Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to “labeling” by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in physiological and pathophysiological processes.

Peroxiredoxins are thiol-specific antioxidant proteins whose expression is elevated in several cancers. We compared the expression and function of Prdx1 and Prdx6 between the MCF-7 mammary adenocarcinoma cell line and the noncancerous MCF-10A cell line. We found elevated Prdx1 expression in MCF-7 cells and comparable expression of Prdx6. Suppression of Prdx1 and/or Prdx6 resulted in a modest increase in peroxide-induced cytotoxicity of MCF-7 cells, and a dramatic increase in MCF-10A cytotoxicity with and without hydrogen peroxide treatment. Our data confirm a cytoprotective role for peroxiredoxins and suggest a synergistic role for Prdx1 and Prdx6 in MCF-10A cells.

AMP-activated protein kinase (AMPK) is the central metabolic regulator of the cell and controls energy consumption based upon nutrient availability. Due to its role in energy regulation, AMPK has been implicated as a barrier for cancer progression and is suppressed in multiple cancers. To examine whether AMPK regulates bladder cancer cell growth, HTB2 and HT1376 bladder cells were treated with an AMPK activator, AICAR. AICAR treatment reduced proliferation and induced the expression of p27Kip1 (CDKN1B), which was mediated through an mTOR-dependent mechanism. Interestingly, AMPKα2 knockdown resulted in reduced p27 levels, whereas AMPKα1 suppression did not. To further determine the exact mechanism by which AMPKa2 regulates p27, HTB2 and HT1376 cells were transduced with a shRNA targeting AMPKα2. Stable knockdown of AMPKα2 resulted in increased proliferation and decreased p27 protein. The reduced p27 protein was determined to be dependent upon SKP2. Additionally, loss of AMPKα2 in a xenograft and a chemical carcinogen model of bladder cancer resulted in larger tumors with less p27 protein and high SKP2 levels. Consistent with the regulation observed in the bladder cancer model systems, a comprehensive survey of human primary bladder cancer clinical specimens revealed low levels of AMPKα2 and p27, and high levels of SKP2. Implications These results highlight the contribution of AMPKα2 as a mechanism for controlling bladder cancer growth by regulating proliferation through mTOR suppression and induction of p27 protein levels, thus indicating how AMPKα2 loss may contribute to tumorigenesis.

<p>Knockdown of SKP2 negates the effects of AMPK�2 mediated control of p27 and cell proliferation.</p>

<p>AMPK activation reduces p27 through mTOR dependent mechanism.</p>