Inhibition of mTOR attenuates the initiation and progression of BRCA1-associated mammary tumors

Abstract

Inherited mutation in breast cancer susceptibility gene 1 (BRCA1) is strongly associated with mammary tumors that exhibit triple-negative characteristics, are insensitive to endocrine-targeted therapies, and show basal-like properties, including aggressive phenotypes [1, 2]. It has been reported that the average cumulative risk of breast cancer for BRCA1 mutation carriers by age 70 years is 57% (95% confidence interval [CI]: 47%-66%) [3]. Despite the high incidence and aggressive characteristics of BRCA1-associated breast cancer, few substantial improvements in preventing or treating this cancer have been made, largely due to the challenges of clinic-based cohort studies. During malignant transformation, cancer progression is facilitated by metabolic reprogramming–one of the hallmark characteristics of cancer. Previously, we found that inhibition of AKT is a potential strategy for the prevention and therapeutic management of Brca1-mutant mammary tumors. However, pharmacological inhibition proved less effective and less safe compared to genetic perturbation, limiting its potential for clinical application [4]. Meanwhile, mTOR, a key regulator of metabolism and a downstream target of the PI3K/AKT signaling pathway, has emerged as a promising therapeutic target for several diseases, including treatment of cancer [5].

In addition to identifying the contribution of mTOR signaling to BRCA1-deficient cells (Supplementary Figure S1), we provide genetic and pharmacological evidence using multi-orthogonal preclinical models [6-8] that mTOR is closely involved in the development and growth of Brca1-mutated mammary tumors (Figure 1A). To investigate the role of mTOR in the absence of BRCA1, we assessed the development of mammary glands in post-pubertal Brca1/Mtor-mutant mice by examining ductal and lobular development of the fourth mammary gland. Measurements of mammary gland density using the Branch software (ver. 1.1 [9]) showed that ductal length and branching were significantly diminished in the mammary glands of Brca1^co/coMtor^co/coMMTV-Cre mice (Figure 1B,C, Supplementary Figure S2). To determine whether mTOR contributes to BRCA1-deficient mammary tumor formation, we examined tumor formation in cohorts of Brca1^co/co (n = 28), Brca1^co/coMtor^co/co (n = 30), Brca1^co/coMMTV-Cre (n = 24), and Brca1^co/coMtor^co/coMMTV-Cre (n = 29) mice (Top left of Figure 1A). Brca1^co/co and Brca1^co/coMtor^co/co mice showed no signs of mammary abnormalities, including tumors, up to 24 months of age. In contrast, Brca1^co/coMMTV-Cre mutant mice developed breast cancer, reaching a high incidence (37.5%; 9/24) by 24 months of age. During the same period, Brca1^co/coMtor^co/coMMTV-Cre mice exhibited a lower incidence of breast cancer (6.9%; 2/29) and significantly better tumor-free survival compared to Brca1^co/coMMTV-Cre mice (P = 0.008, log-rank test) (Figure 1D). Next, we examined whether mTOR inhibition using a clinically applicable pharmacological approach would produce similar effects as genetic ablation. To test pharmacological inhibition of mTOR, we administered everolimus (20 mg/kg, oral, 5 times/week) or vehicle to 4-month-old Brca1^co/coMMTV-Cre mice for 11 months (lower left of Figure 1A, Supplementary Figure S3). During this period, Brca1^co/coMMTV-Cre mice in both groups spontaneously developed palpable mammary tumors. At the end of the study period (15 months of age), vehicle-treated Brca1^co/coMMTV-Cre mice showed a high incidence of mammary tumors (93%; 13 of 14). During the same period, everolimus-treated Brca1^co/coMMTV-Cre mice exhibited a breast cancer incidence of 46% (5 of 11) and significantly longer tumor-free survival compared to their vehicle-treated counterparts (P = 0.0117, log-rank test) (Figure 1E). Moreover, while multiple tumors were found in 2 of 14 (14%) vehicle-treated mice, no cases of multiple tumors were detected in everolimus-treated mice (Figure 1F). In addition to tumor formation, whole-mount analysis of non-tumor–bearing mammary glands revealed that everolimus treatment reduced total ductal length and branch number by approximately 30% compared to vehicle treatment (Supplementary Figure S3). Notably, everolimus treatment significantly reduced the formation of abnormal hyperplastic foci (0.6 vs. 5.5 foci/mammary gland; P < 0.01) in non-tumor–bearing mammary glands of Brca1-mutant mice (Figure 1G,H). Taken together, these results suggest that genetic ablation and pharmacological inhibition of mTOR signaling prevents the proliferation of mammary epithelial cells and reduce tumor formation in Brca1-mutant mice.

To determine whether mTOR inhibition also suppresses the progression of BRCA1-associated breast cancer, we tested the efficacy of everolimus on spontaneously developed mammary tumors in Brca1^co/coMMTV-Cre mice through periodic observation and palpation. Tumor-bearing mice (size < 0.5 cm³) were then randomized to receive either vehicle or everolimus via oral gavage (Top right of Figure 1A, Supplementary Figure S4). Tumor volumes at baseline and during progression were measured weekly using magnetic resonance imaging (MRI) until the tumors reached a volume of ∼3 cm³ (Figure 1I). Tumors in vehicle-treated mice grew more rapidly than those in everolimus-treated mice (Figure 1J, left panel). An analysis of weekly progression showed that 73% (28 of 38) of tumors in vehicle-treated mice exhibited greater than 50% progression, compared to only 33% (46 of 141) in everolimus-treated mice (Figure 1J, right panel; P = 0.0002, chi-square test). Additionally, the weekly increase in tumor volume in the everolimus-treated group (51.0%, 95% CI: 40.3%-61.8%) was significantly lower (P < 0.001) than that in the vehicle-treated group (89.1%, 95% CI: 70.4%-107.7%). Moreover, everolimus-treated mice showed significantly longer survival (2.8-fold on average) compared to vehicle-treated mice (Figure 1K; P < 0.001). Importantly, while everolimus treatment significantly improved therapeutic outcomes in Brca1-mutant tumors, responses to everolimus showed heterogeneity among individual mice. Specifically, 8 of the 15 mice, designated as responder mice, exhibited a significant reduction in the ratio of tumor volume (RTV) in response to everolimus. In contrast, the remaining 7 mice, designated as non-responders, displayed a higher RTV than responders and vehicle-treated mice (Figure 1L,M). Additionally, the survival of responders (13.0 weeks) was nearly double that of non-responders (6.6 weeks) (Figure 1N). To further examine the therapeutic efficacy of everolimus, we employed an engraft model for preclinical evaluation. Tumor tissues were collected from 22 individual spontaneously developed mammary tumors in Brca1^co/coMMTV-Cre mice, orthotopically transplanted into nude female mice, amplified, re-transplanted, and subsequently treated with either vehicle or everolimus. Tumor progression was monitored (bottom right of Figure 1A, Supplementary Figure S5), and all mice were sacrificed when any tumors in either vehicle- or everolimus-treated group reached ∼3 cm³ (Figure 1O, Supplementary Figure S6). Tumors from everolimus-treated mice showed significant reductions in RTV (43%) and weight (38%) compared to tumors from vehicle-treated mice (Figure 1P, Supplementary Figure S5). These findings suggest effective management of BRCA1-associated breast cancer by pharmacological mTOR inhibition.

To explore this heterogeneity, we conducted global proteome and phosphoproteome profiling of vehicle- and everolimus-treated allograft tumors (Supplementary Figure S7A). Two distinct sample clusters (Sub1 and Sub2) were identified using both protein and phosphopeptide data (Supplementary Figure S7B,C). We identified 304 and 323 proteins that were upregulated, and 251 and 291 phosphopeptides that were upregulated, in Sub1 and Sub2, respectively (Figure 1Q). Sub2, characterized by higher RTVs and weights, represented the non-responders, whereas Sub1 corresponded to the responders (Figure 1R). The upregulated proteins and phosphoproteins in Sub2 were associated with neutrophil extracellular trap formation (NETosis) and leukotriene metabolism (Figure 1S). Enzymes involved in phosphatidylinositol and arachidonic acid formation/metabolism were upregulated in non-responders, leading to the release of leukotrienes (Figure 1T). Upon leukotriene binding, (1) proteins and phosphorylations mediating actin polymerization required for neutrophil migration, and (2) proteins involved in NETosis, were upregulated in non-responders (Figure 1T). Western blotting and immunohistochemistry confirmed the upregulation of representative markers of the leukotriene and NETosis pathways (Figure 1U, Supplementary Figure S7D,E).

Therefore, our findings provide preclinical evidence that targeting mTOR inhibition is a potential strategy for the prevention and therapeutic management of BRCA1-associated breast cancer. Additionally, activation of the leukotriene-neutrophil activation axis can serve as a predictive marker of resistance to targeted mTOR inhibition. We further discussed the leukotriene signaling as a predictive biomarker and potential clinical translational value of this study in supplementary information.

Chang-il Hwang, Daehee Hwang, and Sang Soo Kim conceived the study, designed, and supervised the experiments. Tae Hyun Kim, Chu-Xia Deng, Sung Chul Lim, Chang-il Hwang, Daehee Hwang, and Sang Soo Kim wrote and revised the manuscript. Eun Jung Park, Tae Hyun Kim, Dong Hoon Shin, Heesun Cheong, Chu-Xia Deng, Sung Chul Lim, Chang-il Hwang, Daehee Hwang, and Sang Soo Kim contributed to the data analysis and interpretation. Hye Jung Baek, Jihao Xu, and Heesun Cheong contributed to the in vitro experiments. Eun Joo Cho, Min Kyung Ki, and Dong Hoon Shin conducted the in vivo animal experiments. Geun Hee Han performed bioinformatical analysis. Tae Hyun Kim executed the statiscal analysis. Sung Chul Lim performed the pathological analysis. Eun Jung Park performed immunological analysis. All authors read and approved the final manuscript.

The authors declare no conflict of interest.

This work was supported by the National Cancer Center of Korea (NCC-2210680/2410880) and the National Research Foundation of Korea (2023R1A2C1004000).

All procedures involving animals and their care were approved by the Institutional Animal Care and Use Committee of the National Cancer Center of Korea (NCC-15-295).