Targeting the mTOR Pathway for the Prevention of ER-Negative Breast Cancer.

PREVENTION RELEVANCE: Our results show that everolimus delays mammary tumor formation in multiple mouse models, suggesting that mTOR inhibitors will be useful for the prevention of ER-negative and triple-negative breast cancer in humans. See related Spotlight, p. 787.

PI4KIIIß is a therapeutic target in chromosome 1q-amplified lung adenocarcinoma.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIß (PI4KIIIß). Molecular, biochemical, and cell biological studies show that PI4KIIIß-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIß-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIß antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIß-dependent secretion for cancer cell survival and tumor progression.

Death-associated protein kinase 1 promotes growth of p53-mutant cancers.

Estrogen receptor-negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor-negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers.

The combination of tamoxifen and the rexinoid LG100268 prevents ER-positive and ER-negative mammary tumors in p53-null mammary gland mice.

In pursuit of effective therapeutic agents for the estrogen receptor (ER)-negative breast cancer, we previously showed that bexarotene reduced mammary tumor development by 75% in ErbB2 mice. To further improve the effectiveness of breast cancer prevention, we have now investigated the effects of a combinatorial therapy consisting of two cancer preventive drugs. On the basis of the hypothesis, rexinoid LG100268 plus tamoxifen would more effectively prevent the development of both ER-positive and ER-negative breast cancer. We treated p53-null mammary gland mice with tamoxifen and LG100268, individually and in combination. By 60 weeks of age, vehicle-treated mice developed tumors in 52% of transplanted mammary glands, whereas mice treated with tamoxifen and LG100268 developed tumors in only 13% of transplanted mammary glands. To further define the mechanistic effects of this combinatorial treatment, we investigated the effects of tamoxifen and LG100268 on mammary tissue biomarkers. In mammary tissue harvested before tumor development, the proliferation markers Ki67 and cyclin D1 were significantly reduced in mice treated with the combination therapy. In addition, the rexinoid target genes ABCA1 and ABCG1 were induced in both the rexinoid and combination treatment groups, whereas expression remained constant in tamoxifen group. These results show that tamoxifen-LG100268 combinatorial treatment is more effective in preventing mammary tumors than either agent alone. In addition, these studies have identified relevant tissue biomarkers that can be used to show the effect of these agents on mammary tissue. These results support the development of clinical trials of antiestrogen and rexinoid combinatorial therapy for the prevention of patients with high-risk breast cancer.

Effect of lapatinib on the development of estrogen receptor-negative mammary tumors in mice.

Lapatinib, a selective orally available inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases, is a promising agent for the treatment of breast cancer. We examined the effect of lapatinib on the development of mammary tumors in MMTV-erbB2 transgenic mice, which express wild-type ErbB2 under the control of the mouse mammary tumor virus promoter and spontaneously develop estrogen receptor (ER)-negative and ErbB2-positive mammary tumors by 14 months of age. Mice were treated from age 3 months to age 15 months with vehicle (n = 17) or lapatinib (30 or 75 mg/kg body weight; n = 16 mice per group) by oral gavage twice daily (6 d/wk). All statistical tests were two-sided. By 328 days after the start of treatment, all 17 (100%) of the vehicle-treated mice vs five (31%) of the 16 mice treated with high-dose lapatinib developed mammary tumors (P < .001). Among MMTV-erbB2 mice treated for 5 months (n = 20 mice per group), those treated with lapatinib had fewer premalignant lesions and noninvasive cancers in their mammary glands than those treated with vehicle (P = .02). Lapatinib also effectively blocked epidermal growth factor-induced signaling through the EGFR and ErbB2 receptors, suppressed cyclin D1 and epiregulin mRNA expression, and stimulated p27 mRNA expression in human mammary epithelial cells and in mammary epithelial cells from mice treated for 5 months with high-dose lapatinib. Thus, cyclin D1, epiregulin, and p27 may represent useful biomarkers of lapatinib response in patients. These data suggest that lapatinib is a promising agent for the prevention of ER-negative breast cancer.

The AP-1 transcription factor regulates postnatal mammary gland development.

The AP-1 transcription factor is activated by multiple growth factors that are critical regulators of breast cell proliferation. We previously demonstrated that AP-1 blockade inhibits breast cancer cell growth in vitro. Yet a specific role of AP-1 in normal mammary gland development has not been studied. Using a bi-transgenic mouse expressing an inducible AP-1 inhibitor (Tam67), we found that the AP-1 factor regulates postnatal proliferation of mammary epithelial cells. Mammary epithelial proliferation was significantly reduced after AP-1 blockade in adult, prepubertal, pubertal, and hormone-stimulated mammary glands. In pubertal mice, mammary cell proliferation was greatly reduced, and the cells that did proliferate failed to express Tam67. We also observed structural changes such as suppressed branching and budding, reduced gland tree size, and less fat pad occupancy in developing mammary glands after AP-1 blockade. We further demonstrated that Tam67 suppressed the expression of AP-1-dependent genes (TIMP-1, vimentin, Fra-1, and fibronectin) and the AP-1-dependent growth regulatory genes (cyclin D1 and c-myc) in AP-1-blocked mammary glands. We therefore conclude that AP-1 factor is a pivotal regulator of postnatal mammary gland growth and development.