A human transcriptome array on ERα-positive breast cancer continuum of risk identified Secreted Frizzled-Related Protein 1 (SFRP1) as decreased during breast cancer progression. In addition, SFRP1 was inversely associated with breast tissue age-related lobular involution, and differentially regulated in women with regard to their parity status and the presence of microcalcifications. The causal role of SFRP1 in breast carcinogenesis remains, nevertheless, not well understood. In this study, we characterized mammary epithelial cells from both nulliparous and multiparous mice in organoid culture ex vivo, in the presence of estradiol (E2) and/or hydroxyapatite microcalcifications (HA). Furthermore, we have modulated SFRP1 expression in breast cancer cell lines, including the MCF10A series, and investigated their tumoral properties. We observed that organoids obtained from multiparous mice were resistant to E2 treatment, while organoids obtained from nulliparous mice developed the luminal phenotype associated with a lower ratio between Sfrp1 and Esr1 expression. The decrease in SFRP1 expression in MCF10A and MCF10AT1 cell lines increased their tumorigenic properties in vitro. On the other hand, the overexpression of SFRP1 in MCF10DCIS, MCF10CA1a, and MCF7 reduced their aggressiveness. Our results support the hypothesis that a lack of SFRP1 could have a causal role in early breast carcinogenesis.

In 2020, breast cancer was the most widespread cancer. This heterogenous disease can be divided into molecular subtypes according to three biomarkers: Estrogen Receptor (ER), Progesterone Receptor (PR) and Epidermal Growth Factor Receptor 2 (HER2). Currently, triple-negative breast cancers (TNBC), negative for all three biomarkers, have no personalized treatment. Also, about 40% of women with breast cancer receiving tamoxifen, a selective estrogen receptor modulator (SERM), have or will develop a resistance. Bazedoxifene (BZA), a SERM, a selective estrogen receptor modulator (SERD) and an indirect Signal transducer and activator of transcription 3 (STAT3) inhibitor, used for treatment of osteoporosis, has exhibited in vitro antitumoral effects. Therefore, BZA efficacy must be further evaluated. We hypothesize that BZA is a therapeutic option for hormone receptor (HR)-positive breast cancers and a novel anticancer agent for TNBC patients. To verify our hypothesis, we performed 2D viability assays on breast cancer cell lines treated with different BZA and hydroxytamoxifen (4OHT) concentrations to calculate the absolute half maximal inhibitory concentration (IC50). We then performed 3D viability assays, which is a first with BZA treatment. We further investigated gene expression of known STAT3 targets in breast cancer subtypes in UALCAN and on survival outcomes in PRECOG and ROCplot. The 72 hours BZA IC50 is respectively 8.038 ± 0.132 µM for TNBC basal-like 2 cell line (SUM149PT) and the 4OHT IC50 for this cell line is 6.095 ± 0.133 µM (p-value= 0.1). Despite IC50 seeming lower for 4OHT, the results are not significant when performing two sided Mann-Whitney. Similarly, for MDA-MB-231, another TNBC cell line, characterized as mesenchymal-like, BZA IC50 (7.988± 0.482 µM) seems higher but is not significantly different to the 4OHT IC50 (7.080± 0.167 µM) (p-value= 0.1). We also found that BZA IC50 and 4OHT IC50 for MCF7 (HR+) are not significantly different (p-value= 0.4). 3D viability assays performed on SUM149PT spheroids treated for 3 days with 2X and 5X BZA IC50 decreased cell viability whilst 2X 4OHT IC50 did not reduce cell viability. Lastly, we determined that BIRC5, a known inhibitor of apoptosis, is more expressed in TNBC patients from TCGA breast cancer cohort than other molecular subtypes, but patients diagnosed with luminal A breast cancer (HR+/HER2-) receiving chemotherapy and experiencing a recurrence have a higher median gene expression of BIRC5. In summary, we have shown that BZA seems less effective to reduce cell viability in 2D assays than 4OHT in TNBC cell line but may be more effective in 3D models. A decrease in phosphorylated STAT3 following BZA treatment could be beneficial to patients with luminal A breast cancer who are not responding to chemotherapy. Citation Format: Juliette Bherer, Alisson Clemenceau, Caroline Diorio, Francine Durocher. Breast cancer treatment: Exploring bazedoxifene, a known SERM, SERD and indirect STAT3 inhibitor [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 1587.

Breast cancer is the leading cause of cancer-related death in women worldwide. Our group previously performed a human transcriptome array on normal tissue, atypical ductal hyperplasia, ductal carcinoma in situ and estrogen receptor-α positive invasive ductal carcinoma. Among the transcripts down-regulated during carcinogenesis: Secreted Frizzled-Related Protein 1 (SFRP1), a Wnt signaling pathway antagonist inversely associated with age-related lobular involution has been identified.Interestingly, SFRP1 expression is higher in presence of hydroxyapatite microcalcifications in non-tumoral breast tissue. SFRP1 is also known to be involved in the increase of bone resorption, notably by downregulating osteoblastic differentiation. This suggests that the lack of SFRP1 could be involved in both osteoblast-like phenotype acquisition and early breast carcinogenesis. Unfortunately, data on the mechanism of microcalcifications formation and their impacts on breast carcinogenesis are still lacking.We hypothesize that loss of SFRP1 is responsible for microcalcifications accumulation and early breast carcinogenesis. Our objectives are:1. To define the impact of microcalcifications on SFRP1 expression in non-tumoral mammary gland and 2. to decipher the causal role of SFRP1 in microcalcifications formation. Organoid culture using freshly purified epithelial cells from nulliparous and multiparous mice mammary glands, in presence or in absence of hydroxyapatite crystals has been performed. Organoids number and phenotype have been studied (aim 1). We also have modulated SFRP1 expression in mammary cell lines by lentiviral transduction and performed proliferation, migration and alizarin assays to assess the tumoral aggressivity and cells abilities to produce calcium deposits in vitro (aim 2). SFRP1 and markers of osteoblastic differentiation (Runt-related transcription factor 2, RUNX2) and bone formation (Alkaline Phosphatase, ALPL; Osteopontin, SPP1; Periostin, POSTN) will also be quantified by quantitative PCR in both mice organoids and human mammary cell lines (aims 1 and 2). 1. The ratio between the mammary gland and body weight obtained from nulliparous mice (n = 12) is significantly lower compared with multiparous mice (n = 12; fold-change = 1.84; p-value < 0.001). Hydroxyapatite crystals induced an increase in the proportion of organoids with lumen and branching compared with the control without hydroxyapatite specifically in organoids obtained from nulliparous mice. Organoid’s size and gene expression profiles are still to be assessed. 2. SFRP1 downregulation in a non-tumoral atypical ductal hyperplasia cell line (MCF10AT1) induced an increase in proliferative and migratory cells abilities. It was also responsible for an increase in estrogen receptor 1 (ESR1) expression, suggesting that SFRP1 is related to luminal carcinogenesis. Results also demonstrate that decreasing SFRP1 in MCF10AT1 induced a profile similar to growth stage in bone tissue, which notably means an increase of expression of the osteoblastic transcription factor RUNX2 and the marker of mineralization SPP1, also involved in lactation-related pink adipose tissue formation. We also observed an increase of POSTN expression, known to be involved in decreased bone mineral density during lactation.SFRP1 upregulation in the invasive luminal A cell line MCF7 reduced cell migration and ESR1 expression. Likewise, it increased MCF7 proliferation compared to control. In addition, it induced an increase of RUNX2 and SPP1 expression, similar to what is reported in matrix mineralization profile in bone. These results support our hypothesis that SFRP1 has a causal role in the acquisition of a malignant phenotype but also in osteoblast-like phenotype in mammary epithelial cells. The cause of SFRP1 downregulation in women remains to be explored. Citation Format: Alisson Clemenceau, Aurélie Lacouture, Juliette Bherer, Audet-Walsh Étienne, Caroline Diorio, Francine Durocher. Secreted frizzled related protein 1: From lobular involution to breast osteoimmunological disorder [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-11.

Breast cancer (BC) is a heterogenous disease classified into four molecular subtypes (Luminal A, Luminal B, HER2 and triple-negative (TNBC)) depending on the expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal receptor 2 (HER2). The development of effective treatments for BC, especially TNBC, remains a challenge. Aminosteroid derivative RM-581 has previously shown an antiproliferative effect in multiple cancers in vitro and in vivo. In this study, we evaluated its effect in BC cell lines representative of BC molecular subtypes, including metastatic TNBC. We found that RM-581 has an antiproliferative effect on all BC molecular subtypes, especially on Luminal A and TNBC, in 2D and 3D cultures. The combination of RM-581 and trastuzumab or trastuzumab-emtansine enhanced the anticancer effect of each drug for HER2-positive BC cell lines, and the combination of RM-581 and taxanes (docetaxel or paclitaxel) improved the antiproliferative effect of RM-581 in TNBC and metastatic TNBC cell lines. We also confirmed that RM-581 is an endoplasmic reticulum (EnR)-stress aggravator by inducing an increase in EnR-stress-induced apoptosis markers such as BIP/GRP78 and CHOP and disrupting lipid homeostasis. This study demonstrates that RM-581 could be effective for the treatment of BC, especially TNBC.