondrial oxygen consumption and reactive oxygen species (ROS) production, thus contributing to the resistance of breast cancer cells to metformin treatment.ConclusionWe have discovered a novel mechanism by which TPD54 regulates pyruvate dehydrogenase and affects the sensitivity of breast cancer to metformin treatment. Our findings highlight the important post-translational regulation of PDK1 on PDH E1α and the potential application of TPD54 as a biomarker for selecting tumors that may be sensitive to metformin therapy. These provide new insights into understanding the regulation of PDH complexes and the resistance mechanisms of cancer cells to metformin treatment.

Synergistic Activity of Paclitaxel, Sorafenib, and Radiation Therapy in advanced Renal Cell Carcinoma and Breast Cancer

Advanced cancer has been shown to be associated with a higher percentage of epigenetic changes than with genetic mutations. Preclinical models have shown that the combination of paclitaxel, sorafenib, and radiation therapy (RT) plays a crucial role in renal cell carcinoma (RCC) and breast cancer. This study aimed to investigate the involvement of mitochondrial cytochrome c–dependent apoptosis in the mechanism of action of a combination of paclitaxel, sorafenib, and RT in RCC and breast cancer. RCC and breast cancer cell lines were exposed to paclitaxel and sorafenib alone or combined in the presence of radiation, and cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The synergistic anticancer effects of the combination therapy on cell cycle and intracellular signaling pathways were estimated using flow cytometry and immunoblot analysis. RCC and breast cancer cell line xenograft models were used to examine the antitumor activity in vivo. Our results suggest that paclitaxel, sorafenib, and RT synergistically decreased the viability of RCC and breast cancer cells and significantly induced their apoptosis, as shown by caspase-3 cleavage. Paclitaxel, sorafenib, and radiation cotreatment reduced antiapoptotic factor levels in these cells and, thereby, significantly reduced the tumor volume of RCC and breast cancer cell xenografts. The current study suggests that paclitaxel, sorafenib, and radiation cotreatment was more effective than cotreatment with paclitaxel or sorafenib and radiation. These findings may offer a new therapeutic approach to RCC and breast cancer.

Loss of the Nuclear Pool of Ubiquitin Ligase CHIP/STUB1 in Breast Cancer Unleashes the MZF1-Cathepsin Pro-oncogenic Program

CHIP/STUB1 ubiquitin ligase is a negative co-chaperone for HSP90/HSC70, and its expression is reduced or lost in several cancers, including breast cancer. Using an extensive and well-annotated breast cancer tissue collection, we identified the loss of nuclear but not cytoplasmic CHIP to predict more aggressive tumorigenesis and shorter patient survival, with loss of CHIP in two thirds of ErbB2+ and triple-negative breast cancers (TNBC) and in one third of ER+ breast cancers. Reduced CHIP expression was seen in breast cancer patient-derived xenograft tumors and in ErbB2+ and TNBC cell lines. Ectopic CHIP expression in ErbB2+ lines suppressed in vitro oncogenic traits and in vivo xenograft tumor growth. An unbiased screen for CHIP-regulated nuclear transcription factors identified many candidates whose DNA-binding activity was up- or downregulated by CHIP. We characterized myeloid zinc finger 1 (MZF1) as a CHIP target, given its recently identified role as a positive regulator of cathepsin B/L (CTSB/L)-mediated tumor cell invasion downstream of ErbB2. We show that CHIP negatively regulates CTSB/L expression in ErbB2+ and other breast cancer cell lines. CTSB inhibition abrogates invasion and matrix degradation in vitro and halts ErbB2+ breast cancer cell line xenograft growth. We conclude that loss of CHIP remodels the cellular transcriptome to unleash critical pro-oncogenic pathways, such as the matrix-degrading enzymes of the cathepsin family, whose components can provide new therapeutic opportunities in breast and other cancers with loss of CHIP expression.Significance: These findings reveal a novel targetable pathway of breast oncogenesis unleashed by the loss of tumor suppressor ubiquitin ligase CHIP/STUB1. Cancer Res; 78(10); 2524-35. ©2018 AACR.

IL6 blockade potentiates the anti-tumor effects of γ-secretase inhibitors in Notch3-expressing breast cancer.

Notch pathways have important roles in carcinogenesis including pathways involving the Notch1 and Notch2 oncogenes. Pan-Notch inhibitors, such as gamma secretase inhibitors (GSIs), have been used in the clinical trials, but the outcomes of these trials have been insufficient and have yielded unclear. In the present study, we demonstrated that GSIs, such as MK-0752 and RO4929097, inhibit breast tumor growth, but increase the breast cancer stem cell (BCSC) population in Notch3-expressing breast cancer cells, in a process that is coupled with IL6 induction and is blocked by the IL6R antagonist Tocilizumab (TCZ). IL6 induction results from inhibition of Notch3-Hey2 signaling through MK-0752. Furthermore, HIF1α upregulates Notch3 expression via direct binding to the Notch3 promoter and subsequently downregulates BCSCs by decreasing the IL6 levels in Notch3-expressing breast cancer cells. Utilizing both breast cancer cell line xenografts and patient-derived xenografts (PDX), we showed that the combination of MK-0752 and Tocilizumab significantly decreases BCSCs and inhibits tumor growth and thus might serve as a novel therapeutic strategy for treating women with Notch3-expressing breast cancers.

Abstract 3106: Targeted delivery via Elastin Like Polypeptide: Biodistribution of a doxorubicin derivative in MDA-MB231 human breast cancer xenografts in mice

Abstract The anticancer agent doxorubicin has been widely used in the treatment of a variety of hematological malignancies and solid tumors. Despite doxorubicin’s efficiency in eradicating tumor cells, severe damage to healthy tissues, along with cardiotoxicity, limits its use in clinics. To overcome adverse side effects, improve patient safety, and enhance therapeutic efficacy, we have designed a thermally responsive biopolymer doxorubicin carrier that can be specifically targeted to tumor tissue by locally applying mild hyperthermia (41 degrees C). The developed drug vehicle is composed of: a cell penetrating peptide (SynB1) to promote tumor and cellular uptake; thermally responsive Elastin like polypeptide (ELP); and the (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH) containing a pH-sensitive hydrazine linker that releases dox in the acidic tumor environment. We used the in vivo imaging system, IVIS, to determine the biodistribution of doxorubicin-delivered ELP in MDA-MB231 triple negative human breast cancer cell line xenografts in nude mice. Tumor bearing mice were treated with a single IV injection of 10 mg/kg doxorubicin equivalent dose with free doxorubicin, thermally responsive SynB1 ELP 1-DOXO, and a thermally nonresponsive control biopolymer, SynB1 ELP 2-DOXO. Following a 2 hour treatment with hyperthermia, tumors showed a 2-fold higher uptake when treated with SynB1 ELP 1-DOXO compared to free doxorubicin. Accumulation of the thermally non-responsive control SynB1 ELP2 -DOXO was comparable to free doxorubicin, indicating that an increase in dox accumulation with ELP is due to aggregation in response to thermal targeting. Higher levels of SynB1 ELP1 -DOXO and SynB1 ELP2 -DOXO with respect to free doxorubicin were observed in kidneys. Fluorescence intensity from hearts of animals treated with SynB1 ELP1 - DOXO show a 5-fold lower accumulation of doxorubicin than the same dose of free doxorubicin. SynB1-ELP1-DOXO biopolymers demonstrated a 6-fold increase in tumor/heart ratio in comparison to free doxorubicin, indicating preferential accumulation of the drug carrier in tumors. Overall, thermally targeted polymers are a promising therapy to enhance tumor targeting and uptake of anticancer drugs and to minimize free drug toxicity in healthy tissues, representing a great potential for clinical application. Citation Format: Sonja Dragojevic, Jung Su Ryu, Felix Kratz, Drazen Raucher. Targeted delivery via Elastin Like Polypeptide: Biodistribution of a doxorubicin derivative in MDA-MB231 human breast cancer xenografts in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3106. doi:10.1158/1538-7445.AM2017-3106

Overcoming resistance to HER2-targeted therapy with a novel HER2/CD3 bispecific antibody

ABSTRACTT-cell-based therapies have emerged as one of the most clinically effective ways to target solid and non-solid tumors. HER2 is responsible for the oncogenesis and treatment resistance of several human solid tumors. As a member of the HER family of tyrosine kinase receptors, its over-activity confers unfavorable clinical outcome. Targeted therapies directed at this receptor have achieved responses, although development of resistance is common. We explored a novel HER2/CD3 bispecific antibody (HER2-BsAb) platform that while preserving the anti-proliferative effects of trastuzumab, it recruits and activates non-specific circulating T-cells, promoting T cell tumor infiltration and ablating HER2(+) tumors, even when these are resistant to standard HER2-targeted therapies. Its in vitro tumor cytotoxicity, when expressed as EC50, correlated with the surface HER2 expression in a large panel of human tumor cell lines, irrespective of lineage or tumor type. HER2-BsAb-mediated cytotoxicity was relatively insensitive to PD-1/PD-L1 immune checkpoint inhibition. In four separate humanized mouse models of human breast cancer and ovarian cancer cell line xenografts, as well as human breast cancer and gastric cancer patient-derived xenografts (PDXs), HER2-BsAb was highly effective in promoting T cell infiltration and suppressing tumor growth when used in the presence of human peripheral blood mononuclear cells (PBMC) or activated T cells (ATC). The in vivo and in vitro antitumor properties of this BsAb support its further clinical development as a cancer immunotherapeutic.