LKB1 loss promotes endometrial cancer progression via CCL2-dependent macrophage recruitment
Christopher G. Peña, … , Rolf A. Brekken, Diego H. Castrillon
Christopher G. Peña, … , Rolf A. Brekken, Diego H. Castrillon
Published September 28, 2015
Citation Information: J Clin Invest. 2015;125(11):4063-4076. https://doi.org/10.1172/JCI82152.
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Research Article Oncology

LKB1 loss promotes endometrial cancer progression via CCL2-dependent macrophage recruitment

Abstract

Endometrial cancer is the most common gynecologic malignancy and the fourth most common malignancy in women. For most patients in whom the disease is confined to the uterus, treatment results in successful remission; however, there are no curative treatments for tumors that have progressed beyond the uterus. The serine/threonine kinase LKB1 has been identified as a potent suppressor of uterine cancer, but the biological modes of action of LKB1 in this context remain incompletely understood. Here, we have shown that LKB1 suppresses tumor progression by altering gene expression in the tumor microenvironment. We determined that LKB1 inactivation results in abnormal, cell-autonomous production of the inflammatory cytokine chemokine (C-C motif) ligand 2 (CCL2) within tumors, which leads to increased recruitment of macrophages with prominent tumor-promoting activities. Inactivation of Ccl2 in an Lkb1-driven mouse model of endometrial cancer slowed tumor progression and increased survival. In human primary endometrial cancers, loss of LKB1 protein was strongly associated with increased CCL2 expression by tumor cells as well as increased macrophage density in the tumor microenvironment. These data demonstrate that CCL2 is a potent effector of LKB1 loss in endometrial cancer, creating potential avenues for therapeutic opportunities.

Authors

Christopher G. Peña, Yuji Nakada, Hatice D. Saatcioglu, Gina M. Aloisio, Ileana Cuevas, Song Zhang, David S. Miller, Jayanthi S. Lea, Kwok-Kin Wong, Ralph J. DeBerardinis, Antonio L. Amelio, Rolf A. Brekken, Diego H. Castrillon

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Figure 1

Discovery and validation of transcripts regulated by LKB1 in endometrial epithelium by gene-expression profiling.

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Discovery and validation of transcripts regulated by LKB1 in endometrial...
(A) Western blot of immortalized, nontransformed EM cells stably transduced with lentivirus encoding either nontarget shRNA or 1 of 2 different LKB1 shRNAs (shRNA1, shRNA2) that resulted in efficient LKB1 knockdown. LKB1 knockdown led to lower pAMPK levels, as expected, and modest effects on the levels of the phosphorylated forms of downstream mTOR-signaling components pS6 or p4EBP1. (B) Venn diagram of stably transduced cell lines showing the number of genes differentially expressed following LKB1 knockdown with the 2 shRNAs at a threshold of 3× or greater. P < 0.05 (Illumina Microarray Human HT-12 v4 BeadChip, n = 3 biological replicates per shRNA). There was significant overlap (n = 35; P < 0.0001 per hypergeometric test) among differentially expressed genes following shRNA1 and shRNA2 knockdown (n = 53 and 121, respectively, among n = 18,281 genes represented in microarray), demonstrating that our experimental strategy was capable of identifying bona fide LKB1 targets. (C) Validation of gene-expression alterations by qRT-PCR, ΔΔCt method, depicting the mean fold change of shRNA1 and shRNA2 per gene analyzed (n = 3 independent samples distinct from those used for microarray expression profiling). Note that all gene-expression changes were consistent with the microarray data and also that LKB1, which is downregulated as expected, serves as an internal control. CCL2 showed the greatest alteration in expression levels per both microarray and RT-PCR among the subset of genes selected for validation. Error bars represent SEM.