Mammary Tumorigenesis Research Articles

Scribble promotes fibrosis-dependent mechanisms of hepatocarcinogenesis by p53/PUMA-mediated glycolysis

Backgrounds and aimsLiver cancer is the sixth most common type of cancer and the fifth leading cause of cancer mortality worldwide. Scribble has been shown to function as a neoplastic tumor suppressor gene in most tumors. Our previous studies reported that down-regulation or mislocalization of Scribble was sufficient to initiate mammary tumorigenesis and NSCLC. Recently, it was reported that Scribble was highly expressed in hepatocellular carcinoma (HCC). We aim to study how it was up-regulated and the contradictory role of Scribble in HCC. Methods and resultsUsing a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis system, we showed that Scribble was over-expressed and which may protect the mice against hepatic fibrosis. Unexpectedly, we found out the potential for Scribble to act as a tumor driver at the advanced stage of N-nitrosodiethylamine (DEN) plus CCl4 induced HCC mice model in vivo. In addition, we observed even higher expression of Scribble in HCC tumors harboring elevated levels of wild-type p53. Most importantly, nuclear translocated Scribble could interact with p53, which lead to enhanced stability and transcriptional activity of p53. Mechanistically, our data suggested that Scribble might drive HCC progression by promoting metabolic regulation of p53 through p53-upregulated modulator of apoptosis (PUMA)-mediated Warburg effect. ConclusionsOur data identified the molecular basis of hepatic fibrosis-specific gene expression of polarity gene, such as Scribble. Interestingly, with the progression from fibrosis to cirrhosis to HCC, its nuclear translocation promoted a wild-type p53-mediated cancer metabolic switch and tumor progression in HCC. Taken together, we demonstrated that Scribble was up-regulated and served a protective role in liver fibrosis, while also apparently acting as a tumor driver in fibrosis-dependent hepatocarcinogenesis.

Safety of lactational exposure to venlafaxine on the rat mammary gland development and carcinogenesis in F1 female offspring

The chronic use of selective serotonin reuptake inhibitors or serotonin-norepinephrine reuptake inhibitors (SNRIs) may result in human gynecomastia, mammoplasia, galactorrhea, and elevated breast cancer risk. As antidepressants are frequently used for postpartum depression (PPD) treatment, this study investigated the adverse effects of lactational exposure to venlafaxine (VENL, a selective SNRI) on mammary gland development and carcinogenesis in F1 female offspring. Thus, lactating Wistar rats (F0) received VENL by oral gavage at daily doses of 3.85, 7.7, or 15.4 mg/kg (N = 9, each group) from lactational day (LD 1) until the weaning of the offspring (LD 21). F1 female offspring were euthanized for mammary gland, and ovary histological analyses on the post-natal day (PND) 22 and 30 (1 pup/litter/period, N = 9, each group). At PND 22, other females (2 pups/litter, N = 18, each group) received a single dose of carcinogen N-methyl-N-nitrosourea (MNU, 50 mg/kg) intraperitoneally (i.p.) for tumor susceptibility assay until PND 250. Tumor incidence and latency were recorded and representative tumor samples were collected for histopathology. The results indicate that lactational exposure to VENL did not alter the development of the mammary gland (epithelial ductal tree or the mean number of terminal end buds), or the ovary (weight and primary, secondary, tertiary, and Graafian follicles) in prepubertal F1 female offspring. In addition, VENL exposure did not influence tumor incidence or tumor latency in adult female offspring that received MNU. Thus, the findings of this animal study indicated that lactational VENL exposure, a period similar to human PPD, did not exert an adverse effect on the mammary gland development at the prepubertal phase or on chemically induced mammary tumorigenesis in adult F1 female rats.

Environmental cadmium exposure facilitates mammary tumorigenesis via reprogramming gut microbiota-mediated glutamine metabolism in MMTV-Erbb2 mice

Cadmium (Cd) is a heavy metal that has been widely reported to be linked to the onset and progression of breast cancer (BC). However, the mechanism of Cd-induced mammary tumorigenesis remains elusive. In our study, a transgenic mouse model that spontaneously develops tumors through overexpression of wild-type Erbb2 (MMTV-Erbb2) was constructed to investigate the effects of Cd exposure on BC tumorigenesis. The results showed that oral exposure to 3.6 mg/L Cd for 23 weeks dramatically accelerated tumor appearance and growth, increased Ki67 density and enhanced focal necrosis and neovascularization in the tumor tissue of MMTV-Erbb2 mice. Notably, Cd exposure enhanced glutamine (Gln) metabolism in tumor tissue, and 6-diazo-5-oxo-l-norleucine (DON), a Gln metabolism antagonist, inhibited Cd-induced breast carcinogenesis. Then our metagenomic sequencing and mass spectrometry-based metabolomics confirmed that Cd exposure disturbed gut microbiota homeostasis, especially Helicobacter and Campylobacter abundance remodeling, which altered the gut metabolic homeostasis of Gln. Moreover, intratumoral Gln metabolism profoundly increased under Cd-elevated gut permeability. Importantly, depletion of microbiota with an antibiotic cocktail (AbX) treatment led to a significant delay in the appearance of palpable tumors, inhibition of tumor growth, decrease in tumor weight, reduction in Ki67 expression and low-grade pathology in Cd-exposed MMTV-Erbb2 mice. Also, transplantation of Cd-modulated microbiota decreased tumor latency, accelerated tumor growth, increased tumor weight, upregulated Ki67 expression and exacerbated neovascularization as well as focal necrosis in MMTV-Erbb2 mice. In summary, Cd exposure induced gut microbiota dysbiosis, elevated gut permeability and increased intratumoral Gln metabolism, leading to the promotion of mammary tumorigenesis. This study provides novel insights into environmental Cd exposure-mediated carcinogenesis.

Glucose-induced CRL4COP1-p53 axis amplifies glycometabolism to drive tumorigenesis

The diabetes-cancer association remains underexplained. Here, we describe a glucose-signaling axis that reinforces glucose uptake and glycolysis to consolidate the Warburg effect and overcome tumor suppression. Specifically, glucose-dependent CK2 O-GlcNAcylation impedes its phosphorylation of CSN2, a modification required for the deneddylase CSN to sequester Cullin RING ligase 4 (CRL4). Glucose, therefore, elicits CSN-CRL4 dissociation to assemble the CRL4COP1 E3 ligase, which targets p53 to derepress glycolytic enzymes. A genetic or pharmacologic disruption of the O-GlcNAc-CK2-CSN2-CRL4COP1 axis abrogates glucose-induced p53 degradation and cancer cell proliferation. Diet-induced overnutrition upregulates the CRL4COP1-p53 axis to promote PyMT-induced mammary tumorigenesis in wild type but not in mammary-gland-specific p53 knockout mice. These effects of overnutrition are reversed by P28, an investigational peptide inhibitor of COP1-p53 interaction. Thus, glycometabolism self-amplifies via a glucose-induced post-translational modification cascade culminating in CRL4COP1-mediated p53 degradation. Such mutation-independent p53 checkpoint bypass may represent the carcinogenic origin and targetable vulnerability of hyperglycemia-driven cancer.

Loss of function of GATA3 regulates FRA1 and c-FOS to activate EMT and promote mammary tumorigenesis and metastasis

Basal-like breast cancers (BLBCs) are among the most aggressive cancers, partly due to their enrichment of cancer stem cells (CSCs). Breast CSCs can be generated from luminal-type cancer cells via epithelial-mesenchymal transition (EMT). GATA3 maintains luminal cell fate, and its expression is lost or reduced in BLBCs. However, deletion of Gata3 in mice or cells results in early lethality or proliferative defects. It is unknown how loss-of-function of GATA3 regulates EMT and CSCs in breast cancer. We report here that haploid loss of Gata3 in mice lacking p18Ink4c, a cell cycle inhibitor, up-regulates Fra1, an AP-1 family protein that promotes mesenchymal traits, and downregulates c-Fos, another AP-1 family protein that maintains epithelial fate, leading to activation of EMT and promotion of mammary tumor initiation and metastasis. Depletion of Gata3 in luminal tumor cells similarly regulates Fra1 and c-Fos in activation of EMT. GATA3 binds to FOSL1 (encoding FRA1) and FOS (encoding c-FOS) loci to repress FOSL1 and activate FOS transcription. Deletion of Fra1 or reconstitution of Gata3, but not reconstitution of c-Fos, in Gata3 deficient tumor cells inhibits EMT, preventing tumorigenesis and/or metastasis. In human breast cancers, GATA3 expression is negatively correlated with FRA1 and positively correlated with c-FOS. Low GATA3 and FOS, but high FOSL1, are characteristics of BLBCs. Together, these data provide the first genetic evidence indicating that loss of function of GATA3 in mammary tumor cells activates FOSL1 to promote mesenchymal traits and CSC function, while concurrently repressing FOS to lose epithelial features. We demonstrate that FRA1 is required for the activation of EMT in GATA3 deficient tumorigenesis and metastasis.

ERα prevents tumorigenesis of both liver and breast cancer cells through CCN5

Estrogen receptor alpha (ERα)-mediated estrogen signaling has also shown to prevent hepatic tumorigenesis in mice. Consistent with this, hormone replacement therapy with estrogen supplementation dramatically reduced the risk of hepatocellular carcinoma. Silencing of ERα is also a key event for the transformation of ERα-positive breast cancer cells into malignant triple-negative breast cancer cells. However, the mechanisms underlying ERα-mediated prevention of both hepatic and mammary tumorigenesis in humans are still unclear. Here, we present a functional genomics study of ERα targeting by comparing human liver cancer cells with human breast cancer cells using “loss or gain of function” genetic assays of ERα in vitro and in vivo. We discover that cellular communication network factor 5 (CCN5) is a direct downstream target of ERα; ERα suppresses growth and prevents tumorigenesis and malignant transformation of both liver and breast cancer cells through CCN5 in humans. The ERα-CCN5 regulatory axis functions as suppressors for both hepatic and mammary tumors, which is a common mechanism of preventing tumorigenesis for both liver cancer and breast cancer in humans.

Allyl isothiocyanate inhibits invasion and angiogenesis in breast cancer via EGFR-mediated JAK-1/STAT-3 signaling pathway.

Angiogenesis, invasion, and metastasis are the main events of cancer cells. JAK-1/STAT-3 is a key intracellular signaling transduction pathway, which controls the growth, differentiation, apoptosis, invasion, and angiogenesis of various cancer cells. The present study explored the impact of allyl isothiocyanate (AITC) on the JAK-1/STAT-3 pathway in DMBA-induced rat mammary tumorigenesis. The mammary tumor was initiated through a single dose of 25mg DMBA/rat by a subcutaneous injection administered near the mammary gland. We observed decreased body weight and increased the total number of tumors, tumor incidence, tumor volume, well-developed tumor, and histopathological abnormalities in DMBA-induced rats that were modulated after being treated with AITC. Staining of mammary tissues showed a high accumulation of collagen in DMBA-induced rats and it was normalized by the AITC treatment. Moreover, DMBA-induced mammary tissues showed up-regulated expressions of EGFR, pJAK-1, pSTAT-3, nuclear fraction of STAT-3, VEGF, VEGFR2, HIF-1α, MMP-2, and MMP-9 and the down-regulated expressions of cytosolic fraction of STAT-3 and TIMP-2. Oral administration of AITC on DMBA-induced rats inhibits angiogenesis and invasion by modifying these angiogenic and invasive markers. The finding of the present study was further confirmed by molecular docking analysis that shows a strong binding interaction between AITC with STAT-3 and cocrystal structure of STAT-3 glide energy of -18.123 and -72.246 (kcal/mole), respectively. Overall, the results suggested that AITC inhibits activation of the JAK-1/STAT-3 pathway, which subsequently prevents angiogenesis and invasion. It was recommended that AITC might develop a beneficial effect against breast cancer.

Protective Effects of Pelargonidin against DMBA-Induced Mammary Tumorigenesis in BALB/c Mice through Reduced Oxidative Stress and Lipid Anomalies

Among luminal types of breast cancers, ER + breast cancer is the most frequently diagnosed cancer globally. ER + breast cancer is commonly treated with SERM drugs that block ER to prevent ER-mediated cancerous growth. Our previous computational screening found pelargonidin (PG) can inhibit ER-signaling with potent bioactivity and satisfactory toxicological features. The present study explored the anti-tumoral prospect of PG against DMBA-induced ER + murine mammary carcinogenesis. The female BALB/c mice were divided into control (A) and DMBA-exposed groups. Following tumor appearance, the DMBA-exposed group was divided into five groups: tumor control, PG-treated (Groups P25, P50, and P100), and tamoxifen-treated (TAM). The results indicated that PG-treatment dose-dependently reduced the mean tumor volume, reinstated body weight loss, and enhanced the percentage survival of tumor-bearing mice. In addition, we recorded a significant reduction in LPO, total cholesterol, and triglycerides and a surge in the activity of antioxidases and phase II detoxifying enzymes in PG-treated animals. PG also dose-dependently increased the serum level of unbound estradiol, an indicator of competitive ER binding by an ER agonist/antagonist. These data suggest that pelargonidin has potent anticancer potential against the animal model of ER + breast cancer that matches the efficiency of tamoxifen with conceivably fewer side effects.

FGFR2 Controls Growth, Adhesion and Migration of Nontumorigenic Human Mammary Epithelial Cells by Regulation of Integrin β1 Degradation

The role of fibroblast growth factor receptor 2 (FGFR2), an important mediator of stromal paracrine and autocrine signals, in mammary gland morphogenesis and breast cancer has been extensively studied over the last years. However, the function of FGFR2 signalling in the initiation of mammary epithelial oncogenic transformation remains elusive. Here, FGFR2-dependent behaviour of nontumorigenic model of mammary epithelial cells was studied. In vitro analyses demonstrated that FGFR2 regulates epithelial cell communication with extracellular matrix (ECM) proteins. Silencing of FGFR2 significantly changed the phenotype of cell colonies in three-dimensional cultures, decreased integrins α2, α5 and β1 protein levels and affected integrin-driven processes, such as cell adhesion and migration. More detailed analysis revealed the FGFR2 knock-down-induced proteasomal degradation of integrin β1. Analysis of RNA-seq databases showed significantly decreased FGFR2 and ITGB1 mRNA levels in breast tumour samples, when compared to non-transformed tissues. Additionally, high risk healthy individuals were found to have disrupted correlation profiles of genes associated with FGFR2 and integrin signalling, cell adhesion/migration and ECM remodelling. Taken together, our results strongly suggest that FGFR2 loss with concomitant integrin β1 degradation is responsible for deregulation of epithelial cell-ECM interactions and this process may play an important role in the initiation of mammary gland epithelial tumorigenesis.

Endogenous myoglobin expression in mouse models of mammary carcinoma reduces hypoxia and metastasis in PyMT mice

Myoglobin (MB) is expressed in different cancer types and may act as a tumor suppressor in breast cancer. The mechanisms by which basal MB expression level impacts murine mammary tumorigenesis are unclear. We investigated how MB expression in breast cancer influences proliferation, metastasis, tumor hypoxia, and chemotherapy treatment in vivo. We crossed PyMT and WapCreTrp53flox mammary cancer mouse models that differed in tumor grade/type and onset of mammary carcinoma with MB knockout mice. The loss of MB in WapCre;Trp53flox mice did not affect tumor development and progression. On the other hand, loss of MB decreased tumor growth and increased tissue hypoxia as well as the number of lung metastases in PyMT mice. Furthermore, Doxorubicin therapy prevented the stronger metastatic propensity of MB-deficient tumors in PyMT mice. This suggests that, although MB expression predicts improved prognosis in breast cancer patients, MB-deficient tumors may still respond well to first-line therapies. We propose that determining the expression level of MB in malignant breast cancer biopsies will improve tumor stratification, outcome prediction, and personalized therapy in cancer patients.

Abstract 339: Exploring the roles of GSDMD in mammary tumorigenesis

Abstract Breast cancer (BC) is the most frequently diagnosed cancer worldwide, with an ever-increasing incidence rate. While screening and treatment methods have improved markedly over the last three decades, BC remains the leading cause of malignant death in women annually. Therefore, there is an urgent need to identify new biomarkers and therapeutic targets for BC. Recently, dysregulated gasdermin D (GSDMD), a central player of inflammation-induced cell death called pyroptosis, has been shown to be implicated in cancer. However, it is unclear whether GSDMD plays important roles in BC. In addition, the molecular mechanism underlying GSDMD-involved tumorigenesis remains unknown. Here, we discovered that GSDMD is significantly upregulated in BC. We have shown that overexpression or knockout of GSDMD increases or decreases BC cell proliferation, respectively. In addition, we have identified and validated several novel GSDMD-interacting proteins that play important roles in breast cancer. These present findings will not only shed light on the roles of GSDMD in BC tumorigenesis but also provide new preclinical data for future successful treatments of BC patients. Citation Format: Derek Yisen Zhang. Exploring the roles of GSDMD in mammary tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 339.

Abstract 85: Senescent cells promote breast cancer tumorigenesis

Abstract Age is the largest risk factor for breast cancer development. While numerous changes arise with age, we posit that the accumulation of p16INKA4 (p16)+ senescent stromal cells—which express cytokines, chemokines, and enzymes together recognized as senescence-associated secretory phenotype (SASP)—promote cancer progression. Indeed, high p16 positivity in stroma strongly correlates with breast cancer recurrence. To investigate how senescent stroma impact tumorigenesis, we crossed MMTV-PyMT (PyMT) mice that spontaneously develop mammary tumors to INK-ATTAC (INK) mice, which allows selective elimination of p16+ cells. Depletion of p16+ stroma in PyMT/INK mice led to a significantly delayed tumor onset and changes in tumor-infiltrating immune cells, both of which could be recapitulated by treating PyMT mice with a senolytic drug. scRNA-seq analyses of murine and human tumor samples revealed that senescence is restricted to a specific cancer associated fibroblasts (CAF) subpopulation that expresses numerous immune and extracellular matrix SASP factors that could impact T cells and Natural Killer (NK) cells. We refer to these cells as senescent CAFs (senCAFs). To assess the impact of senCAFs on T and NK cells’ anti-tumor effect, we depleted each population and found that the elimination of senCAFs no longer restricted tumor development in the absence of NK cells. We thus focused on addressing how senCAFs modulate NK cell function. Together our data highlight how senescent CAFs contribute to mammary gland tumorigenesis by modifying tumor microenvironment. Citation Format: Jiayu (Jennifer) Ye, Ana Paula Delgado, Qihao Ren, Robert S. Powers, Sheila A. Stewart. Senescent cells promote breast cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 85.

Abstract 4237: Social isolation induces gut dysbiosis, mitochondrial metabolic dysfunction, and infiltration of tumor immunosuppressive cells: Do they explain enhanced mammary tumorigenesis

Abstract Social isolation (SI), are associated with increased mortality from many diseases, including breast cancer. Up to 41% of breast cancer patients are estimated to feel socially isolated when assessed between 6 months and 2 years following their cancer diagnosis. Since socially isolated breast cancer survivors are at a 64% higher risk of breast cancer-specific mortality than socially integrated survivors, the biological mechanisms which mediate the effects of SI need to be identified to prevent increased mortality. In a preclinical model, we found that SI caused recurrence of mammary cancers which had been treated with hormone therapy and thus led cancer cells to emerge from dormancy. We further found that mammary glands of SI rats exhibited upregulation of IL6/JAK/STAT3 signaling pathway and impaired oxidative phosphorylation (OXPHOS), compared with group housed (GH) rats. We investigated if these changes could be mediated through the gut-brain-axis. Stress can affect the gut microbiota, and the gut microbiota is critical for regulating the host immunity. In four separate experiments, the effect of SI on the gut microbiota composition was assessed in C57BL/6 mice that were either group housed or housed singly in SI for 4 weeks. In each study replicate, several differences in the gut microbiota composition were seen, but these differences were mostly unique to each experiment. Beta-diversity was increased in three of the four experiments in SI mice. Nevertheless, at the genus level, SI suppressed the abundance of Akkermansia in all four replicates and increased Acetatifactor in three of the four replicates. These two bacterial changes are expected to disrupt OXPHOS, perhaps by resulting a suppression in short-chain fatty acid production. Further, low Akkermansia and high Acetatifactor are expected to increase inflammation. Thus, it is possible that the changes in the composition of the gut microbiota of SI animals explain an increase in IL6 signaling and a suppression in OXPHOS in their mammary gland. We also have studied immune cells in the spleen in SI and GH mice. SI increased the frequency of pro-inflammatory CD4+RORy+ cells, and the immunosuppressive Treg cells (CD4+Foxp3+) and myeloid derived suppressor cells (PMN-MDSCs). SI also increased PD1 expression in Foxp3+ cells. In an on-going study, we found that fecal microbiota transplant (FMT) from SI mice increased E0771 mammary tumor growth in antibiotic treated GH recipient mice, compared with mice receiving FMT from GH mice. Whether the FMT also altered IL6 and OXPHOS signaling in the mammary glands and tumors of recipient mice is currently being investigated. Our findings suggest that social isolation may increase breast cancer through inducing gut dysbiosis. Citation Format: Fabia de Oliveira Andrade, Maddie McDermott, Lu Jin, Vivek Verma, Karla Andrade de Oliveira, Christopher Staley, Leena Hilakivi-Clarke. Social isolation induces gut dysbiosis, mitochondrial metabolic dysfunction, and infiltration of tumor immunosuppressive cells: Do they explain enhanced mammary tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4237.

Abstract 5280: O-GlcNAcylation of progesterone receptor promotes mammary tumorigenesis

Abstract The role of hormone receptors in breast cancer has been well established as a key contributor to breast cancer tumorigenesis. Estrogen receptor and progesterone receptors are present in nearly 70% of breast cancers. The role of estrogen receptor and estrogens in breast cancer has been extensively studied and targeted effectively through endocrine therapy, however the impact of progesterone and the progesterone receptor (PR) independent of estrogen is not well understood. We have previously demonstrated that PR promotes tumor growth and drives an immune suppressive environment, therefore understanding the biology of PR in breast cancer is critical. We have also demonstrated that PR attenuates type 1 interferon signaling via inhibition of STAT1 phosphorylation and increased degradation of STAT2. Post-translational modifications such as phosphorylation and SUMOylation can influence PR target gene promoter selectivity thereby altering its function. Our recent work indicates PR is modified by O-GlcNAc, a single N-acetyl-glucosamine sugar that cycles on and off and serine or threonine amino acids in nuclear, cytoplasmic and mitochondrial proteins. Levels of total O-GlcNAc staining are higher in breast cancer tissue compared to adjacent normal tissue. Active O-GlcNAcylation, as evidenced by immunohistochemistry staining of O-GlcNAc-transferase (OGT), is elevated in patients with PR+ tumors compared to PR-. Using T47D breast cancer cells (an ER/PR-positive tumor line), mass spectrometry analysis revealed an O-GlcNAc site at S499 on PR. We then used a naturally occurring PR-negative variant of T47D cells to introduce stable expression of a mutant PR with serine to alanine substitution at the O-GlcNAc site (S499A), thereby blocking O-GlcNAc flux, in addition to wt PR as a control. RNA-Seq analysis of these cells following treatment with progesterone revealed several significant differential gene expression patterns, including rescue of PR dependent interferon signaling attenuation. T47D-mutant PR cell lines implanted into immune deficient mice had significantly decreased growth as compared to WT-PR control tumors. In syngeneic mice harboring PR-positive and PR-negative tumors, loss of O-GlcNAc flux and increased O-GlcNac levels increased tumor growth only when tumors were expressing PR, corroborating the influence of O-GlcNAc in immunodeficient mice. Together these findings indicate O-GlcNAcylation of PR is potentially a mechanism of PR driven breast cancer tumorigenesis and immune evasion. Citation Format: Harmony Ivanna Saunders, Sean M. Holloran, Gloria Trinca, Chad Slawson, Christy Hagan. O-GlcNAcylation of progesterone receptor promotes mammary tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5280.

Abstract 2367: Hedgehog signaling influences tumor immunogenicity by the modulation of T cell populations in breast cancer

Abstract Breast cancer is becoming more prevalent as it now makes up 30% of all new cancer cases yearly for U.S. women. Additionally, highly aggressive triple-negative breast cancer (TNBC) presents with immunologically “cold” tumors characterized by limited infiltration of anti-tumorigenic lymphocytes and the promotion of immunosuppressive cell populations. Therefore, it is essential to consider possible combinational therapies in treating primary tumors and in preventing metastases and recurrence. In the breast tumor microenvironment (TME), the presence and activity of regulatory T cells (Tregs) have been associated with poorer prognoses in breast cancer due to their ability to promote the progression of tumor growth and metastasis. Our group has extensively studied how the Hedgehog (Hh) signaling pathway can modulate the aggressiveness and the immune portfolio of the mammary TME, and we have recently reported the role of this pathway in regulating Treg activity and abundance. We have discovered that Hh inhibition can influence Treg-to-Th17 plasticity, promoting Tregs into a pro-inflammatory Th17-like phenotype (Tr17), therefore, affecting the immunogenicity of the mammary TME. As Tregs are highly influential to other immune populations of the TME, it is imperative to determine how this phenotypic change in Tregs after Hh inhibition influences other T cell populations of the mammary TME. We hypothesize that systemic Hh inhibition reshapes the tumor-immune microenvironment to be tumor-eradicating by influencing key immune populations of the mammary TME. Therefore, we further investigated how CD4+ and CD8+ T cells are influenced by systemic Hh blockade in a mammary carcinoma mouse model using single-cell RNA-Sequencing (scRNA-Seq), multiparameter flow cytometry, and R programming for data analyses and visualization. We sought to interpret how Hh inhibition influences key T cell populations that can dictate the immunogenicity of the primary tumor and to determine if these changes may be a result of the Treg-to-Th17 transition in response to Hh manipulation. In these investigations, we found that systemic Hh inhibition modified the abundance, metabolic signatures, and effector functions of CD4+ and CD8+ T cell populations within the primary tumor. As Tregs can impair essential tumor-infiltrating lymphocytes and promote the recruitment of other immunosuppressive constituents to the primary mammary tumor and Th17s can promote inflammatory and cytotoxic immune populations, these studies will expand on the mechanism by which Hh signaling modulates mammary tumorigenesis holistically, as well as specifically, through Tregs. This comprehensive work revealing the mechanistic role of Hh signaling in Treg plasticity and other T cell subsets will also inform innovative combinational therapeutic strategies to treat highly aggressive TNBC. Citation Format: Courtney A. Swain, Dominique C. Hinshaw, Brandon J. Metge, Heba M. Alsheikh, Lalita A. Shevde-Samant. Hedgehog signaling influences tumor immunogenicity by the modulation of T cell populations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2367.

Z-Endoxifen prevents aggressive mammary cancers in mice by inhibiting cell proliferation and creating a tumor suppressive microenvironment.

Aggressive estrogen receptor (ER) positive breast cancer is frequently tamoxifen-resistant; alternative endocrine approaches exist for therapy, but not for prevention, particularly in premenopausal women. We examined the efficacy of the selective ER modulator (Z-endoxifen) as monotherapy and in combination with the selective progesterone receptor modulators (onapristone and ulipristal acetate) in the tamoxifen-insensitive C3(1)/SV40TAg mouse mammary tumorigenesis model. Unlike tamoxifen at human equivalent dose (HED) 101 mg/day, endoxifen at HED 24 mg/day significantly increased latency and reduced tumor growth relative to untreated controls. Ulipristal acetate (UPA) at HED 81 mg/day also significantly increased latency however failed to inhibit tumor growth, while onapristone (HED 98 mg/day) had no tumor prevention efficacy in this model. Addition of UPA to endoxifen did not enhance preventive efficacy over endoxifen alone. The expression of genes associated with cell cycle, cell proliferation and extracellular matrix remodeling was similarly repressed by endoxifen and UPA however only endoxifen significantly downregulated prominent genes associated with poor prognosis (Col11a1, Il17b, Pdgfa, Tnfrsf11a). Our results indicate that endoxifen can prevent breast cancers, even when tamoxifen-resistant, through its role in favorable tissue remodeling and immunomodulation.

Loss of Kmt2c in vivo leads to EMT, mitochondrial dysfunction and improved response to lapatinib in breast cancer

Deep sequencing of human tumours has uncovered a previously unappreciated role for epigenetic regulators in tumorigenesis. H3K4 methyltransferase KMT2C/MLL3 is mutated in several solid malignancies, including more than 10% of breast tumours. To study the tumour suppressor role of KMT2C in breast cancer, we generated mouse models of Erbb2/Neu, Myc or PIK3CA-driven tumorigenesis, in which the Kmt2c locus is knocked out specifically in the luminal lineage of mouse mammary glands using the Cre recombinase. Kmt2c knock out mice develop tumours earlier, irrespective of the oncogene, assigning a bona fide tumour suppressor role for KMT2C in mammary tumorigenesis. Loss of Kmt2c induces extensive epigenetic and transcriptional changes, which lead to increased ERK1/2 activity, extracellular matrix re-organization, epithelial-to-mesenchymal transition and mitochondrial dysfunction, the latter associated with increased reactive oxygen species production. Loss of Kmt2c renders the Erbb2/Neu-driven tumours more responsive to lapatinib. Publicly available clinical datasets revealed an association of low Kmt2c gene expression and better long-term outcome. Collectively, our findings solidify the role of KMT2C as a tumour suppressor in breast cancer and identify dependencies that could be therapeutically amenable.

Kindlin-1 regulates IL-6 secretion and modulates the immune environment in breast cancer models.

The adhesion protein Kindlin-1 is over-expressed in breast cancer where it is associated with metastasis-free survival; however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in mouse models of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells led to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs. Similar changes in T cell populations were seen following depletion of Kindlin-1 in the polyomavirus middle T antigen (PyV MT)-driven mouse model of spontaneous mammary tumorigenesis. There was a significant increase in IL-6 secretion from Met-1 cells when Kindlin-1 was depleted and conditioned media from Kindlin-1-depleted cells led to a decrease in the ability of Tregs to suppress the proliferation of CD8+ T cells, which was dependent on IL-6. In addition, deletion of tumor-derived IL-6 in the Kindlin-1-depleted tumors reversed the reduction of tumor-infiltrating Tregs. Overall, these data identify a novel function for Kindlin-1 in regulation of anti-tumor immunity, and that Kindlin-1 dependent cytokine secretion can impact the tumor immune environment.

Abstract P5-15-01: Rediscovering IRF5: A prognostic indicator with novel roles in mammary gland development, ribosome biogenesis, tumor initiation, and metastasis

Abstract Breast cancer is the second leading cause of death for women in the United States. However, only 10% of breast cancers have been linked to inherited genomic mutations. Thus, identifying biomarkers to predict cancer progression and metastasis remains a clear need in the research and medical world. Here, we report a novel role for Interferon Regulatory Factor 5 (IRF5) in mammary gland development, tumorigenesis, and metastasis. Historically, IRF5 has been studied as a transcription factor in the context of genetic risk for autoimmune diseases. However, mining of The Cancer Genome Atlas revealed that loss of IRF5 expression in human breast cancer is significantly linked with progression to high grade carcinoma, increased metastasis, and decreased overall and recurrence-free survival. Supporting these analyses, we demonstrated that female Irf5-/- BALB/c mice have higher incidence of spontaneous atypical ductal hyperplasia (ADH), increased progression to DCIS, and ultimately, increased incidence of IDC. Using qPCR, FISH and IHC, we confirmed that IRF5 is expressed in both luminal and basal myoepithelial cells, but expression is higher in basal cells. Histologic analysis of whole mount preparations of Irf5-/- mammary glands revealed aberrant ductal morphogenesis, characterized by expansion of luminal and basal myoepithelial cells with a loss of organized glandular structure. RNAseq of primary mammary epithelial cells from wild type and Irf5-/- littermate mice showed Irf5-/- mammary epithelial cells to be enriched in ribosome biogenesis pathways, the physiologic consequences of which were demonstrated through increased rates of protein synthesis. Transferring our studies in vivo, we demonstrated that loss of tumor IRF5 expression resulted in decreased tumor-infiltrating lymphocytes and increased pulmonary metastasis in the murine orthotopic 4T1 implantation model. Mechanistically, we found that—as in our studies utilizing primary mammary epithelial cells—IRF5 expression in tumors regulated protein translation and ribosome biogenesis. In light of these findings, we propose IRF5 as a novel prognostic biomarker, loss of which alters mammary gland development, drives tumor initiation and metastasis, and dysregulates mammary epithelial cell protein synthesis. Citation Format: Betsy Barnes, Zarina Brune, Matthew Rice, Carter Somerville. Rediscovering IRF5: A prognostic indicator with novel roles in mammary gland development, ribosome biogenesis, tumor initiation, and metastasis [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 P5-15-01.

Abstract P6-05-16: Social isolation induces gut dysbiosis, mitochondrial metabolic dysfunction, and infiltration of tumor immunosuppressive cells: do they explain enhanced mammary tumorigenesis?

Abstract Inadequate social contacts and loneliness, often referred to as social isolation (SI), are associated with increased mortality from many diseases, including breast cancer. Up to 41% of breast cancer patients have been identified as feeling socially isolated. Moreover, socially isolated breast cancer survivors have a 43% higher risk of recurrence than socially integrated survivors. To prevent increased mortality, biological mechanisms which mediate the effects of SI on cancer need to be identified. One unexplored, but possible mechanism is through the gut microbiota. Through bidirectional interactions, the gut is affected by stress and the gut microbiota in turn can modulate stress response, host immunity and metabolism. Here we tested the hypothesis that SI induces gut dysbiosis. In our study, repeated in four separate experiments, adult female mice were divided into two groups – those kept group housed (GH, 4 mice per cage) and those housed singly in SI for 4 weeks. Several differences in the gut microbial family, genus and species levels were seen, but the differences were mostly unique to each of the four experiment. Beta-diversity was increased in three of the four studies in SI mice. Since beta-diversity is increased by aging, SI may accelerate the aging process. At the genus level, SI significantly suppressed the abundance of Akkermansia in all four studies and increased Acetatifactor in three studies. These two bacterial changes are expected to disrupt mitochondrial oxidative phosphorylation (OXPHOS), most likely by suppressing the short-chain fatty acid production. Further, low Akkermansia and high Acetatifactor are expected to increase inflammation. In a separate study, we discovered that SI impaired OXPHOS and activated inflammatory pathways in the mammary gland. We also have assessed immune cells in the spleen. SI increased the frequency of pro-inflammatory CD4+RORy+ cells, and the immunosuppressive Treg (CD4+Foxp3+) and PMN-MDSCs cells. In addition, SI increased PD1 expression in Foxp3+ cells, suggesting that anti-PD1 therapy might adversely affect socially isolated breast cancer patients by invigorating Treg cells. We are currently studying if the changes in the gut microbiota in SI mice are causally linked to their impaired mitochondrial metabolism, immunosuppression and increased mammary cancer mortality. We also plan to investigate if dietary modifications can reverse gut dysbiosis in SI mice and prevent their increased mortality from mammary cancer. Citation Format: Fabia de Oliveira Andrade, Lu Jin, Vivek Verma, Maddie McDermott, Chris Staley, Leena Hilakivi-Clarke. Social isolation induces gut dysbiosis, mitochondrial metabolic dysfunction, and infiltration of tumor immunosuppressive cells: do they explain enhanced mammary tumorigenesis? [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 P6-05-16.