Obesity increases both the risk and mortality associated with many types of cancer including that of the breast. In mice, obesity increases both incidence of spontaneous tumors and burden of transplanted tumors. Our findings identify leptin, an adipose secreted cytokine, in promoting increased mammary tumor burden in obese mice and provide a link between this adipokine and cancer. Using a transplantable tumor that develops spontaneously in the murine mammary tumor virus-Wnt-1 transgenic mice, we show that tumors transplanted into obese leptin receptor (LepRb)-deficient (db/db) mice grow to eight times the volume of tumors transplanted into lean wild-type (WT) mice. However, tumor outgrowth and overall tumor burden is reduced in obese, leptin-deficient (ob/ob) mice. The residual tumors in ob/ob mice contain fewer undifferentiated tumor cells (keratin 6 immunopositive) compared with WT or db/db mice. Furthermore, tumors in ob/ob mice contain fewer cells expressing phosphorylated Akt, a growth promoting kinase activated by the LepRb, compared with WT and db/db mice. In vivo limiting dilution analysis of residual tumors from ob/ob mice indicated reduced tumor initiating activity suggesting fewer cancer stem cells (CSCs). The tumor cell populations reduced by leptin deficiency were identified by fluorescence-activated cell sorting and found to express LepRb. Finally, LepRb expressing tumor cells exhibit stem cell characteristics based on the ability to form tumorspheres in vitro and leptin promotes their survival. These studies provide critical new insight on the role of leptin in tumor growth and implicate LepRb as a CSC target.
Qiao Zheng, Sarah M Dunlap, Jinling Zhu, Erinn Downs-Kelly, Jeremy Rich, Stephen D Hursting, Nathan A Berger and Ofer Reizes
Praveena S Thiagarajan, Qiao Zheng, Manvir Bhagrath, Erin E Mulkearns-Hubert, Martin G Myers, Justin D Lathia and Ofer Reizes
Leptin (LEP) binds to the long form of the leptin receptor (LEPRb), leading to the activation of multiple signaling pathways that are potential targets for disrupting the obesity–breast cancer link. In triple-negative breast cancer (TNBC), LEP is hypothesized to predominantly mediate its tumorigenic effects via a subpopulation of LEPRb-positive tumor cells termed cancer stem cells (CSCs) that can initiate tumors and induce tumor progression. Previously, we showed that LEP promotes CSC survival in vivo. Moreover, silencing LEPRb in TNBC cells compromised the CSC state. The mechanisms by which LEPRb regulates TNBC CSC intracellular signaling are not clear. We hypothesized that activation of LEPRb signaling is sufficient to drive CSC maintenance in TNBC. Here, we show that activation of LEPRb in non-CSCs isolated using our CSC reporter system resulted in a transition to the stem cell state. In CSCs, LEP induced STAT3 phosphorylation, whereas LEP did not induce STAT3 phosphorylation in non-CSCs. Introduction of constitutively active STAT3 into LEPRb-transfected non-CSCs significantly induced NANOG, SOX2 and OCT4 expression compared with control non-CSCs. To determine the intracellular phospho-tyrosine residue of LEPRb that is necessary for the induction of the stem cell state in non-CSCs, we transfected the tyrosine residue point mutants L985, F1077 and S1138 into non-CSCs. Non-CSCs transfected with the L985 mutant exhibited increased STAT3 phosphorylation, increased SOCS3 expression and an induction of GFP expression compared with non-CSCs expressing the F1077 and S1138 mutants. Our data demonstrate that LEPRb-induced STAT3 activation is essential for the induction and maintenance of TNBC CSCs.
Emily L Esakov, James Hale, Elliott G Richards, Luke Torre-Healy, Keerthi Gullapalli, Div Trivedi, Anastasia Chumakova, Oliver Wessely, Jan Jensen, Justin Lathia and Ofer Reizes
Breast cancer is the most prevalent malignancy and second leading cause of death in women worldwide, with hormone receptor-positive luminal breast cancers being the most widespread subtype. While these tumors are generally amenable to endocrine therapy, cellular heterogeneity and acquired ability of tumor cells to undergo cell state switching makes these populations difficult to be fully targeted and eradicated through conventional methods. We have leveraged a quality-by-design (QbD) approach that integrates biological responses with predictive mathematical modeling to identify key combinations of commercially available drugs to induce estrogen receptor expression for therapeutic targeting. This technology utilizes a high level of automation through a custom-built platform to reduce bias as well as design-of-experiments methodology to minimize the experimental iterations required. Utilizing this approach, we identified a combination of clinical compounds, each at concentrations well below their efficacious dose, able to induce the expression of estrogen receptor alpha (ESR1) in hormone-positive breast cancer cells. Induction of ESR1 in luminal cells leads to chemosensitization. These findings provide proof of concept for the utility of the QbD strategy and identify a unique drug cocktail able to sensitize breast cancer cells to tamoxifen.
Qiao Zheng, Lauren Banaszak, Sarah Fracci, Diana Basali, Sarah M Dunlap, Stephen D Hursting, Jeremy N Rich, Anita B Hjlemeland, Amit Vasanji, Nathan A Berger, Justin D Lathia and Ofer Reizes
Despite new therapies, breast cancer continues to be the second leading cause of cancer mortality in women, a consequence of recurrence and metastasis. In recent years, a population of cancer cells has been identified, called cancer stem cells (CSCs) with self-renewal capacity, proposed to underlie tumor recurrence and metastasis. We previously showed that the adipose tissue cytokine LEPTIN, increased in obesity, promotes the survival of CSCs in vivo. Here, we tested the hypothesis that the leptin receptor (LEPR), expressed in mammary cancer cells, is necessary for maintaining CSC-like and metastatic properties. We silenced LEPR via shRNA lentivirus transduction and determined that the expression of stem cell self-renewal transcription factors NANOG, SOX2, and OCT4 (POU5F1) is inhibited. LEPR-NANOG signaling pathway is conserved between species because we can rescue NANOG expression in human LEPR-silenced cells with the mouse LepR. Using a NANOG promoter GFP reporter, we showed that LEPR is enriched in NANOG promoter active (GFP+) cells. In lineage tracing studies, we showed that the GFP+ cells divide in a symmetric and asymmetric manner. LEPR-silenced MDA-MB-231 cells exhibit a mesenchymal to epithelial transition morphologically, increased E-CADHERIN and decreased VIMENTIN expression compared with control cells. Finally, LEPR-silenced cells exhibit reduced cell proliferation, self-renewal in tumor sphere assays, and tumor outgrowth in xenotransplant studies. Given the emergence of NANOG as a pro-carcinogenic protein in multiple cancers, these studies suggest that inhibition of LEPR may be a promising therapeutic approach to inhibit NANOG and thereby neutralize CSC functions.