Mammary Tumor Model Research Articles

P21CIP1 Promotes Mammary Cancer-Initiating Cells via Activation of Wnt/TCF1/CyclinD1 Signaling.

Cancer stem cells (CSC) generate and sustain tumors due to tumor-initiating potential, resulting in recurrence or metastasis. We showed that knockout of the cell-cycle inhibitor, p21CIP1, in the PyMT mammary tumor model inhibits metastasis; however the mechanism remained unknown. Here, we show a pivotal role for p21 in potentiating a cancer stem-like phenotype. p21 knockout in PyMT mammary tumor cells caused dramatic suppression of CSC properties involving tumorsphere formation, ALDH1 activity, and tumor-initiating potential, which were in turn rescued by p21 overexpression into PyMT/p21 knockout cells. Interestingly, p21 knockout dramatically suppresses Wnt/β-catenin signaling activity, leading to striking inhibition of LEF1 and TCF1 expression. TCF1 knockdown in PyMT cells suppressed tumorsphere formation due to Cyclin D1 attenuation. These data demonstrate that p21 promotes a CSC-like phenotype via activation of Wnt/TCF1/Cyclin D1 signaling. IMPLICATIONS: p21 is a strong promoter of mammary CSCs.

Abstract 1993: Gluthathione peroxidase 2 downregulation in the PyMT mammary tumor model leads to an aggressive phenotype due to stimulation of angiogenesis and tumor stemness via HIF1 alpha signaling

Abstract Breast tumors are known to undergo high levels of mitochondrial oxidative phosphorylation leading to accumulation of reactive oxygen species (ROS), which in turn potentiate cancer stem cells and chemoresistance. This was shown to be due to HIF1α stabilization by ROS; however, the signaling events downstream of HIF1α regulating the cancer stem cell (CSC) phenotype remain unknown. Kaplan Meier survival curves upon the gene profiling database of breast cancer patients showed that reduced levels of the ROS scavenging enzyme, glutathione peroxidase 2 (GPx2) in breast cancers was associated with shorter patient survival duration. Consistent with this idea, we found that GPx2 was downregulated in metastatic mammary tumor cells, suggesting GPx2 loss enhances metastasis. Indeed, GPx2 knockdown in poorly metastatic cells increased ROS production, shortened tumor latency, and stimulated dramatic mammary tumor growth and metastasis. These effects were associated with marked increases in angiogenesis, CSC-like properties, and chemoresistance. Here, we show that GPx2 downregulation in mammary epithelial cells increases ROS production and HIF1α stabilization which were accompanied by VEGF upregulation and enhanced angiogenesis. On the other hand, GPx2 loss caused dramatic mammosphere formation and resistance to doxorubicin, implying effects on stemness leading to chemoresistance. These data suggest that GPx2 loss is a critical event that drives mammary tumorigenesis and metastasis via HIF1α signaling leading to stimulation of angiogenesis and stemness properties. Understanding the ramifications of GPx2 loss in breast cancer will allow us to identify key regulators of stemness and chemoresistance, with the goal of identifying novel drug targets in metastasis. Citation Format: Zuen Ren, Huizhi Liang, Outhiriaradjou Benard, Kimita Suyama, Joseph M. Albanese, Larry Norton, Wei Zheng, Rachel B. Hazan. Gluthathione peroxidase 2 downregulation in the PyMT mammary tumor model leads to an aggressive phenotype due to stimulation of angiogenesis and tumor stemness via HIF1 alpha signaling [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 1993.

PD-1 Blockade During Post-partum Involution Reactivates the Anti-tumor Response and Reduces Lymphatic Vessel Density.

Post-partum breast cancer patients, or breast cancer patients diagnosed within 10 years of last childbirth, are ~3–5 times more likely to develop metastasis in comparison to non-post-partum, or nulliparous, patients. Additionally, post-partum patients have increased tumor-associated lymphatic vessels and LN involvement, including when controlled for size of the primary tumor. In pre-clinical, immune-competent, mouse mammary tumor models of post-partum breast cancer (PPBC), tumor growth and lymphogenous tumor cell spread occur more rapidly in post-partum hosts. Here we report on PD-L1 expression by lymphatic endothelial cells and CD11b+ cells in the microenvironment of post-partum tumors, which is accompanied by an increase in PD-1 expression by T cells. Additionally, we observed increases in PD-L1 and PD-1 in whole mammary tissues during post-partum mammary gland involution; a known driver of post-partum tumor growth, invasion, and metastasis in pre-clinical models. Importantly, implantation of murine mammary tumor cells during post-partum mammary gland involution elicits a CD8+ T cell population that expresses both the co-inhibitory receptors PD-1 and Lag-3. However, upon anti-PD-1 treatment, during post-partum mammary gland involution, the involution-initiated promotional effects on tumor growth are reversed and the PD-1, Lag-3 double positive population disappears. Consequently, we observed an expansion of poly-functional CD8+ T cells that produced both IFNγ and TNFα. Finally, lymphatic vessel frequency decreased significantly following anti-PD-1 suggesting that anti-PD-1/PD-L1 targeted therapies may have efficacy in reducing tumor growth and dissemination in post-partum breast cancer patients.

High-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity

BackgroundDespite promising treatments for breast cancer, mortality rates remain high and treatments for metastatic disease are limited. High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation technique that utilizes high-frequency bipolar electric pulses to destabilize cancer cell membranes and induce cell death. However, there is currently a paucity of data pertaining to immune system activation following H-FIRE and other electroporation based tumor ablation techniques. MethodsHere, we utilized the mouse 4T1 mammary tumor model to evaluate H-FIRE treatment parameters on cancer progression and immune system activation in vitro and in vivo. FindingsH-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment. We further show that local treatment with H-FIRE significantly reduces 4T1 metastases. H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo. Our data indicate that the level of tumor ablation correlates with increased activation of cellular immunity. Likewise, we show that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression. InterpretationCell death and tumor ablation following H-FIRE treatment activates the local innate immune system, which shifts the tumor microenvironment from an anti-inflammatory state to a pro-inflammatory state. The non-thermal damage to the cancer cells and increased innate immune system stimulation improves antigen presentation, resulting in the engagement of the adaptive immune system and improved systemic anti-tumor immunity.

Pro-Apoptotic and Anti-Angiogenesis Effects of Olive Leaf Extract on Spontaneous Mouse Mammary Tumor Model by Balancing Vascular Endothelial Growth Factor and Endostatin Levels

It has been proven that olive associated products such as olive leaf extract (OLE) causes significant reduction in cancer cells viability and proliferation. Female BALB/c adult mice were divided into four groups. Three days prior to oral treatments, tumors were transplanted. First group were treated with distilled water and other three groups were received, respectively, 75, 150, and 225 mg/kg/day of OLE for three weeks. For assessment of anti-angiogenesis and pro-apoptotic effect of OLE on tumor tissue, tumor volume, cell mitosis and apoptosis, and also vascular endothelial growth factor (VEGF) and endostatin levels were assessed. OLE treatment with 150 and 225 mg/kg/day lead to significant reduction in tumor volume and cell mitosis compared with the control group, while the same doses significantly increase tumor cell apoptosis. OLE treatment with 150 mg/kg/day increase endostatin levels, while the same dose did not significantly decrease VEGF levels. The VEGF level is significantly reduced by the treatment with OLE 225 mg/kg/day for three weeks. Although, further studies are needed to clarify anti-angiogenesis and anti-apoptotic mechanism of OLE, consumption of OLE polyphenols after tumor transplantation reduced spontaneous mouse mammary tumor growth.

Integrin α9 depletion promotes β-catenin degradation to suppress triple-negative breast cancer tumor growth and metastasis.

Although integrin α9 (ITGA9) is known to be involved in cell adhesion and motility, its expression in cancer and its role in tumor growth and metastasis remain largely unknown. Our study was designed to investigate the role of ITGA9 in triple-negative breast cancer (TNBC). ITGA9 expression in TNBC cells was knocked out (KO) using CRISPR/Cas9 technology. Four orthotopic mouse mammary xenograft tumor models coupled with cell culture studies were performed to determine the effect of ITGA9 depletion on TNBC tumor growth and metastasis and the underlying mechanism. Bioinformatics analysis showed that ITGA9 level is significantly higher in TNBC than other breast cancer subtypes, and higher ITGA9 level is associated with significantly worse distant metastasis-free survival and recurrence-free survival in TNBC patients. Experimentally, ITGA9 KO significantly reduced TNBC cell cancer stem cell (CSC)-like property, tumor angiogenesis, tumor growth and metastasis by promoting β-catenin degradation. Further mechanistic studies revealed that ITGA9 KO causes integrin-linked kinase (ILK) relocation from the membrane region to the cytoplasm, where it interacts with protein kinase A (PKA) and inhibits PKA activity leading to increased activity of glycogen synthase kinase 3 (GSK3) and subsequent β-catenin degradation. Overexpressing β-catenin in ITGA9 KO cells reversed the inhibitory effect of ITGA9 KO on tumor growth and metastasis. Furthermore, ITGA9 downregulation in TNBC tumors by nanoparticle-mediated delivery of ITGA9 siRNA drastically decreased tumor angiogenesis, tumor growth and metastasis. These findings indicate that ITGA9 depletion suppresses TNBC tumor growth and metastasis by promoting β-catenin degradation through the ILK/PKA/GSK3 pathway.

Identification of a Rat Mammary Tumor Risk Locus That Is Syntenic with the Commonly Amplified 8q12.1 and 8q22.1 Regions in Human Breast Cancer Patients.

Breast cancer risk is 31% heritable, yet the majority of the underlying risk factors remain poorly defined. Here, we used F2-linkage analysis in a rat mammary tumor model to identify a novel 11.2 Mb modifier locus of tumor incidence and burden on rat chromosome 5 (chr5: 15.4 – 26.6 Mb). Genomic and RNA sequencing analysis identified four differentially expressed candidates: TMEM68, IMPAD1, SDCBP, and RBM12B. Analysis of the human syntenic candidate region revealed that SDCBP is in close proximity to a previously reported genetic risk locus for human breast cancer. Moreover, analysis of the candidate genes in The Cancer Genome Atlas (TCGA) revealed that they fall within the commonly amplified 8q12.1 and 8q22.1 regions in human breast cancer patients and are correlated with worse overall survival. Collectively, this study presents novel evidence suggesting that TMEM68, IMPAD1, SDCBP, and RBM12B are potential modifiers of human breast cancer risk and outcome.

Targeted Delivery of Chlorin e6 via Redox Sensitive Diselenide-Containing Micelles for Improved Photodynamic Therapy in Cluster of Differentiation 44-Overexpressing Breast Cancer.

The off-target activation of photosensitizers is one of the most well-known obstacles to effective photodynamic therapy (PDT). The selected activation of photosensitizers in cancer cells is highly desired to overcome this problem. We developed a strategy that enabled diselenide bonds to link hyaluronic acid (HA) and photosensitizer chlorin e6 (Ce6) to assemble the micelles (HA-sese-Ce6 NPs) that can target cancer and achieve a redox responsive release of drugs to enhance the PDT efficiency in breast cancer. The HA was used to form a hydrophilic shell that can target cluster of differentiation 44 (CD44) on the cancer cells. The selenium-containing core is easily dissembled in a redox environment to release Ce6. The triggered release of Ce6 in a redox condition and the positive feedback release by activated Ce6 were observed in vitro. In cytotoxicity assays and in vitro cellular uptake assays, the increased PDT efficiency and targeted internalization of HA-sese-Ce6 NPs in the cells were verified, compared to a free Ce6 treated group. Similar results were showed in the therapeutic study and in vivo fluorescence imaging in an orthotopic mammary fat pad tumor model. In addition, a significant inhibition of metastasis was found after the HA-sese-Ce6 NPs treatment. In general, this study promises an ingenious and easy strategy for improved PDT efficiency.

Diverse Roles of the Plasminogen Receptor, Plg‐RKT, in Mammary Tumor Development and Metastasis

ObjectivesPlg‐RKT is a unique transmembrane plasminogen receptor, highly expressed on tumor cells and macrophages. We investigated the role of Plg‐RKT in mammary tumor incidence, development, growth and metastasis in a PyMT transgenic mouse mammary tumor model.MethodsPlg‐RKT deficiency was bred into PyMT mice to compare mammary tumor development in PyMT/Plg‐RKT+/+ and PyMT/Plg‐RKT−/− female littermates.ResultsMultifocal mammary tumor development was observed in all animals with no difference between genotypes in time of first appearance of palpable tumors. Mice in both groups developed mammary tumors in 3 – 10 mammary glands per animal. However, the mean number of tumors in PyMT/Plg‐RKT−/− mice was significantly less than in PyMT/Plg‐RKT+/+ mice. Tumors in PyMT/Plg‐RKT−/− mice grew more slowly than tumors in PyMT/Plg‐RKT+/+ mice. At 20 weeks of age, cumulative mammary tumor volumes and tumor weights were significantly less for PyMT/Plg‐RKT−/− mice. Histological examination revealed abundant fibrin deposition in mammary tumors in PyMT/Plg‐RKT−/− mice but not in PyMT/Plg‐RKT+/+ mice, suggesting dysregulated fibrin surveillance and markedly dysregulated plasminogen activation due to the absence of Plg‐RKT. Early mammary tumors also differed markedly in their morphology: mammary foci in PyMT/Plg‐RKT−/− mice were primarily adenomas and were surrounded by a pronounced collagen‐rich extracellular matrix; whereas PyMT/Plg‐RKT+/+ mice had large, hyperproliferative tumor nodules that were not completely surrounded by extracellular matrix.We compared lung metastasis in PyMT/Plg‐RKT−/− and PyMT/Plg‐RKT+/+ mice, expecting fewer metastases in PyMT/Plg‐RKT−/− mice due to their decreased mammary tumor burden. Surprisingly, at 20 weeks of age PyMT/Plg‐RKT−/− mice had more metastatic lung tumors and a higher metastatic burden when tumor cells in the lungs were quantified by PCR for PyMT transcript expression. To evaluate the influence of mammary tumor burden on metastasis, we analyzed metastasis in a small cohort (n=8) of PyMT/Plg‐RKT−/− mice that at 22 weeks of age, had developed a mammary tumor burden similar to PyMT/Plg‐RKT+/+ mice at 20 weeks. When comparing metastasis in these mice with comparable tumor size, the larger metastatic burden in PyMT/Plg‐RKT−/− mice was even more striking. Staining of lung tissues for the pan‐macrophage marker (F4/80) revealed marked infiltration of macrophages both in the lung parenchyma and peritumoral to lung foci in PyMT/Plg‐RKT+/+ but not PyMT/Plg‐RKT−/− mice.ConclusionsTogether these results show that Plg‐RKT supports mammary tumor development and growth, and suggest that Plg‐RKT alters the ECM to promote tumorigenesis. Conversely, Plg‐RKT inhibits tumor metastasis, possibly because Plg‐RKT on non‐tumor cells, i.e., monocytes/macrophages, contributes to an anti‐metastatic environment in the target organ.Support or Funding InformationHL 081046, CA166473, Merit Rev. #5I01BX002026This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NIR-Triggered Phototherapy and Immunotherapy via an Antigen-Capturing Nanoplatform for Metastatic Cancer Treatment.

Combined phototherapy and immunotherapy demonstrates strong potential in the treatment of metastatic cancers. An upconversion nanoparticle (UCNP) based antigen‐capturing nanoplatform is designed to synergize phototherapies and immunotherapy. In particular, this nanoplatform is constructed via self‐assembly of DSPE‐PEG‐maleimide and indocyanine green (ICG) onto UCNPs, followed by loading of the photosensitizer rose bengal (RB). ICG significantly enhances the RB‐based photodynamic therapy efficiency of UCNP/ICG/RB‐mal upon activation by a near‐infrared (NIR) laser, simultaneously achieving selective photothermal therapy. Most importantly, tumor‐derived protein antigens, arising from phototherapy‐treated tumor cells, can be captured and retained in situ, due to the functionality of maleimide, which further enhance the tumor antigen uptake and presentation by antigen‐presenting cells. The synergized photothermal, photodynamic, and immunological effects using light‐activated UCNP/ICG/RB‐mal induces a tumor‐specific immune response. In the experiments, intratumoral administration of UCNP/ICG/RB‐mal, followed by noninvasive irradiation with an NIR laser, destroys primary tumors and inhibits untreated distant tumors, using a poorly immunogenic, highly metastatic 4T1 mammary tumor model. With the simultaneous use of anti‐CTLA‐4, about 84% of the treated tumor‐bearing mice achieve long‐term survival and 34% of mice develop tumor‐specific immunity. Overall, this antigen‐capturing nanoplatform provides a promising approach for the treatment of metastatic cancers.

Endothelial miR-30c suppresses tumor growth via inhibition of TGF-β-induced Serpine1.

In tumors, extravascular fibrin forms provisional scaffolds for endothelial cell (EC) growth and motility during angiogenesis. We report that fibrin-mediated angiogenesis was inhibited and tumor growth delayed following postnatal deletion of Tgfbr2 in the endothelium of Cdh5-CreERT2 Tgfbr2fl/fl mice (Tgfbr2iECKO mice). ECs from Tgfbr2iECKO mice failed to upregulate the fibrinolysis inhibitor plasminogen activator inhibitor 1 (Serpine1, also known as PAI-1), due in part to uncoupled TGF-β-mediated suppression of miR-30c. Bypassing TGF-β signaling with vascular tropic nanoparticles that deliver miR-30c antagomiRs promoted PAI-1-dependent tumor growth and increased fibrin abundance, whereas miR-30c mimics inhibited tumor growth and promoted vascular-directed fibrinolysis in vivo. Using single-cell RNA-Seq and a NanoString miRNA array, we also found that subtypes of ECs in tumors showed spectrums of Serpine1 and miR-30c expression levels, suggesting functional diversity in ECs at the level of individual cells; indeed, fresh EC isolates from lung and mammary tumor models had differential abilities to degrade fibrin and launch new vessel sprouts, a finding that was linked to their inverse expression patterns of miR-30c and Serpine1 (i.e., miR-30chi Serpine1lo ECs were poorly angiogenic and miR-30clo Serpine1hi ECs were highly angiogenic). Thus, by balancing Serpine1 expression in ECs downstream of TGF-β, miR-30c functions as a tumor suppressor in the tumor microenvironment through its ability to promote fibrin degradation and inhibit blood vessel formation.

Blood Interactions, Pharmacokinetics, and Depth-Dependent Ablation of Rat Mammary Tumors with Photoactivatable, Liposomal Doxorubicin.

Photosensitizers can be integrated with drug delivery vehicles to develop chemophototherapy agents with antitumor synergy between chemo- and photocomponents. Long-circulating doxorubicin (Dox) in porphyrin-phospholipid (PoP) liposomes (LC-Dox-PoP) incorporates a phospholipid-like photosensitizer (2 mole %) in the bilayer of Dox-loaded stealth liposomes. Hematological effects of endotoxin-minimized LC-Dox-PoP were characterized via standardized assays. In vitro interaction with erythrocytes, platelets, and plasma coagulation cascade were generally unremarkable, whereas complement activation was found to be similar to that of commercial Doxil. Blood partitioning suggested that both the Dox and PoP components of LC-Dox-PoP were stably entrapped or incorporated in liposomes. This was further confirmed with pharmacokinetic studies in Fischer rats, which showed the PoP and Dox components of the liposomes both had nearly identical, long circulation half-lives (25-26 hours). In a large orthotopic mammary tumor model in Fischer rats, following intravenous dosing (2 mg/kg Dox), the depth of enhanced Dox delivery in response to 665 nm laser irradiation was over 1 cm. LC-Dox-PoP with laser treatment cured or potently suppressed tumor growth, with greater efficacy observed in tumors 0.8 to 1.2 cm, compared with larger ones. The skin at the treatment site healed within approximately 30 days. Taken together, these data provide insight into nanocharacterization and photo-ablation parameters for a chemophototherapy agent.

Abstract PD7-07: Synergistic targeting of CDK4/6 and BCL-2 pathways in estrogen receptor positive breast cancer

Abstract Background: Despite incremental advances in chemotherapy and endocrine therapy, survival outcomes for patients with ER-positive (ER+) metastatic breast cancer (MBC) remain poor. The majority of relapsing tumors exhibit deregulation of the cyclin-dependent kinase 4 and 6 (CDK4/6)/cyclin D1 (CCND1)/Rb signaling pathway. CDK4/6 inhibitors (such as palbociclib) in combination with endocrine therapy have been shown to significantly improve progression free survival in patients who are in 1st or 2nd line relapse, although overall survival benefit has yet to be demonstrated. This may reflect their largely cytostatic mechanism of action, with minimal induction of tumor cell death. Thus, combinatorial strategies that also induce apoptosis could be beneficial. Notably, the pro-survival protein BCL-2 is overexpressed in the majority of ER+ tumors and the potent and specific BCL-2 inhibitor venetoclax (ABT-199) has been found to synergize with endocrine therapy in patient derived xenograft (PDX) models. Promising activity has also been observed in an early phase clinical trial. We therefore investigated dual targeting of the CDK4/6 and BCL-2 pathways in pre-clinical models of ER+ and BCL-2+ breast cancer. Results: We first examined endocrine sensitive or resistant cell-lines and found that pro-survival BCL-2 proteins were upregulated in resistant cells. BCL-2 family protein levels were also found to be elevated in palbociclib resistant cells, suggesting that BCL-2 could represent a therapeutic target. We next determined whether venetoclax improved response to dual therapy comprising the selective estrogen receptor degrader fulvestrant and palbociclib. In clonogenic assays of endocrine sensitive breast cancer cell lines, triple therapy containing venetoclax significantly reduced the number and size of colonies, when compared to double therapy. The addition of venetoclax to fulvestrant/palbociclib also augmented cell death in tumor organoid models derived from either ER+ BCL-2+ primary tumors or PDX models. Moreover, triple therapy improved tumor response and overall survival in mice bearing ER+ BCL-2+ PDX tumors. Mechanistically, this was accompanied by increased apoptosis and reduced cellular proliferation (as determined by cleaved caspase-3 and Ki67 levels, respectively). As CDK4/6 inhibitors have recently been shown to promote anti-tumor immunity, we evaluated immune modulation using the ER+ 67NR cell line in a syngeneic (BALB/c) mouse mammary tumor model. Similar to the PDX models, triple therapy comprising fulvestrant, palbociclib and venetoclax was more effective than double therapy comprising either fulvestrant/palbociclib or fulvestrant/venetoclax. Flow cytometric analysis of tumors revealed that this was accompanied by a reduced intratumoral FOXP3+:cytotoxic CD8 T-cell ratio. Conclusions: The addition of the BCL-2 inhibitor venetoclax to conventional therapy comprising endocrine therapy and a CDK4/6 inhibitor augments tumor response and elicits a favorable intratumoral immune profile. Collectively, these findings support investigation of combination therapy in the clinic for patients with ER+ BCL-2+ MBC. Citation Format: Whittle JR, Vaillant F, Policheni AN, Liu K, Pal B, Giner G, Fernandez K, Gray DH, Caldon CE, Smyth GK, Visvader JE, Lindeman GJ. Synergistic targeting of CDK4/6 and BCL-2 pathways in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-07.

Abstract P6-20-05: Therapeutic targeting of BRCA1 and TP53 mutant breast cancer through mutant p53 reactivation

Abstract Triple negative breast cancer (TNBC) is an aggressive subset for which novel therapeutic approaches are needed. A significant proportion of TNBC patients harbor either germline or somatic mutations in BRCA1, or epigenetic silencing of BRCA1, which renders them deficient in DNA repair. Virtually all BRCA1 deficient breast cancers harbor mutations in TP53 suggesting that inactivation of p53 is a requirement for tumor progression in the setting of BRCA1 deficiency. Due to this dependency, we hypothesized that restoring wild type p53 function in BRCA1 deficient breast cancer would be therapeutic. The majority of TP53 mutations are missense, which generate a defective protein that potentially can be targeted with small molecules. Zinc Metallochaperones (ZMCs) are a new class of anti-cancer drugs that reactivate a class of zinc deficient mutant TP53 alleles by restoring zinc binding. Using ZMC1 in human breast cancer cell lines expressing the zinc deficient p53R175H, we demonstrate that loss of BRCA1 sensitizes cells to mutant p53 reactivation. Using genetically engineered murine mammary tumor models with Brca1 deficiency, we demonstrate that ZMC1 significantly improves survival in mice bearing tumors harboring the zinc deficient Trp53R172H allele but not the Trp53 null allele. We synthesized a novel formulation of ZMC1 (Zn-1), in which the drug is made in complex with zinc to improve zinc delivery, and demonstrate that Zn-1 has increased efficacy over ZMC1. Furthermore, we show that ZMC1 plus olaparib is a highly effective combination for tumors expressing the p53R172H mutant. In conclusion, we have validated preclinically a novel therapeutic approach for BRCA1 deficient breast cancer through reactivation of mutant p53. Citation Format: Na B, Yu X, Wither T, Gilleran J, Yao M, Foo TK, Chen C, Moore D, Xia B, Lin Y, Kimball D, Ganesan S, Carpizo D. Therapeutic targeting of BRCA1 and TP53 mutant breast cancer through mutant p53 reactivation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-20-05.

Pyranocarbazole derivatives as potent anti-cancer agents triggering tubulin polymerization stabilization induced activation of caspase-dependent apoptosis and downregulation of Akt/mTOR in breast cancer cells

A series of new pyranocarbazole derivatives were synthesized via semi-synthetic modification of koenimbine (1a) and koenidine (1b) isolated from the leaves of Murraya koenigii. Among all, compound 3bg displayed significant anti-cancer activity against MDA-MB-231, DU145 and PC3 cell lines with the IC50 values of 3.8, 7.6 and 5.8 μM, respectively. It was also observed that the halogenated-benzyl substitution at N-9 position, C-3 Methyl and C-7 methoxy group on carbazole motif are favoured for anti-cancer activity. The detailed investigation was carried out with compound 3bg and its SEDDS (self-emulsifying drug delivery systems) formulation 3bgF. The in vivo drug release behavior study showed that the formulation enhanced slow release and better bioavailability at a tumor site. Compound 3bg and its formulation (3bgF) significantly inhibited cell proliferation and colony formation, induced G2/M arrest, reduced cellular ROS generation and induced caspase-dependent apoptosis in MDA-MB-231 cells. 3bg also induced significant alteration of Bax/Bcl expression ratio suggesting involvement of mitochondrial apoptosis. Additionally, 3bg caused down-regulation of mTOR/Akt survival pathway. 3bg do not bind to DNA, but interacts with tubulin as observed with in silico molecular docking studies. This interaction results in stabilization of tubulin polymerization similar to paclitaxel as detected in cell-free assay. Oral administration of 3bgF for 30 days at dose rate of 10 and 20 mg/kg body weight significantly reduced tumor growth in syngenic rat LA-7 mammary tumor model. These results indicated that the pyranocarbazole natural product based N-substituted analogues can act as potential anti-cancer lead.

Author Correction: Every step of the way: integrins in cancer progression and metastasis.

In the originally published article, pertuzumab was incorrectly described as an anti-PI3K therapy in the section 'Integrins in anticancer therapy'. The sentence should read 'In mouse mammary tumour models, increased collagen levels and increased β1 integrin and SRC activity have been demonstrated to accompany, and promote, combined resistance to anti-human epidermal growth factor receptor 2 (HER2; also known as ERBB2) (trastuzumab and pertuzumab) and anti-PI3K (buparlisib) therapies164.' This has now been corrected in all versions of the original article.

TPEN Exerts Antitumor Efficacy in Murine Mammary Adenocarcinoma Through an H2O2 Signaling Mechanism Dependent on Caspase-3.

Breast cancer is the second most common cancer worldwide. N, N, N', N'-Tetrakis (2-pyridylmethyl)-ethylenediamine (TPEN) is a lipid-soluble zinc metal chelator that induces apoptosis in cancer cells through oxidative stress (OS). However, the effectiveness and the mechanisms involved in TPENinduced cell death in mammary adenocarcinoma cells in vitro and in vivo are still unclear. This study aimed to evaluate the cytotoxic effect of TPEN in mouse embryonic fibroblasts (MEFs, as normal control cells) and mammary adenocarcinoma cancer cells (TS/A cells) in vitro and in a mammary tumor model in vivo. Cells were treated with TPEN (0-3 µM), and changes in nuclear chromatin and DNA, mitochondrial membrane potential (ΔΨm), and intracellular reactive oxygen species (ROS) levels were determined by both fluorescence microscopy and flow cytometry. Cell proliferation and the cell cycle were also analyzed. Cellular markers of apoptosis were evaluated by Western blot. Finally, the effect of TPEN in a mammary adenocarcinoma tumor model in vivo was determined by immunohistological analyses. TPEN induced apoptosis in TS/A cells in a dose-dependent manner, increasing nuclear chromatin condensation, DNA fragmentation, cell cycle arrest and ΔΨm loss. Additionally, TPEN increased dichlorofluorescein fluorescence (DCF+) intensity, indicative of ROS production; increased DJ-1-Cys106-sulfonate expression, a marker of intracellular H2O2 stress; induced p53 and PUMA upregulation; and activated caspase-3. Moreover, TPEN induced mammary cancer cell elimination and tumor size reduction in vivo 48 h after treatment through an OS-induced apoptotic mechanism. TPEN selectively induces apoptosis in TS/A cells through an H2O2-mediated signaling pathway. Our findings support the use of TPEN as a potential treatment for breast cancer.

Dynamic role of the codon 72 p53 single-nucleotide polymorphism in mammary tumorigenesis in a humanized mouse model

Female breast cancer (BrCa) is the most common noncutaneous cancer among women in the United States. Human epidemiological studies reveal that a p53 single-nucleotide polymorphism (SNP) at codon 72, encoding proline (P72) or arginine (R72), is associated with differential risk of several cancers, including BrCa. However, the molecular mechanisms by which these variants affect mammary tumorigenesis remain unresolved. To investigate the effects of this polymorphism on susceptibility to mammary cancer, we used a humanized p53 mouse model, homozygous for either P72 or R72. Our studies revealed that R72 mice had a significantly higher mammary tumor incidence and reduced latency in both DMBA-induced and MMTV-Erbb2/Neu mouse mammary tumor models compared to P72 mice. Analyses showed that susceptible mammary glands from E-R72 (R72 x MMTV-Erbb2/Neu) mice developed a senescence-associated secretory phenotype (SASP) with influx of proinflammatory macrophages, ultimately resulting in chronic, protumorigenic inflammation. Mammary tumors arising in E-R72 mice also had an increased influx of tumor-associated macrophages, contributing to angiogenesis and elevated tumor growth rates. These results demonstrate that the p53 R72 variant increased susceptibility to mammary tumorigenesis through chronic inflammation.

Deregulation of VEGFR-2 and PDGFR Expression and Microvascular Density in a Triple-Negative Model of Canine Malignant Mammary Tumors with Lymph Node or Lung Metastasis

Canine mammary tumors (CMT) represent the most common cancer in noncastrated female dogs. Interestingly, triple-negative tumors are the most common molecular subtype in female dogs. In this study, we proposed to evaluate the expression of vascular endothelial growth factor receptor 2 (VEGFR-2), Platelet-derived growth factor receptor (PDGFR), and microvascular density (MVD) in a group of metastatic and nonmetastatic triple-negative CMT and compare the expression based on clinical parameters. Twenty-six female dogs with triple-negative mammary tumors were divided into three groups: nonmetastatic tumors (NMT) (n = 11), tumors with lymph node metastasis (LNM) (n = 10), and tumors with lung metastasis (LM) (n = 5). We observed increased VEGFR-2 expression in LNM compared with NMT and a positive correlation between tumor grade and VEGFR-2 expression. A positive correlation was noted between VEGFR-2 and PDGFR expression. Regarding microvascular density (MVD), we identified a higher number of vessels in primary tumors with lymph node metastasis and lung metastasis compared with tumors with no metastasis. The primary tumors with lung metastasis exhibited an increased MVD compared with carcinoma with lymph node metastasis. Overall, our results suggest a deregulation of VEGFR-2 and PDGFR and high MVD in metastatic tumors, indicating a role for angiogenesis in tumor progression.

Emerging Role of Novel Biomarkers of Ly6 Gene Family in Pan Cancer.

Stem cell antigen-1 (Sca-1) is the first identified member of mouse Ly6 gene family. We discovered that Sca-1 disrupts TGFβ signaling and enhances mammary tumorigenesis in a DMBA-induced mammary tumor model. Sca-1 gene is lost during evolution in humans. Human Ly6 genes Ly6D, LyE, LyH, and LyK on human chromosome 8q24.3 genes are syntenic to the mouse chromosome 15 where Sca-1 is located. We found that Ly6D, E, H, and K are upregulated in human cancer compared to normal tissue and that the increased expression of these genes are associated with poor prognosis of multiple types of human cancer. Several other groups have indicated increased expression of Ly6 genes in human cancer. Here we described the relevance of expression of human Ly6D, LyE, LyH, and LyK in functioning of normal tissues and tumor progression.