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 AMPK alpha were quantified in a cohort of primary human bladder cancers and adjacent nontumor tissues. The levels of p-AMPK alpha, AMPK alpha 1, AMPK alpha 2, and total AMPK alpha were significantly suppressed in both lowand high-grade disease when compared with nontumor tissue. To elucidate the AMPK alpha 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 AMPK alpha protein. Additionally, macrophage CM treatment of HTB2 and HT1376 bladder cells for various times also reduced AMPK alpha protein but not mRNA levels. Direct TNF alpha treatment also suppressed AMPKa 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 AMPK alpha levels. In summary, these data demonstrate the potential role for inflammation and inflammatory cytokines in regulating the levels of AMPK alpha and promoting mTORC1 activation in bladder cancer.