Stat3 is required to maintain the full differentiation potential of mammary stem cells and the proliferative potential of mammary luminal progenitors.

Stat3 has a defined role in mammary gland where it is a critical mediator of cell death during post-lactational regression. On the other hand, Stat3 is required for the self-renewal of embryonic stem cells and is sufficient for the induction of a naïve pluripotent state in epiblast stem cells. Mammary stem cells (MaSCs) have a high capacity for self-renewal and can grow robustly in transplantation experiments in vivo. However, a role for Stat3 in MaSCs has not been investigated. Here we show that depletion of Stat3 from basal cells results in reduced primary transplantation efficiency and diminishes the potential to generate ductal, but not alveolar, outgrowths. In addition, Stat3 is required for maximal proliferation of luminal progenitors

Combined Inhibition of mTOR and CDK4/6 Is Required for Optimal Blockade of E2F Function and Long-term Growth Inhibition in Estrogen Receptor-positive Breast Cancer.

The cyclin dependent kinase (CDK)-retinoblastoma (RB)-E2F pathway plays a critical role in the control of cell cycle in estrogen receptor-positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor-resistant cell lines reactivate the CDK-RB-E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908-20. ©2018 AACR.CRUK Cambridge Institute, Cambridge, UK (Jason Carroll, Igor Chernukhin, Rasmus Siersbæk3) Novo Nordisk Fonden (NNF 14136) (Rasmus Siersbæk

Stat3 Controls Lysosomal-Mediated Cell Death In Vivo

It is well established that lysosomes play an active role during the execution of cell death. A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme. However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions. Here we show that the physiological process of post-lactational regression of the mammary gland is accomplished through a non-classical, lysosomal-mediated pathway of cell death. We found that, during involution, lysosomes in the mammary epithelium undergo widespread LMP. Furthermore, although cell death through LMP is independent of executioner caspases 3, 6 and 7, it requires Stat3, which upregulates the expression of lysosomal proteases cathepsin B and L, while downregulating their endogenous inhibitor Spi2A (ref.8). Our findings report a previously unknown, Stat3-regulated lysosomal-mediated pathway of cell death under physiological circumstances. We anticipate that these findings will be of major importance in the design of treatments for cancers such as breast, colon and liver, where cathepsins and Stat3 are commonly overexpressed and/or hyperactivated respectively. © 2011 Macmillan Publishers Limited. All rights reserved

BLG-Cre mediated deletion of Stat3 affects repopulating frequency of stem cells and outgrowth phenotype.

<p>(A) Whole mount staining of mammary outgrowths originating from CD24⁺ CD49f^hi basal cells sorted from mammary glands of <i>Stat3^fl/fl,BLG-Cre⁻</i> and <i>Stat3^fl/fl;BLG-Cre⁺</i> females four weeks after natural weaning. (B) Limiting dilution analysis of the repopulating frequency of the mammary stem cell-enriched population sorted from mammary glands of <i>Stat3</i>^fl/fl,BLG-Cre<i>⁻</i> and <i>Stat3</i>^fl/fl;BLG-Cre⁺ females four weeks after natural weaning. Number of outgrowths is shown per number of transplanted fat pads. CI: confidence interval.</p

<i>Stat3^fl/fl;BLG-Cre⁺</i> glands show incomplete involution and luminal progenitors have reduced proliferative capacity.

<p>(A) RT-PCR analysis of Stat3 expression in FACS sorted populations of mammary epithelial cells. MRU: mammary repopulating units. (B, C) H&E staining of sections of <i>Stat3^fl/fl;BLG-Cre⁻</i> and <i>Stat3^fl/fl;BLG-Cre⁺</i> mammary glands collected at day 5 of the second gestation (B) or four weeks after natural weaning (C). (D) Western blot analysis of four <i>Stat3^fl/fl;BLG-Cre⁻</i> and five <i>Stat3^fl/fl;BLG-Cre⁺</i> mammary glands four weeks after natural weaning for the expression or activation of Stat5, Erk, Akt, β-casein and WAP. β-actin was used as a loading control. (E) Immunohistochemistry staining for pStat5 (red) and E-cadherin (green) in mammary gland sections from <i>Stat3^fl/fl;BLG-Cre⁻</i> and <i>Stat3^fl/fl;BLG-Cre⁺</i> mice collected four weeks after natural weaning. Nuclei were stained with Hoechst 33342 (blue). (F) Flow cytometry analysis of luminal progenitors isolated from mammary glands of <i>Stat3^fl/fl;BLG-Cre⁻</i> and <i>Stat3^fl/fl;BLG-Cre⁺</i> females four weeks after natural weaning. (G) <i>In vitro</i> colony forming analysis performed on CD24⁺ CD49f^hi CD61⁺ luminal progenitor cells sorted from <i>Stat3^fl/fl;BLG-Cre⁻</i> and <i>Stat3^fl/fl;BLG-Cre⁺</i> mammary glands. Points represent the value for each mouse and lines depict mean values for each group. p value was determined using Student’s t test, * p<0.05.</p

Stat3 is required to maintain the multipotency of mammary stem cells and their proliferative potential.

<p>(A) Whole mount staining of mammary outgrowths originating from CD24⁺ CD49f^hi basal cells sorted from mammary glands of 5-week-old <i>Stat3^fl/fl,K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females. (B) Limiting dilution analysis to assess the repopulating frequency of the mammary stem cell-enriched population sorted from mammary glands of 5-week-old <i>Stat3^fl/fl,K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females. Number of outgrowths per number of transplanted fat pads and percentage of fat pad filled ± standard error of the mean are shown. CI: confidence interval. (C) H&E staining of mammary outgrowths originating from CD24⁺ CD49f^hi basal cells sorted from mammary glands of 5-week-old <i>Stat3^fl/fl;K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females. (D, E) Immunohistochemistry staining for pStat5 (red, D), Ki67 (red, E) and E-cadherin (green) in mammary outgrowths originating from CD24⁺ CD49f^hi cells from mammary glands of 5-week-old <i>Stat3^fl/fl,K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females. Nuclei were stained with Hoechst 33342 (blue).</p

Normal mammary gland development in Stat3 depleted glands.

<p>(A) Whole mount staining of mammary glands of 5-week-old <i>Stat3^fl/fl;K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females. (B) Flow cytometry analysis of luminal and basal cells isolated from mammary glands of <i>Stat3^fl/fl;K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females four weeks after natural weaning. p value was determined using Student’s t test. ns: not significant.</p

Stat3 controls lysosomal-mediated cell death in vivo

It is well established that lysosomes play an active role during the execution of cell death1. A range of stimuli can lead to lysosomal membrane permeabilization (LMP), thus inducing programmed cell death without involvement of the classical apoptotic programme2, 3. However, these lysosomal pathways of cell death have mostly been described in vitro or under pathological conditions4, 5, 6, 7. Here we show that the physiological process of post-lactational regression of the mammary gland is accomplished through a non-classical, lysosomal-mediated pathway of cell death. We found that, during involution, lysosomes in the mammary epithelium undergo widespread LMP. Furthermore, although cell death through LMP is independent of executioner caspases 3, 6 and 7, it requires Stat3, which upregulates the expression of lysosomal proteases cathepsin B and L, while downregulating their endogenous inhibitor Spi2A (ref. 8). Our findings report a previously unknown, Stat3-regulated lysosomal-mediated pathway of cell death under physiological circumstances. We anticipate that these findings will be of major importance in the design of treatments for cancers such as breast, colon and liver, where cathepsins and Stat3 are commonly overexpressed and/or hyperactivated respectively1, 9, 10

CD70-Driven Chronic Immune Activation Is Protective against Atherosclerosis

Chronic infection and inflammation are strongly associated with the development of atherosclerosis. To investigate whether chronic inflammation in the absence of an infectious cause also predisposes to the development of atherosclerosis, we used a mouse model in which sterile inflammation is driven by enhanced costimulation. Constitutive triggering of CD27 on T cells through overexpression of CD70 on B cells increases the numbers of IFN gamma-producing effector T cells, which reduces the numbers of B cells. However, despite these pro-atherogenic features, we found that CD70-transgenic (CD70TG) mice on an ApoE*3-Leiden background were strongly protected against the induction of atherosclerotic lesions, with a normal increase in serum cholesterol level and the absence of atheroprotective antibodies. We found that circulating monocytes in CD70TG mice were activated and increased in numbers, in particular the pool of inflammatory (Ly6C(+)) monocytes. Importantly, monocytes from CD70TG mice had no defects in phagocytosis nor in TNF alpha production, but they were more prone to apoptosis, which was IFN gamma-dependent. These data indicate that sterile pro-inflammatory conditions can be protective against atherosclerosis development, possibly due to a reduced viability of circulating monocytes. This unexpected outcome provides a new insight into the consequences of costimulatory signals and their impact on innate immunity. Copyright (C) 2010 S. Karger AG, Base