Stavros Kopsiaftis

AMP-Activated Protein Kinase α 2 Isoform Suppression in Primary Breast Cancer Alters AMPK Growth Control and Apoptotic Signaling

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. AMPK-dependent 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 G1-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.

Increased expression of L-selectin (CD62L) in high-grade urothelial carcinoma: A potential marker for metastatic disease.

INTRODUCTION L-Selectin (CD62L) is a vascular adhesion molecule constitutively expressed on leukocytes with a primary function of directing leukocyte migration and homing of lymphocytes to lymph nodes. In a gene expression microarray study comparing laser-captured microdissected 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 University of Connecticut Health Center tumor bank. Tumor cells were isolated from frozen tumor tissue sections by laser-captured microdissected followed by RNA isolation. Quantitative polymerase chain reaction was used to validate the level of CD62L transcripts. Immunohistochemistry and enzyme-linked immunosorbent assay 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 lymph node specimens from patients with high-grade MIBC and known nodal involvement. Up-regulated 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 nonmetastatic 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.

AMPKalpha2 Regulates Bladder Cancer Growth Through SKP2-mediated Degradation of p27

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, 5-aminoimidazole-4-carboxamide ribonucleotide (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 an 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. Mol Cancer Res; 14(12); 1182–94. ©2016 AACR.

AMPKα Is Suppressed in Bladder Cancer through Macrophage-Mediated Mechanisms

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.

Abstract P4-08-01: AMPK facilitates breast cancer cell survival by modulating microenvironmental stress

Recurrent and metastatic breast cancers are responsible for the majority of breast-cancer related deaths. These cancer cells are able to adapt to stressors within the tumor microenvironment including hypoxia, low nutrient levels, and chemotherapy-induced toxicities. Breast cancer cells can respond to these microenvironmental stressors through a variety of mechanisms, including cell cycle inhibition and metabolic alteration. Tumor cell survival is dependent on the ability to alter these mechanisms in response to stress. AMPK (AMP-activated protein kinase) is the main metabolic sensor of the cell, and both its expression and activity have been reported to be altered in breast cancer. Moreover, there are two isoforms of the catalytic subunit (α1 and α2), and differential functionality of these isoforms has been suggested. Using estrogen receptor-positive human breast cancer cell lines, we investigated the effect of differential AMPKα isoform expression on breast cancer cell survival. We found that over-expression of AMPKα2 in MCF-7 cells resulted in decreased ATP production in response to low glucose levels, while the knockdown of AMPKα2 in HCC1500 cells ablated this response to low glucose conditions. A similar difference in response was also seen when the cells were treated with a combination of nutrient stress and the estrogen receptor alpha (ERα) inhibitor, ICI182780. In response to this finding, we compared the glycolytic and oxygen consumption rates of our MCF-7 GFP and MCF-7 AMPKα2 cells. We found that in response to low glucose stress, AMPKα2 expressing MCF-7 cells maintained both a higher glycolytic rate and a higher oxygen consumption rate as compared to GFP cells. Furthermore, these cells seem to alter their cellular signaling in response to metabolic stress faster than GFP cells. To evaluate this differential response to microenvironmental stress in vivo, MCF-7 cells expressing either GFP or AMPKα2 were injected into athymic nude mice previously implanted with slow-release estradiol pellets. After one week, the estradiol pellets were removed to induce cellular dormancy for thirty days. Analysis of tumors at this time indicated that more of the AMPKα2 expressing cells survived estradiol deprivation than did the control cells. Analysis of proliferation by Ki67 staining indicated that the GFP cells maintained proliferation during deprivation, while AMPKα2 cells were largely negative for proliferation. ApoTag staining revealed a similar trend for apoptotic cells. This suggests that an inability to control cell cycle resulted in a decreased survival of the GFP cells under estradiol deprivation. Following the deprivation period, estradiol pellets were re-implanted and residual dormant tumors resumed growth. AMPKα2 tumors grew to roughly double the size of GFP tumors. Interestingly, AMPKα2 tumors had a higher number of mitotic events than did GFP tumors as visualized by Ki67 staining. This could be due to more viable cells being present following estradiol deprivation. We conclude that the expression of AMPKα2 promotes long-term breast cancer survival in estrogen-sensitive cells, due to their increased ability to sense and respond to changes in their microenvironment, which therefore increases their chances for survival. Citation Format: Sullivan KL, Kopsiaftis S, Phoenix KN, Fox MM, Tsurutani N, Vella AT, Claffey KP. AMPK facilitates breast cancer cell survival by modulating microenvironmental stress. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-01.

Abstract 1138: AMPK promotes survival of breast cancer cells by modulating metabolic stress

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Breast cancer cells can respond to microenvironmental stressors by becoming dormant, that is inhibiting cell proliferation until the environment becomes growth-permissive. One of the survival pathways implicated in dormant cancer cells is the p38MAPK pathway. Modulation of signaling to combat metabolic stressors could provide survival benefits to dormant breast cancer cells. AMPK (AMP-activated protein kinase) is the central metabolic regulator of the cell, and its expression is altered in breast cancer. Moreover, there are two isoforms of the catalytic subunit (α1 and α2), and differential functionality of these isoforms has been reported. Using estrogen-receptor positive human breast cancer cell lines, we investigated the effect of differential AMPKα isoform expression on breast cancer survival. We found that over-expression of AMPKα2 in MCF-7 cells resulted in stronger p38MAPK activation in response to chemical AMPK activation or metabolic stress. Moreover, the same signaling was observed in HCC1500 cells, which endogenously express AMPKα2. Additionally, we cultured our cell lines as spheroids in order to mimic a tumor microenvironment. MCF-7 AMPKα2 cells formed larger, more viable spheres than control cells. In addition, the expression of AMPKα2 facilitated spheroid survival under hypoxic conditions. Finally, activation of p38MAPK was seen most abundantly in the MCF-7 AMPKα2 spheres. Our in vitro studies indicate an AMPKα2-dependent regulation of p38MAPK in response to metabolic stress in order to promote cancer cell survival. To evaluate cancer dormancy in vivo, MCF-7 cells expressing either GFP or AMPKα2 were injected into athymic nude mice previously implanted with slow-release estradiol pellets. After one week, the estradiol pellets were removed to induce cellular dormancy for thirty days. Analysis of tumors at this time indicated that more of the AMPKα2 expressing cells survived estradiol deprivation than did the control cells. Analysis of proliferation by Ki67 staining indicated that the GFP cells maintained proliferation during deprivation, while AMPKα2 cells were largely negative for proliferation. ApoTag staining revealed a similar trend for apoptotic cells. This suggests an inability to control cell cycle resulted in a decreased survival of the GFP cells under estradiol deprivation. Consistent with our observed in vitro cell signaling, AMPKα2 expressing tumors expressed higher levels of phospho-p38MAPK than GFP expressing tumors. Following the deprivation period, estradiol pellets were re-implanted and residual dormant tumors resumed growth. AMPKα2 tumors grew to roughly double the size of GFP tumors. Interestingly, AMPKα2 tumors had a higher number of mitotic events than did GFP tumors as visualized by Ki67 staining. This could be due to more viable cells being present following estradiol deprivation. We conclude that the expression of AMPKα2 promotes long-term breast cancer survival in estrogen-sensitive cells. Citation Format: Katie L. Sullivan, Stavros Kopsiaftis, Kathryn N. Phoenix, Melissa M. Fox, Kevin P. Claffey. AMPK promotes survival of breast cancer cells by modulating metabolic stress. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1138. doi:10.1158/1538-7445.AM2015-1138

Abstract 4209: AMPK suppression in bladder tumorigenesis

Bladder cancer is the fifth most diagnosed cancer in the US. In 2013 there were approximately 72,570 newly diagnosed cases and 15,210 deaths attributing to this disease where the average age of onset for this disease is 65 and remains one of the most expensive cancers to treat due to lifelong surveillance and invasive procedures. Bladder cancer is defined by two distinct pathways which result in either low grade non-invasive or high grade invasive cancer. Although these are marked by distinct mutations, it has been observed that both display mTORC1 activation. This suggests that activated mTORC1 may be an early contributing factor in bladder tumorigenesis. Additionally, rapamycin, an inhibitor of mTORC1, inhibited tumorigenesis in a mouse model of bladder cancer further implicating mTORC1 as an essential pathway in tumorigenesis. To elucidate the mechanism of mTORC1 activation in bladder cancer, AMP-activated protein kinase (AMPK), a negative regulator of mTORC1, was investigated. AMPK is a critical metabolic regulator that suppresses cellular growth in response to metabolic stress. Additionally it has been observed that AMPK activation is suppressed in breast and hepatocellular carcinoma and the AMPKα2 isoform is suppressed in breast and gastric cancer. To investigate whether levels or activation of AMPK are altered in bladder cancer a pilot cohort of adjacent non-tumor and bladder tumor human samples was obtained as was a human bladder cancer tissue array. In both the pilot cohort and the array, the levels of AMPKα1 and AMPKα2 were suppressed at statistically significant levels in low and high grade bladder cancer when compared to adjacent non-tumor tissue. Messenger RNA expression for both isoforms in bladder tumors revealed that AMPKα2 may be selectively suppressed at the mRNA level. However AMPKα1 suppression appeared to be due to translational or post translational regulatory mechanisms. It was also observed that AMPKα1 was located predominately in the cytoplasm while AMPKα2 was located in both the nuclear and cytoplasmic compartments. This suggests that AMPKα isoforms may have distinct roles in the bladder and may be suppressed by different mechanisms. To test the relevance of AMPK in bladder tumorigenesis the mouse BBN bladder carcinogenesis model was employed. BBN was supplemented in the drinking water of wild-type and AMPKα2-/- mice at a concentration of .05% over the course of 20 weeks. AMPKα2-/- mice displayed a significant increase in bladder weight indicating the presence of larger tumors. There was also an increase in KI67 positive cells in the tumors of AMPKα2-/- mice indicating increased proliferation with no appreciable apoptosis observed in any tumors. Ongoing studies in which BBN is supplemented for 16 weeks and then removed for the duration aim to validate the role of AMPKα2 in bladder tumor initiation and/or progression. In summary, loss of AMPK activity, especially AMPKα2, can promote bladder tumorigenesis through increased proliferation via mTORC1 activity. Citation Format: Stavros Kopsiaftis, Kathryn N. Phoenix, Katie L. Sullivan, John A. Taylor, Kevin P. Claffey. AMPK suppression in bladder tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4209. doi:10.1158/1538-7445.AM2014-4209

Abstract P5-04-15: Investigating the role of the AMPK signaling cascade in breast cancer tumor dormancy

Breast cancer can recur in patients long after treatment for initial disease. The mechanisms underlying recurrence as well as the characteristics of these dormant tumor cells remain poorly understood. AMPK (AMP-activated protein kinase) is the central metabolic regulator of the cell, and has two catalytic isoforms, a1 and α2. The loss of AMPK, more specifically AMPKα2, is observed in primary breast cancer and its expression could function to limit tumor expansion in vivo. We hypothesize that the expression of AMPKα2 will confer a survival advantage in dormant breast cancer cells by upregulating the p38MAPK signaling axis. In order to investigate breast cancer dormancy mechanisms in vitro, we generated mammospheres using the estrogen-dependent MCF-7 human breast cancer cell line, which has low expression of AMPKα2. Control MCF-7 cells expressing GFP and MCF-7 cells overexpressing the AMPKα1 or AMPKα2 isoforms were cultured as mammospheres and exposed to various metabolic stressors including estrogen, glucose, and serum deprivation. Spheroids were evaluated for size, viability, gene expression at the RNA level by qRT-PCR, and intracellular signaling by immunoblot as well as immunohistochemistry. AMPKα2 expressing mammospheres were larger and more viable than either the GFP or AMPKα1 expressing spheres. In addition, these cells were resistant to serum deprivation, and upregulated members of the p38MAPK signaling axis, a key player in tumor dormancy. To evaluate estrogen deprivation-induced dormancy in vivo, MCF-7 cell lines expressing either GFP or AMPKα2 embedded in a matrigel matrix were implanted in athymic nude mice containing supplemental slow-release estradiol pellets. After one week, estradiol pellets were removed to induce a dormant period for roughly thirty days. Evaluation of matrigel plugs at this time indicated that AMPKα2 expressing cells demonstrated significant survival whereas GFP expressing cells were significantly depleted. Estadiol pellets were then re-implanted for five weeks to determine if the AMPKα2 expressing cells still responded to estradiol supplementation.This resulted in the AMPKα2 expressing tumors rapidly expanding to a maximal size as compared to the GFP expressing tumors. Consistent with our findings in vitro using mammosphere cultures, the AMPKα2 expressing cells had higher levels of phospho-p38MAPK than the GFP expressing cells, as seen by immunofluorescence. We conclude that the expression of AMPKα2 promotes both survival and dormancy in estrogen-sensitive breast tumor cells, thus implying a significant role in breast cancer recurrence. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-15.

Abstract 1092: AMPK alpha 2 promotes tumor cell survival in models of breast cancer dormancy.

Breast cancer can recur after standard of care treatment, even after many years. As a result, there is a need to identify new ways to treat metastatic recurrence, and to understand the mechanisms behind long-term cancer cell dormancy. AMP-dependent kinase (AMPK) has been implicated in tumor cell survival pathways and is under investigation as a potential target for breast cancer treatment. A defining characteristic of dormant tumor cells has been shown to be a reduction in mitogenic ERK1/2 signaling with a concomitant activation of stress-induced p38MAPK signaling. This study aimed to investigate the role of AMPKα2 in establishing and/or maintaining a dormant state. Two estrogen-dependent human breast cancer cell lines that express different levels of AMPKα2 were used. MCF-7 cells, with little to no AMPKα2 expression, were transfected to express GFP control or AMPKα2, while ZR-75-1 cells, with high AMPKα2 expression, were transduced with lentiviral shAMPKα2 or shGFP control particles. Stable cell lines were plated into mammospheres and were exposed to stress conditions such as hypoxia, depletion of essential growth factors, or low glucose. These mammospheres were evaluated by direct cell count, size, and immunofluoresence. In addition, single cell suspensions were mixed with matrigel and injected into athymic nude mice containing slow-release estradiol cholesterol pellets. Estradiol pellets were removed one week after implantation, and re-introduced six weeks later. Matrigel plugs and tumors were harvested and analyzed for cell survival by histological analysis, as well as immunofluorescent analysis of the tumor cells for ERK/p38 activation, epithelial cadherin, differentiation proteins, and autophagy markers. In order to investigate the survival of MCF-7 cells with and without AMPKα2, the amount of cells remaining after prolonged estrogen deprivation was evaluated. There were twice as many viable AMPKα2 expressing cells as GFP cells. Re-exposure of these animals to estradiol resulted in a rapid expansion of the remaining GFP and AMPKα2 tumor cells. Notably, AMPKα2 tumors maintained a significant growth advantage and were double the size of the GFP control tumors at harvest. Evaluation of the p38MAPK pathway by immunoblot revealed that those cells expressing increased amounts of AMPKα2 exhibited high levels of phospho-p38MAPK. Furthermore, those cells that expressed AMPKα2 also expressed increased amounts of LC3-II and autophagosome formation, standard autophagy markers. In conclusion, AMPK promotes breast cancer cell survival under stress in models of tumor dormancy potentially through promotion of the p38MAPK pathway with concomitant reduction in MAPK signaling, and/or autophagy pathways. This study suggests that AMPKα2 contributes to breast cancer recurrence by promotion of tumor cell dormancy and may be a novel therapeutic target. Citation Format: Katie L. Sullivan, Kathryn N. Phoenix, Melissa M. Fox, Stavros Kopsiaftis, Kevin P. Claffey. AMPK alpha 2 promotes tumor cell survival in models of breast cancer dormancy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1092. doi:10.1158/1538-7445.AM2013-1092

Abstract 310: Role of AMPKalpha isoforms in bladder tumorigenesis.

Bladder cancer is the fifth most diagnosed cancer and currently is one of the most expensive cancers to treat due to lifelong surveillance. Bladder cancer is marked by two distinct pathways that result in either noninvasive low grade or invasive high grade cancer. Although low and high grade bladder cancers are defined by driver mutations, similarities such as mTORC1 pathway activation exist. Along these lines rapamycin treatment, an mTORC1 inhibitor, has been shown to suppress tumorigenesis in a mouse model of bladder cancer. These data suggest that mTORC1 activation may be an early tumorigenic event in the bladder. In order to elucidate the mechanism by which the mTORC1 signaling is increased, AMPK (AMP-activated protein kinase), a critical negative regulator of the pathway, was further investigated. AMPK governs a cell9s response to metabolic stressors by suppressing anabolic pathways and inhibiting cell growth. Furthermore, it has been observed that AMPK activation is diminished in primary breast cancer. In order to determine whether AMPK activation and/or levels are altered in bladder cancer, a pilot cohort of human samples was obtained and the expression of both AMPKa catalytic isoforms was assessed. Both AMPKa1 and AMPKa2 were significantly suppressed in bladder cancer. AMPKa isoform levels in adjacent non-tumor bladder epithelium and bladder cancer were evaluated in matched patient samples and this revealed cancer-specific AMPKa suppression does indeed exist. Additionally, AMPKa1 protein levels were partially suppressed in low grade cancer and further suppressed in high grade cancer. In contrast, AMPKa2 levels were uniformly suppressed in both high and low grade of cancer. Interestingly, it was observed that AMPKa1 is localized predominantly to the cytoplasm while AMPKa2 was present in cytoplasmic and nuclear compartments. This data suggests that the AMPKa isoforms may have distinct roles in the bladder. To test whether loss of AMPKa2 function promoted bladder tumorigenesis, wild-type and AMPKa2-/- mice received 0.05% BBN [n-butyl-n-(4 hydroxybutyl)] nitrosamine, a rodent bladder carcinogen, in drinking water over the course of 20 weeks. Significant tumors were observed at 20 weeks in both wild-type and AMPKa2-/- mice and bladder weights were significantly higher in AMPKa2-/- mice correlating with larger bladder tumor mass. Additionally, there was a significant increase in the amount of proliferating tumor cells as assessed by Ki67 in the AMPKa2-/- mice. This study suggests that AMPKa isoforms are suppressed in bladder cancer and that AMPKa may play a critical role in bladder cancer progression by loss of expression and tumor suppressive function. Citation Format: Stavros Kopsiaftis, Kathryn N. Phoenix, Katie L. Sullivan, Poornima Hegde, John A. Taylor, Kevin P. Claffey. Role of AMPKalpha isoforms in bladder tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 310. doi:10.1158/1538-7445.AM2013-310