Adenosine monophosphate-activated protein kinase (AMPK) is a metabolic regulator that promotes energy conservation and restoration when cells are exposed to nutrient stress. Given the high metabolic requirement of cancer cells, AMPK activation has been suggested as a potential preventative and therapeutic target. However, previous findings have shown that AMPK activity is diminished in some cancers. Expression of the 2 catalytic isoforms, AMPKα1 and AMPKα2, was evaluated in primary breast cancer and matched nontumor-adjacent tissue samples using immunohistochemistry. AMPKdependent growth signaling events were examined in primary human mammary epithelial cells (HMECs) using RNAi to understand the importance of AMPKα2 in normal growth regulation. To test whether AMPKα2 would reinstate growth control and apoptotic mechanisms in breast cancer cells, metabolic stress assays and tumor xenografts were performed in MCF-7 cells, expressing low levels of AMPKα2, with stable transfection of either green fluorescent protein (GFP) or AMPKα2 expression constructs. AMPKα2 was found to be significantly suppressed in breast cancer tissue samples, whereas AMPKα1 was not. In normal HMECs, low glucose stress resulted in AMPK-driven growth inhibition. Interestingly, this response was ablated when AMPKα2 was silenced. Metabolic stress assays in MCF-7 cells indicated that AMPKα2 expression reduced both mTOR signaling and cyclin D1 expression, contributing to G 1 -phase cell cycle arrest. Cells expressing AMPKα2 underwent apoptosis more readily than GFP control cells. Xenograft studies demonstrated that MCF-7 tumors expressing AMPKα2 display reduced proliferation and increased apoptotic events. Furthermore, AMPKα2 xenografts exhibited diminished cyclin D1 levels along with an increased amount of nuclear p53, thereby implicating the AMPKα2-p53 signaling axis as a mediator of cell apoptosis. Together, these results highlight the significance of reduced AMPK activity contributing to human carcinogenesis and, specifically, the role of AMPKα2 with respect to its control of normal mammary epithelial cell growth and its reduced expression in breast cancer.

Introduction L-selectin (CD62L) is a vascular adhesion molecule constitutively expressed on leucocytes with a primary function of directing leucocyte migration and homing of lymphocytes to lymph nodes (LNs). In a gene expression microarray study comparing laser captured micro-dissected (LCM) high grade muscle invasive bladder cancer (MIBC) without prior treatment and low grade bladder cancer (LGBC) human samples, we found CD62L to be the highest differentially expressed gene. We sought to examine the differential expression of CD62L in MIBCs and its clinical relevance. Methods Unfixed fresh and formalin fixed paraffin-embedded human bladder cancer specimens and serum samples were obtained from the UCHC tumor bank. Tumor cells were isolated from frozen tumor tissue sections by LCM followed by RNA isolation. qPCR was used to validate the level of CD62L transcripts. Immunohistochemistry and ELISA were performed to evaluate the CD62L protein localization and expression level. Flow cytometry was used to identify the relative number of cells expressing CD62L in fresh tumor tissue. In silico studies were performed using the Oncomine Database. Results Immunostaining showed a uniformly higher expression of CD62L in MIBC specimens vs. LGBCs specimens. Further, CD62L localization was seen in foci of metastatic tumor cells in LN specimens from patients with high grade MIBC and known nodal involvement. Upregulated expression of CD62L was also observed by flow cytometric analysis of freshly isolated tumor cells from biopsies of high grade cancers vs. LGBC specimens. Circulating CD62L levels were also found to be higher in serum samples from patients with high grade metastatic vs. high grade non-metastatic MIBC. In addition, in silico analysis of Oncomine Microarray Database showed a significant correlation between CD62L expression and tumor aggressiveness and clinical outcomes. Conclusion These data confirm the expression of CD62L on urothelial carcinoma cells and suggest that CD62L may serve as biomarker to predict the presence of or risk for developing metastatic disease in patients with bladder cancer.

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.

Bladder cancer presents as either low- or high-grade disease, each with distinct mutational profiles; however, both display prominent mTORC1 activation. One major negative regulator of mTORC1 is AMPK, which is a critical metabolic regulator that suppresses cellular growth in response to metabolic stress by negatively regulating mTORC1. Alterations in the activation and protein levels of AMPK have been reported in breast, gastric, and hepatocellular carcinoma. To investigate whether AMPK suppression is responsible for mTOR activation in bladder cancer, the levels of AMPKα were quantified in a cohort of primary human bladder cancers and adjacent nontumor tissues. The levels of p-AMPKα, AMPKα1, AMPKα2, and total AMPKα were significantly suppressed in both low- and high-grade disease when compared with nontumor tissue. To elucidate the AMPKα suppression mechanism, we focused on inflammation, particularly tumor-infiltrating macrophages, due to their reported role in regulating AMPK expression. Treatment of HTB2 cancer cells with varying doses of differentiated U937 macrophage conditioned medium (CM) demonstrated a dose-dependent reduction of AMPKα protein. Additionally, macrophage CM treatment of HTB2 and HT1376 bladder cells for various times also reduced AMPKα protein but not mRNA levels. Direct TNFα treatment also suppressed AMPKα at the protein but not RNA level. Finally, staining of the human cohort for CD68, a macrophage marker, revealed that CD68+ cell counts correlated with reduced AMPKα levels. In summary, these data demonstrate the potential role for inflammation and inflammatory cytokines in regulating the levels of AMPKα and promoting mTORC1 activation in bladder cancer.

LAP, latency-associated peptide of TGFβ1, is a known marker of regulatory T cells in the tumor microenvironment. TGFβ is a major tolerogenic cytokine that is co-opted by tumors to evade the immune system and is implicated in resistance to checkpoint inhibitors. LAP cages TGFβ in an inactive, latent state until it is activated and released by integrins, MMPs or other activators. Anti-LAP antibodies inhibit the release of TGFβ, inhibit tumor growth in mouse models (Gabriely et al., 2017), and have promise as novel cancer therapeutics. Anti-LAP antibodies are thought to mediate anti-tumor activity both by inhibiting the release of active TGFβ and by reducing the number of LAP+ immunosuppressive cells in the tumor microenvironment (TME). To provide insight into the cellular sources of TGFβ in the TME and further delineate the mechanism of action of anti-LAP antibodies in tumor growth inhibition we determined the expression profile of LAP on the surface of both Treg and other immune cell subsets derived from tumor, spleen, and human PBMCs. LAP expression on the surface of various mouse and human immune cell subsets was determined using flow cytometry. Tumor infiltrating leukocytes were profiled from CT26 and 4T1 syngeneic mouse tumor models. Expression of LAP was detected on a subset of regulatory T cells, M2 macrophages, dendritic cells, G-MDSC and M-MDSC populations in both CT26 and 4T1 tumors suggesting that each of these cell types are a potential source for TGFβ activation in the tumor microenvironment. Striking differences were detected in LAP expression on immune cell subsets in the TME versus spleens of CT26 tumor bearing mice, consistent with TGFβ expression being increased upon immune cell activation. Additionally, CD4+ cells were isolated from human PBMCs from normal healthy donors and activated using CD3/CD28 +IL2 stimulation. Consistent with previous reports, activated Foxp3+ T cells were positive for cell surface LAP. In addition, LAP expression was also assessed on various human macrophage subsets (M1, M2a, M2b and M2c) that were skewed from CD14+ cells isolated from PBMCs. LAP expression was detected in varying amounts on the surface of the different macrophage subsets with M2a macrophages demonstrating the highest expression. Taken together these results demonstrate that LAP is expressed on a variety of immune cells with known immunosuppressive function. The location of the LAP-TGFβ1 complex is of critical biological and clinical importance because, once the mature TGFβ1 cytokine, which has a short half-life in solution, is released, it acts locally, either in an autocrine or near paracrine fashion. These results warrant further research to determine the effectiveness of anti-LAP in inhibiting the release of TGFβ and its effects on immunosuppression in the tumor microenvironment. Citation Format: Stavros Kopsiaftis, Patricia E. Rao, Randall Burton, Jessie M. English, Barbara S. Fox, Kenneth J. Simon. Expression of LAP, latency-associated peptide of TGFb, on immune cell subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2794.