Triple-negative Mammary Carcinoma Research Articles

Multifaceted antineoplastic curative potency of novel water-soluble methylimidazole-based oxidovanadium (IV) complex against triple negative mammary carcinoma

A bunch of complexes harboring vanadium as metal centers have been reported to exhibit a wide array of antineoplastic properties that come under non‑platinum metallodrug series and emerge to offer alternative therapeutic strategies from the mechanistic behaviors of platinum-drugs. Though antineoplastic activities of vanado-complexes have been documented against several animal and xenografted human cancers, the definite mechanism of action is yet to unveil. In present study, a novel water soluble 1-methylimidazole substituted mononuclear dipicolinic acid based oxidovanadium (IV) complex (OVMI) has been evaluated for its antineoplastic properties in breast carcinoma both in vitro and in vivo. OVMI has been reported to generate cytotoxicity in human triple negative breast carcinoma cells, MDA-MB-231 as well as in mouse 4T1 cells by priming them for apoptosis. ROS-mediated, mitochondria-dependent as well as ER-stress-evoked apoptotic death seemed to be main operational hub guiding the cytotoxicity of OVMI in vitro. Moreover, OVMI has been noticed to elicit antimetastatic effect in vitro. Therapeutic application of OVMI has been extended on 4T1-based mammary tumor of female Balb/c mice, where it has been found to reduce tumor size, volume and restore general tissue architecture successfully to a great extent. Apart from that, OVMI has been documented to limit 4T1-based secondary pulmonary metastasis along with being non-toxic and biocompatible in vivo.

Tupistra chinensis a traditional Chinese plant suppresses tumor growth and metastasis in vitro against ER positive and triple negative breast cancer

AimTo investigate the effects of Tupichinol E, an alkaloid present in Tupistra chinensis BAKER (Liliaceae), on the metastasis of triple-negative mammary carcinoma in vitro and elucidate the underlying mechanisms. Tupichinol E binding to EGFR stabilizes the protein structure and is a pharmacologically active substance that can be used against cancer due to its potent activity. MethodsThe growth of MCF-7 and MDA-MB-231 cells was assessed using the MTT assay. The apoptosis and cell cycle of MCF-7 cells were evaluated with flow cytometry, and the related proteins were examined using Western blotting. ResultsTupichinol E (50–200 μmol/L) inhibits the growth of MCF-7 cells in a dose- and time-dependent manner (the IC50 values are 85.05 ± 1.08 and 50.52 ± 1.06 μmol/L, respectively, at 48 and 72 h). Treatment of MCF-7 cells with Tupichinol E (50–200 μmol/L) dose-dependently induced apoptosis of MCF-7 cells, accompanying the activation of caspase 3. The cells treated with Tupichinol E (100 and 200 μmol/L) significantly increased the percentage of cells in the G2/M phase with a reduction in the expression of cyclin B1. ConclusionThese results demonstrated that Tupichinol E is an effective anti-tumor compound in vitro and has the potential to be developed as a new anticancer drug.

Immunoregulatory and Anti-cancer Activities of Combination Treatment of Novel Four-Herb Formula and Doxorubicin in 4T1-Breast Cancer Bearing Mice.

To investigate the in vivo immunomodulatory and anti-tumor mechanisms of the combined treatment of novel Four-Herb formula (4HF) and doxorubicin in triple-negative breast cancer (TNBC). Murine-derived triple-negative mammary carcinoma cell line, 4T1 cells, was cultured and inoculated into mouse mammary glands. Sixty-six mice were randomly assigned into 6 groups (n=11 in ench): naïve, control, LD 4HF (low dose 4HF), HD 4HF (high dose 4HF), LD 4HF + D (low dose and doxorubicin), and D (doxorubicin). Apart from the naïve group, each mouse received subcutaneous inoculation with 5 × 105 4T1 cells resuspended in 100 µL of normal saline in the mammary fat pads. Starting from the day of tumor cell inoculation, tumors were grown for 6 days. The LD and HD groups received daily oral gavage of 658 and 2,630 mg/kg 4HF, respectively. The LD 4HF+D group received daily oral gavage of 658 mg/kg 4HF and weekly intraperitoneal injection of doxorubicin (5 mg/kg). The D group received weekly intraperitoneal injections of doxorubicin (5 mg/kg). The treatment naïve mice received daily oral gavage of 0.2 mL double distilled water and 0.1 mL normal saline via intraperitoneal injection once a week. The control group received daily oral gavage of 0.2 mL double-distilled water. The treatment period was 30 days. At the end of treatment, mice organs were harvested to analyze immunological activities via immunophenotyping, gene and multiplex analysis, histological staining, and gut microbiota analysis. Mice treated with the combination of 4HF and doxorubicin resulted in significantly reduced tumor and spleen burdens (P<0.05), altered the hypoxia and overall immune lymphocyte landscape, and manipulated gut microbiota to favor the anti-tumor immunological activities. Moreover, immunosuppressive genes, cytokines, and chemokines such as C-C motif chemokine 2 and interleukin-10 of tumors were significantly downregulated (P<0.05). 4HF-doxorubicin combination treatment demonstrated synergetic activities and was most effective in activating the anti-tumor immune response (P<0.05). The above results provide evidence for evaluating the immune regulating mechanisms of 4HF in breast cancer and support its clinical significance in its potential as an adjunctive therapeutic agent or immune supplement.

Boosting Ferroptosis Therapy with Iridium Single-Atom Nanocatalyst in Ultralow Metal Content.

Nanocatalysts are promising tumor therapeutics due to their ability to induce reactive oxygen species in the tumor microenvironment. Although increasing metal loading can improve catalytic activity, the quandary of high metal content versus potential systemic biotoxicity remains challenging. Here, a fully exposed active site strategy by site-specific anchoring of single iridium (Ir) atoms on the outer surface of a nitrogen-doped carbon composite (Ir single-atom catalyst (SAC)) is reported to achieve remarkable catalytic performance at ultralow metal content (≈0.11%). The Ir SAC exhibits prominent dual enzymatic activities to mimic peroxidase and glutathione peroxidase, which catalyzes the conversion of endogenous H2 O2 into •OH in the acidic TME and depletes glutathione (GSH) simultaneously. With an advanced support of GSH-trapping platinum(IV) and encapsulation with a red-blood-cell membrane, this nanocatalytic agent (Pt@IrSAC/RBC) causes intense lipid peroxidation that boosts tumor cell ferroptosis. The Pt@IrSAC/RBC demonstrates superior therapeutic efficacy in a mouse triple-negative mammary carcinoma model, resulting in complete tumor ablation in a single treatment session with negligible side effects. These outcomes may provide valuable insights into the design of nanocatalysts with high performance and biosafety for biomedical applications.

Evaluation of the in vitro and in vivo effect of liposomal doxorubicin along with oncolytic Newcastle disease virus on 4T1 cell line: Animal preclinical research.

Breast cancer is one of the most common malignancies in women, with one in 20 globally. Oncolytic viruses have recently been the first step in the biological treatment of cancer, either genetically engineered or naturally occurring. They increase specifically inside cancer cells and destroy them without damaging normal tissues or producing a host immune response against tumour cells or expressing transgenes. One of the most known members of this family is the Newcastle disease virus (NDV), a natural oncolytic virus that selectively induces apoptosis and DNA fragmentation in human cancer cells. This study performed biochemical and molecular investigations with variable doses of NDV (32, 64, 128 HAU) and liposomal doxorubicin (9mg/kg) on mouse triple-negative mammary carcinoma cell line 4T1 and BALB/c models tumours for the first time. Real-time quantitative PCR analysis in NDV-treated animal tumours showed increased expression of P21, P27 and P53 genes and decreased expression of CD34, integrin Alpha 5, VEGF and VEGF-R genes. Additional assessments in treated mouse models also showed that NDV increased ROS production, induced apoptosis, reduced tumour size and significantly improved prognosis, with no adverse effect on normal tissues. These findings all together might indicate that NDV in combination with chemotherapy drugs could improve prognosis in cancer patients although many more conditions should be considered.

Circadian disruption and cisplatin chronotherapy for mammary carcinoma

Solid tumors are commonly treated with cisplatin, which can cause off-target side effects in cancer patients. Chronotherapy is a potential strategy to reduce drug toxicity. To determine the effectiveness of timed-cisplatin treatment in mammals, we compared two conditions: clock disrupted jet-lag and control conditions. Under normal and disrupted clock conditions, triple-negative mammary carcinoma cells were injected subcutaneously into eight-week-old NOD.Cg-Prkdcscid/J female mice. Tumor volumes and body weights were measured in these mice before and after treatment with cisplatin. We observed an increase in tumor volumes in mice housed under disrupted clock compared to the normal clock conditions. After treatment with cisplatin, we observed a reduced tumor growth rate in mice treated at ZT10 compared to ZT22 and untreated cohorts under normal clock conditions. However, these changes were not seen with the jet-lag protocol. We also observed greater body weight loss in mice treated with ZT10 compared to ZT22 or untreated mice in the jet-lag protocol. Our observations suggest that the effectiveness of cisplatin in mammary carcinoma treatment is time-dependent in the presence of the circadian clock.

Abstract 1390: A combination of natural polyphenols, a NAD+ booster, and a Toll-like receptor 2/6 agonist exerts high radioprotection in normal tissues without interfering with the anticancer efficacy of ionizing radiations

Abstract Ionizing radiation damages cells via direct ionization of DNA and other cellular targets as well as by indirect effects through reactive oxygen species. The response to radiation exposure depends on the cell type and dose of radiation, inherent tissue sensitivity and repair, and modulating intracellular factors that include cell cycle status, O2 pressure, and levels of thiols and other antioxidants. Potentially protective agents against exposure to harmful radiation have been investigated for decades. However, no ideal radioprotector is currently available. A wide range of phytochemicals are antioxidants and, thus, potentially radioprotective. Topical administration of Pterostilbene (3,5-dimethoxy-4'-hydroxystilbene, PTER, a natural stilbene originally isolated from the heartwood of red sandalwood) protects against chronic UVB radiation-induced skin carcinogenesis, and protects the skin against the burning and irritation effects of acute UVB doses. The protection elicited by PTER is not due to physico-chemical screen or direct antioxidant effects. PTER induces an increase in our overall physiological antioxidant defences. Thus, it is plausible that PTER, could also exert protection against other types of ionizing radiation. We investigated the effect of PTER against ionizing radiation and found that the combination of PTER and silibinin [SIL, (2R,3R)-3,5,7-trihydroxy-2-[(2R,3R)-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-2,3-dihydrochromen-4-one, a natural flavanone isolated from the silybum marianum (milk thistle)] exerts radioprotection against γ radiation injury under in vivo conditions. NAD+ is an essential coenzyme in ATP production, and NAD+ consuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. Nicotinamide, an NAD+ precursor, has been shown to improve the post-radiation repair capacity in cells in a concentration-dependent manner. Hence, NAD+ precursors could function as potential radiomitigators in vivo. Furthermore, replenishment of hematopoietic sites is critical for recovery following radiation exposure. Interestingly, a recent report shows that the Toll-like receptor (TLR) 2/6 agonist, FSL (fibroblast-stimulating lipopeptide)-1 (FSL-1), therapeutically mitigates acute radiation syndrome. We found the combination of PTER, SIL, nicotinamide riboside (a NAD+ precursor) and FSL-1 favoured the long-term survival of irradiated mice. Moreover, this radioprotecting/radiomitigating combination did not reduce the anticancer efficacy of X rays in growing human A549 (lung adenocarcinoma) and MDA-MB-231 (triple negative mammary carcinoma) xenografts. Citation Format: Elena Obrador, Rosario Salvador, Rafael Lopez-Blanch, Ali Jihad-Jebbar, José I. Villaescusa, Alegria Montoro, Ryan W. Dellinger, Jose M. Estrela. A combination of natural polyphenols, a NAD+ booster, and a Toll-like receptor 2/6 agonist exerts high radioprotection in normal tissues without interfering with the anticancer efficacy of ionizing radiations [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 1390.

Palmatine Modulates Triple Negative Mammary Carcinoma by Regulating the Endogenous Function of p53, p21 and Mdm2

Natural products and their bioactive constituents have been investigated for centuries and recognized as a source of valuable therapeutic candidates in the development of contemporary anticancer drugs. Triple negative breast cancers are a sub type of malignant cells formed in the breast tissue caused by uncontrolled and abnormal division. Palmatine, a naturally occurring alkaloid extracted from several medicinal plants in West Africa,has not been extensively investigated for its anti-breast cancer properties especially in triple-negative mammary carcinoma. The 4T1 triple-negative breast cancer cells were transplanted orthotopically into the mammary fat pad of the female balb/c mice. Tumor volume,tumor weight, histology and immunohistochemical analysis were carried out.After 28 days, palmatine (1, 5 and 10 mg/kg) in a dose-dependent manner decreased tumor volume (190.80 ±19.14, 25.40 ± 2.82, 14.20 ± 1.85), reduced tumor weight (1.035± 0.04, 0.8027± 0.01, 0.5090±0.04), inhibited tumor growth (31%, 46%, 66%) and protected against morphological dysplasia induced by the carcinoma (3.50 ± 0.29, 2.25 ± 0.25, 1.75 ± 0.25)respectively. Also, palmatine increased the activity of the tumor protein p53, cyclin-dependent kinase inhibitor 1 (p21) and mouse double minute 2(Mdm2) compared to the untreated carcinoma bearingmice. Overall, palmatine protected against triple negative mammary carcinoma and can be a valuable anticancer compound to treat breast diseases.

Normalizing glucose levels reconfigures the mammary tumor immune and metabolic microenvironment and decreases metastatic seeding.

Obesity and diabetes cumulatively create a distinct systemic metabolic pathophysiological syndrome that predisposes patients to several diseases including breast cancer. Moreover, diabetic and obese women with breast cancer show a significant increase in mortality compared to non-obese and/or non-diabetic women. We hypothesized that these metabolic conditions incite an aggressive tumor phenotype by way of impacting tumor cell-autonomous and tumor cell non-autonomous events. In this study, we established a type 2 diabetic mouse model of triple-negative mammary carcinoma and investigated the effect of a glucose lowering therapy, metformin, on the overall tumor characteristics and immune/metabolic microenvironment. Diabetic mice exhibited larger mammary tumors that had increased adiposity with high levels of O-GlcNAc protein post-translational modification. These tumors also presented with a distinct stromal profile characterized by altered collagen architecture, increased infiltration by tumor-permissive M2 macrophages, and early metastatic seeding compared to non-diabetic/lean mice. Metformin treatment of the diabetic/obese mice effectively normalized glucose levels, reconfigured the mammary tumor milieu, and decreased metastatic seeding. Our results highlight the impact of two metabolic complications of obesity and diabetes on tumor cell attributes and showcase metformin's ability to revert tumor cell and stromal changes induced by an obese and diabetic host environment.

Abstract 1423: A lncRNA MEG9 protects vasculature from DNA damage

Abstract DNA damaging agents including ionizing radiation (IR) therapy are widely used in cancer patients. Previous studies have suggested that the tumor vasculature, specifically endothelial cell (ECs) dictate responses to IR. We recently showed how IR, among other DNA damaging agents, induced changes in microRNA programs and impacted tumor angiogenesis. Here, we describe how different DNA damaging agents affect long noncoding RNA (lncRNA) programs in vascular cells. Specifically, we identify lncRNA MEG9 (Maternally Expressed Gene 9 in humans, Mirg in mouse) as one of the most differentially expressed lncRNAs in angiogenic stimuli and stress responses. MEG9 is robustly induced by DNA damaging agents and suppressed by vascular growth factors. MEG9 is located in the DLK1-DIO3 imprinted cluster. Previous studies have shown how DLK1-DIO3 genes are associated with stem cell biology and with the pathogenesis of several diseases, such as cancer. Other studies have shown that microRNAs in this cluster are regulated by methylation. Consistent with this observation, we found that inhibition of DNA methylation either via 5-azacytidine or silencing of DNMT3b significantly upregulated de novo expression of MEG9. Gain and loss of function assays indicated an anti-apoptotic role of MEG9 via caspase 3/7 pathways in cultured ECs. Similarly, MEG9 inhibition decreased angiogenesis in a 3D sprouting angiogenesis assay. Interestingly, MEG9 inhibition in ECs accelerated fibrin formation suggesting a regulatory role for this lncRNA in thrombosis. Moreover, we observed that MEG9 inhibition affects vascular permeability in vitro and in vivo in bFGF-Matrigel plugs. Finally, systemic delivery of RNAi targeting Mirg (mouse MEG9) decreased tumor burden in a mouse model of triple negative mammary carcinoma. Our mechanistic studies indicated that MEG9 in ECs impacts pathways related to inflammation and thrombosis, both critical mediators of tumor responses to radiation. Taken together, our results show MEG9 as a novel DNA damage induced lncRNA with a protective role in the tumor vasculature functioning via multiple distinct mechanisms. Therefore, we propose that disruption of lncRNA MEG9 is a potent anti-angiogenic strategy that can synergize with DNA damaging agents. Citation Format: Cristina Espinosa-Diez, Eugenia Fraile-Bethencourt, Clayton Hudson, Rebecca Ruhl, Sudarshan Anand. A lncRNA MEG9 protects vasculature from DNA damage [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 1423.

Abstract 5695: Induction of lymphangiogenesis in breast cancer enhances responsiveness to immunotherapies

Abstract Growth and remodeling of tumor-associated lymphatics, termed lymphangiogenesis, has been known to increase metastasis rate in cancer patients. However, a recent study from our group has shown that tumor-associated lymphatics can improve anti-tumor immunity in melanoma upon treatment with immunotherapies. The role of lymphangiogenesis in shaping anti-tumor immune responses in breast cancer, however, remains unexplored. Furthermore, recently approved checkpoint inhibitor immunotherapies have had limited success in breast cancer patients. A better understanding of the role of tumor-associated lymphatics in shaping anti-tumor immune responses could lead to more informed decisions when treating breast cancer patients with immunotherapies. In this study, we investigated the role of induced tumor lymphangiogenesis in modulating responsiveness to different immunotherapies in a murine triple-negative mammary carcinoma model. Spontaneously metastatic 4T1 tumor cells were transduced to over-express the pro-lymphangiogenic growth factor VEGF-C (VC) to induce tumor lymphangiogensis in vivo. Flow cytometry and immunohistochemistry analyses confirmed that VEGF-C over-expressing tumors have an increase in tumor-associated lymphatics than control ones. Higher infiltrations of conv CD4 T cells and macrophages were observed in lymphangiogenic 4T1 tumors. Lymphangiogenic tumors treated with aPD-1 and aCTLA-4 checkpoint inhibitors, or an agonist of the Stimulator of Interferon Genes (STING) pathway, showed improved responsiveness to the therapy compared to the control tumors, as indicated by a reduced tumor growth rate. Moreover, positive correlations between VEGF-C expression and CD4 T cell and macrophage gene expressions were observed in primary human triple-negative breast tumor samples from The Cancer Genome Atlas (TCGA). In conclusion, our study shows that increase in tumor-associated lymphatics in triple-negative breast cancer enhances responsiveness to immunotherapies. Citation Format: Peyman Hosseinchi, Aslan Mansurov, Léa Maillat, Lambert Potin, Gavin Fujimori, Jeffrey A. Hubbell, Melody A. Swartz. Induction of lymphangiogenesis in breast cancer enhances responsiveness to immunotherapies [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 5695.

Abstract P3-08-58: Ratio of CD8 +/ FOXP3, number of FOXP3 + cells and the presence of PTEN labeling in tumor cells and association with survival in triple negative breast cancer

Abstract Introduction: Triple-negative mammary carcinomas (CMTN) correspond to about 15 to 20%, the subtype being more aggressive when compared to the others. The interaction between immune system cells and cancer plays a crucial role in tumor development and progression. However, it is still unclear whether the presence of CD8 cytotoxic lymphocytes provides any prognostic information in breast cancer. Objective: To evaluate the prognostic significance of tumor-associated FOXP3 regulatory T cells (Tregs) and CD8 cytotoxic T lymphocytes (CTLs) in invasive breast carcinomas Method: This is an observational, descriptive retrospective cohort study were evaluated 76 patients diagnosed with non metastatic (stages I, II and III) TNBC. A tissue microarray (TMA) was constructed containing two cores from each tumor block. The expression of CD4, CD8, FOXP3, CD20, CD68, CD163, PD-1, PD-L1, PTEN and phosphoSTAT1 was determined by immunohistochemistry in the TMA. Results: We observed that the inflammatory infiltrate in TNBC is enriched for M2 macrophages and T lymphocytes (CD4+, CD8+ and FOXP3+). In our sample, we observed better survival associated with a higher CD8 + (p = 0.054) and CD4 + (p = 0.082) cell counts, but with no statistical significance in both situations. However, when we analyzed the association of overal survival (OS) with the ratio between the number of CD8 + cells and FOXP3 + (p = 0.007) and CD4 + and FOXP3 + (p = 0.034) and CD4 (p = 0.058) cells, we found that a higher relative proportion of CD8 + or CD4 + lymphocytes in relation to lymphocytes FOXP3 + was associated with an increase in OS. Together, these data suggest that infiltration by T lymphocytes into the tumor microenvironment should exhibit antirumor activity and promote growth control and tumor progression, as opposed to events favoring immunosuppression (regulatory lymphocyte infiltration). The number of FOXP3+ cells and the presence of PTEN staining in tumor cells were associated with OS. Conclusion: We did not observe improvement in survival, with an increase in CD8 and CD4 alone, so we found an improvement in survival, the higher the ratio between CD8 / FOXP3 being statistically significant. The number of FOXP3+ cells and the presence of PTEN staining in tumor cells were associated with OS. Citation Format: Monique C Tavares, Geraldine E Lima, Cristina D Sampaio, Victor P Andrande, Daniel G Gonçalves, Mariana P Macedo, Vladmir C Lima. Ratio of CD8 +/ FOXP3, number of FOXP3 + cells and the presence of PTEN labeling in tumor cells and association with survival in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-58.

Androgen receptor and FOXA1 coexpression define a \u201cluminal-AR\u201d subtype of feline mammary carcinomas, spontaneous models of breast cancer

BackgroundInvasive mammary carcinomas that spontaneously develop in female cats are associated with high mortality, and resemble the most aggressive human breast cancers, especially triple-negative breast cancer (TNBC). Transcriptome studies showed that TNBCs are a heterogeneous group that includes a potentially hormone-dependent subtype named luminal-AR. Some authors proposed an immunohistochemical definition of the luminal-AR subtype, which is not only positive for Androgen Receptor (AR), but also either positive for the transcription factor Forkhead box A1 (FOXA1), or negative for basal markers. The objectives of this study were to describe AR and FOXA1 expressions in feline mammary carcinomas (FMCs), their prognostic value, and if their coexpression could define a “luminal-AR” subtype of triple-negative mammary carcinomas in cats.MethodsIn a previously described retrospective cohort of 180 female cats with FMCs, with a 2-year follow-up post-mastectomy, we assessed AR, FOXA1, ER, PR, Ki-67, HER2, and CK14 expressions by automated immunohistochemistry.ResultsOf the 180 FMCs, 57 (32%) were luminal; i.e., ER and/or PR positive, and 123 (68%) were triple-negative (ER–, PR– and HER2–) FMCs. AR overexpression (found in 33 cases/180, 18%) and FOXA1 index ≥1% (64/180, 36%) were associated with a longer disease-free interval, overall survival, and cancer-specific survival in cats with FMC. Analysis of AR, FOXA1 and CK14 coexpression in triple-negative FMCs showed that AR+ triple-negative FMCs were heterogeneous: there existed an AR+ FOXA1+ CK14– subgroup (n = 7) associated with a better cancer-specific survival by multivariate survival analysis (HR = 0.26, 95% CI: 0.07–0.89, p = 0.03) compared to AR+ FOXA1–CK14+ triple-negative FMCs (n = 46) (HR = 1.00), independently of the pathologic tumor size and pathologic nodal stage. The non-basal-like subtype of triple-negative FMCs that coexpresses AR and FOXA1 (the AR+ FOXA1+ CK14– subgroup) could represent the equivalent of the luminal-AR subgroup of human triple-negative breast cancer.ConclusionsWe identified an AR+ FOXA1+ CK14– subgroup of triple-negative FMCs that might correspond to the luminal-AR subgroup of human triple-negative breast cancers. Cats with FMC may be interesting spontaneous animal models to investigate new strategies targeting the androgen receptor, especially in the aggressive subtype of AR+ basal-like triple-negative mammary carcinomas with loss of FOXA1 expression (the AR+ FOXA1–CK14+ subgroup).

Absence of integrin \u03b13\u03b21 promotes\xa0the progression of HER2-driven breast cancer in vivo

BackgroundHER2-driven breast cancer is correlated with poor prognosis, especially during its later stages. Numerous studies have shown the importance of the integrin α3β1 during the initiation and progression of breast cancer; however, its role in this disease is complex and often opposite during different stages and in different types of tumors. In this study, we aim to elucidate the role of integrin α3β1 in a genetically engineered mouse model of HER2-driven mammary tumorigenesis.MethodsTo investigate the role of α3β1 in HER2-driven tumorigenesis in vivo, we generated a HER2-driven MMTV-cNeu mouse model of mammary tumorigenesis with targeted deletion of Itga3 (Itga3 KO mice). We have further used several established triple-negative and HER2-overexpressing human mammary carcinoma cell lines and generated ITGA3-knockout cells to investigate the role of α3β1 in vitro. Invasion of cells was assessed using Matrigel- and Matrigel/collagen I-coated Transwell assays under static or interstitial fluid flow conditions. The role of α3β1 in initial adhesion to laminin and collagen was assessed using adhesion assays and immunofluorescence.ResultsTumor onset in mice was independent of the presence of α3β1. In contrast, the depletion of α3β1 reduced the survival of mice and increased tumor growth and vascularization. Furthermore, Itga3 KO mice were significantly more likely to develop lung metastases and had an increased metastatic burden compared to WT mice. In vitro, the deletion of ITGA3 caused a significant increase in the cellular invasion of HER2-overexpressing SKBR3, AU565, and BT474 cells, but not of triple-negative MDA-MB-231. This invasion suppressing function of α3β1 in HER2-driven cells depended on the composition of the extracellular matrix and the interstitial fluid flow.ConclusionDownregulation of α3β1 in a HER2-driven mouse model and in HER2-overexpressing human mammary carcinoma cells promotes progression and invasiveness of tumors. The invasion-suppressive role of α3β1 was not observed in triple-negative mammary carcinoma cells, illustrating the tumor type-specific and complex function of α3β1 in breast cancer.

Abstract 1753: SIRPa inhibition monotherapy leads to dramatic change in solid tumor microenvironment and prevents metastasis development

Abstract Targeting immune checkpoints of the adaptive immunity has shown great therapeutic efficacy to fight cancers, but in a limited proportion of patients. Myeloid cells represent a major immune cell type infiltrating many solid tumors, and are often associated with a poor outcome. While TAMs (mainly MDSCs and macrophages) are involved in the regulation of immune responses and in tissue repair in healthy individuals, they can be co-opted by cancer cells to exert suppressor functions, preventing action of other immune cells. In addition to their suppressor functions, TAMs can also participate in tumor growth and metastasis through several other mechanisms. SIRPalpha (SIRPa) is an immune checkpoint expressed by nearly all myeloid cells which interacts with the ubiquitous receptor CD47 and is now well described to regulate macrophage functions, including phagocytosis. Here we evaluated the impact of SIRPa signaling inhibition on tumor microenvironment and metastasis development in orthotopic tumor models in immunocompetent mice. We first found that administration of an antagonist anti-SIRPa monoclonal antibody (mAb) in monotherapy significantly reduces tumor growth in an orthotopic mouse triple-negative mammary carcinoma (4T1) model (n=20, p&amp;lt;0.01) and significantly reduces lung metastasis development (p&amp;lt;0.05). Tumor microenvironment analysis by flow cytometry at early time-point (10 days post-treatment) revealed significant increased frequency of M1 inflammatory macrophages (including M1/M2 ratio), NK cells and both increased of intra-tumoral activated CD8+ T cells and memory CD4+ T lymphocytes. Similarly, using SIRPa mutant mice (deletion of the intracellular signaling domain) in an orthotopic mesothelioma (AK7) model, we found that absence of SIRPa signaling significantly reduces tumor growth as compared to wild-type mice (n= 8, p&amp;lt;0.01). Tumor microenvironment analysis by flow cytometry revealed also significant accumulation of intra-tumoral F4/80+ macrophages, CD11c+ dendritic cells, CD8+ T cells and increased CD8/MDSC ratio. Intra-tumoral CD8+ T lymphocytes significantly over-expressed activation markers (e.g. CD69) in SIRPa mutant mice. Finally, using the orthotopic B16F10 melanoma model, we found that curative treatment (initiated two weeks after tumor inoculation) with the antagonist anti-SIRPa monoclonal antibody significantly reduced tumor growth in this aggressive model (n=7, p&amp;lt;0.05), with two mice treated with the anti-SIRPa mAb in complete remission and two others in partial remission at the end of the study. In conclusion, we found that inhibition of SIRPa signaling in monotherapy rewire innate and adaptive immune cells in cancer. Such straight modifications in the tumor microenvironment led to a reduction in tumor growth in different orthotopic solid tumor models and prevention of metastasis development. Citation Format: Justine Durand, Vanessa Gauttier, Aurore Morello, Sabrina Pengam, Bernard Vanhove, Nicolas Poirier. SIRPa inhibition monotherapy leads to dramatic change in solid tumor microenvironment and prevents metastasis development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1753.

Characterization of the inflammatory infiltrate and association with expression of PD-1, PDL1 and PDL2 and survival in triple-negative mammary carcinoma.

1099Background: However, recent evidences suggest that lymphocyte infiltration in tumors (LIT), which is presented in breast cancer prior to the treatment, may predict response to the therapy and a...

P53 deficiency induces cancer stem cell pool expansion in a mouse model of triple-negative breast tumors.

Triple-negative breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Triple-negative tumors often display activated Wnt/β-catenin signaling and most have impaired p53 function. We studied the interplay between p53 loss and Wnt/β-catenin signaling in stem cell function and tumorigenesis, by deleting p53 from the mammary epithelium of K5ΔNβcat mice displaying a constitutive activation of Wnt/β-catenin signaling in basal cells. K5ΔNβcat transgenic mice present amplification of the basal stem cell pool and develop triple-negative mammary carcinomas. The loss of p53 in K5ΔNβcat mice led to an early expansion of mammary stem/progenitor cells and accelerated the formation of triple-negative tumors. In particular, p53-deficient tumors expressed high levels of integrins and extracellular matrix components and were enriched in cancer stem cells. They also overexpressed the tyrosine kinase receptor Met, a feature characteristic of human triple-negative breast tumors. The inhibition of Met kinase activity impaired tumorsphere formation, demonstrating the requirement of Met signaling for cancer stem cell growth in this model. Human basal-like breast cancers with predicted mutated p53 status had higher levels of MET expression than tumors with wild-type p53. These results connect p53 loss and β-catenin activation to stem cell regulation and tumorigenesis in triple-negative cancer and highlight the role of Met signaling in maintaining cancer stem cell properties, revealing new cues for targeted therapies.

Abstract 4124: Sub-cytotoxic concentrations of a novel polyphenol fatty acid ester derivative arrest breast cancer cell proliferation and metastasis

Abstract In this study, the antiproliferative and antimetastatic properties of phloridzin docosahexaenoate (PZ-DHA), a novel polyphenol fatty acid ester derivative, were explored using in vitro and in vivo models of mammary carcinoma. PZ-DHA combines phloridzin (PZ, a dihydrochalcone found in apple peels) with docosahexaenoic acid (DHA, an omega-3 fatty acid). The regioselective acylation reaction was catalyzed by lipase B enzyme from Candida antarctica. PZ-DHA-induced selective cytotoxicity towards breast cancer cells (triple-negative mammary carcinoma cells, MDA-MB-231, MDA-MB-468, and 4T1; estrogen receptor-positive mammary carcinoma cells, MCF-7 and T-47D) was observed compared to human mammary epithelial cells (HMECs and MCF 10A) using the MTS assay. Significantly less (p&amp;lt;0.05) cytotoxicity of PZ-DHA for normal cells was also confirmed using flow cytometric analysis of annexin-V-FLUOS/propidium iodide-stained MCF 10A and human dermal fibroblasts compared to MDA-MB-231 cells. Flow cytometric analysis of Oregon Green 488-stained MDA-MB-231 cells showed an antiproliferative effect of PZ-DHA at sub-cytotoxic concentrations (10 to 30 μM). Cell cycle analysis showed that MDA-MB-231 replication was arrested at the G2/M phase of the cell cycle following treatment with PZ-DHA at sub-cytotoxic concentrations. PZ-DHA suppressed the migration of MDA-MB-231 and 4T1 cells in vitro in wound healing and cell migration assays. Reduced expression of proteins (β-catenin, slug, Zinc finger E-box-binding homeobox 1, vimentin and matrix metalloproteinase-2) involved in the epithelial-to-mesenchymal transition was demonstrated by western blot analysis of PZ-DHA-treated MDA-MB-231 cell lysates. Finally, 4T1 tumor bearing-female BALB/c mice that received intra-peritoneal injections of PZ-DHA showed a significant reduction (p&amp;lt;0.05, n = 9) in primary tumor volume at the mammary fat pad and fewer metastatic lesions in the lungs compared to the saline-treated control mice, suggesting an in vivo antimetastatic effect of PZ-DHA. In conclusion, findings of this study reveal that PZ-DHA suppresses mammary carcinoma cell proliferation and metastasis, suggesting a potential clinical application to prevent breast cancer progression in patients. Citation Format: Wasundara Fernando, Melanie R. Power Coombs, David W. Hoskin, H. P. Vasantha Rupasinghe. Sub-cytotoxic concentrations of a novel polyphenol fatty acid ester derivative arrest breast cancer cell proliferation and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4124.

Abstract LB-330: Four individually druggable Met hotspots mediate HGF-driven tumor progression

Abstract Activation of Met by HGF plays a key role in tumor progression. Using a newly developed llama platform generating human-like immunoglobulins, we selected 68 different antibodies that competed with HGF for binding to Met. HGF-competing antibodies recognized four hotspots localized in different domains of Met. One hotspot coincides with the previously identified HGF β-chain binding site on blades 2-3 of the SEMA β-propeller. Two hotspots lie within SEMA blade 5 and IPT domains 2-3, respectively, where the HGF α-chain is thought to bind. The last hotspot identifies a new region across the PSI-IPT 1 domains. Individual or combined targeting of these hotspots effectively interrupted HGF-Met signaling in multiple biochemical and biological assays. Selected antibodies directed against SEMA blades 2-3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neo-adjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of Met responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-Met interaction and providing new molecular tools for targeting Met activity in cancer. Citation Format: Anna Hultberg, Cristina Basilico, Cristophe Blanchetot, Natalie De Jonge, Valérie Hanssens, Gitte De Boeck, Alessia Mira, Manuela Cazzanti, Virginia Morello, Torsten Dreier, Michael Saunders, Hans De Haard, Paolo Michieli. Four individually druggable Met hotspots mediate HGF-driven tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-330. doi:10.1158/1538-7445.AM2014-LB-330

Four individually druggable MET hotspots mediate HGF-driven tumor progression.

Activation of MET by HGF plays a key role in tumor progression. Using a recently developed llama platform that generates human-like immunoglobulins, we selected 68 different antibodies that compete with HGF for binding to MET. HGF-competing antibodies recognized 4 distinct hotspots localized in different MET domains. We identified 1 hotspot that coincides with the known HGF β chain binding site on blades 2-3 of the SEMA domain β-propeller. We determined that a second and a third hotspot lie within blade 5 of the SEMA domain and IPT domains 2-3, both of which are thought to bind to HGF α chain. Characterization of the fourth hotspot revealed a region across the PSI-IPT 1 domains not previously associated with HGF binding. Individual or combined targeting of these hotspots effectively interrupted HGF/MET signaling in multiple cell-based biochemical and biological assays. Selected antibodies directed against SEMA blades 2-3 and the PSI-IPT 1 region inhibited brain invasion and prolonged survival in a glioblastoma multiforme model, prevented metastatic disease following neoadjuvant therapy in a triple-negative mammary carcinoma model, and suppressed cancer cell dissemination to the liver in a KRAS-mutant metastatic colorectal cancer model. These results identify multiple regions of MET responsible for HGF-mediated tumor progression, unraveling the complexity of HGF-MET interaction, and provide selective molecular tools for targeting MET activity in cancer.