Epithelial IL-2 is critical for NK cell-mediated cancer immunosurveillance in mammary glands.

Interleukin 2 (IL-2) is the first identified cytokine and its interaction with receptors has been known to shape the immune responses in many lymphoid or non-lymphoid tissues for more than four decades. Active T cells are the primary cellular source for IL-2 production and epithelial cells have never been considered the major cellular source of IL-2 under physiological conditions. It is, however, tempting to speculate that epithelial cells could potentially express IL-2 that regulates the intricate interactions between epithelial cells and lymphocytes. Datamining our recently published single-cell RNAseq in the mouse mammary gland identified IL-2 expression in mammary epithelial cells, which is induced by prolactin via the STAT5 signaling pathway. Furthermore, epithelial IL-2 plays a crucial role in maintaining the physiological functions of natural killer (NK) cells within the mammary glands. IL-2 deletion in the mammary epithelial cells leads to a significant reduction in the number and function of NK cells, which in turn results in defective immunosurveillance, expansion of luminal epithelial cells, and tumor development. Interestingly, T cells in the mammary glands are not changed, indicating the specific regulation of NK cells by epithelial IL-2 production. In agreement, we also found that human epithelial cells express IL-2 and NK cells express the highest level of IL2RB among all the immune cells. Here, we provide the first evidence that epithelial cells produce IL-2, which is critical for maintaining the physiological functions of NK cells in immunosurveillance.

Correction: ERK1/2 Signaling Plays an Important Role in Topoisomerase II Poison-Induced G2/M Checkpoint Activation.

[This corrects the article DOI: 10.1371/journal.pone.0050281.].

ERK1/2 signaling plays an important role in topoisomerase II poison-induced G2/M checkpoint activation.

Topo II poisons, which target topoisomerase II (topo II) to generate enzyme mediated DNA damage, have been commonly used for anti-cancer treatment. While clinical evidence demonstrate a capability of topo II poisons in inducing apoptosis in cancer cells, accumulating evidence also show that topo II poison treatment frequently results in cell cycle arrest in cancer cells, which was associated with subsequent resistance to these treatments. Results in this report indicate that treatment of MCF-7 and T47D breast cancer cells with topo II poisons resulted in an increased phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and an subsequent induction of G2/M cell cycle arrest. Furthermore, inhibition of ERK1/2 activation using specific inhibitors markedly attenuated the topo II poison-induced G2/M arrest and diminished the topo II poison-induced activation of ATR and Chk1 kinases. Moreover, decreased expression of ATR by specific shRNA diminished topo II poison-induced G2/M arrest but had no effect on topo II poison-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling had little, if any, effect on topo II poison-induced ATM activation. In addition, ATM inhibition by either incubation of cells with ATM specific inhibitor or transfection of cells with ATM specific siRNA did not block topo II poison-induced G2/M arrest. Ultimately, inhibition of ERK1/2 signaling greatly enhanced topo II poison-induced apoptosis. These results implicate a critical role for ERK1/2 signaling in the activation of G2/M checkpoint response following topo II poison treatment, which protects cells from topo II poison-induced apoptosis.

RAC1 GTPase plays an important role in γ-irradiation induced G2/M checkpoint activation.

INTRODUCTION: In response to gamma-irradiation (IR)-induced double-strand DNA breaks, cells undergo cell-cycle arrest, allowing time for DNA repair before reentering the cell cycle. G2/M checkpoint activation involves activation of ataxia telangiectasia mutated (ATM)/ATM- and rad3-related (ATR) kinases and inhibition of Cdc25 phosphatases, resulting in inhibition of Cdc2 kinase and subsequent G2/M cell-cycle arrest. Previous studies from our laboratory showed that the G2/M checkpoint activation after IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. In the present studies, we investigated the role of Ras-related C3 botulinum toxin substrate 1 (Rac1) guanosine triphosphatase (GTPase) in IR-induced G2/M checkpoint response and ERK1/2 activation, as well as in cell survival after IR. METHODS: With Rac1-specific inhibitor, dominant negative mutant Rac1 (N17Rac1) and specific small interfering RNA, the effect of Rac1 on IR-induced G2/M checkpoint response and ERK1/2 activation was examined in human breast cancer cells. In addition, the effect of Rac1 on cell survival after irradiation was assessed by using Rac1-specific inhibitor. RESULTS: IR exposure of MCF-7 breast cancer cells was associated with a marked activation of Rac1 GTPase. Furthermore, inhibition of Rac1 by using specific inhibitor, dominant-negative Rac1 mutant, or specific siRNA resulted in attenuation of IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ATM, ATR, Chk1, and Chk2 kinases, as well as phosphorylation of Cdc2-Tyr15. Moreover, Rac1 inhibition or decreased Rac1 expression also abrogated IR-induced phosphorylation of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) and ERK1/2. Ultimately, inhibition of Rac1 markedly increased cellular sensitivity to IR exposure, which involves induction of apoptosis. CONCLUSION: Studies in this report suggest that Rac1 GTPase plays an essential role in the activation of IR-induced ERK1/2 signaling and subsequent G2/M checkpoint response. Furthermore, results also support a role for Rac1 in promoting cell survival after irradiation treatment.

Gamma-irradiation-induced DNA damage checkpoint activation involves feedback regulation between extracellular signal-regulated kinase 1/2 and BRCA1.

Previous studies from our laboratory have shown that the activation of G(2)-M checkpoint after exposure of MCF-7 breast cancer cells to gamma-irradiation (IR) is dependent on the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Studies presented in this report indicate that IR exposure of MCF-7 cells is associated with a marked increase in expression of breast cancer 1 (BRCA1) tumor suppressor, an effect that requires ERK1/2 activation and involves posttranscriptional control mechanisms. Furthermore, reciprocal coimmunoprecipitation, as well as colocalization studies, indicate an interaction between BRCA1 and ERK1/2 in both nonirradiated and irradiated cells. Studies using short hairpin RNA targeting BRCA1 show that BRCA1 expression is necessary for IR-induced G(2)-M cell cycle arrest, as well as ERK1/2 activation in MCF-7 cells. Although BRCA1 expression is not required for IR-induced phosphorylation of ataxia telangiectasia mutated (ATM)-Ser1981, it is required for ATM-mediated downstream signaling events, including IR-induced phosphorylation of Chk2-Thr68 and p53-Ser20. Moreover, BRCA1 expression is also required for IR-induced ATM and rad3 related activation and Chk1 phosphorylation in MCF-7 cells. These results implicate an important interaction between BRCA1 and ERK1/2 in the regulation of cellular response after IR-induced DNA damage in MCF-7 cells.