Abstract B043: Cancer stem cell selection drives adverse response to androgen-deprivation therapy
Authors: Sha Kai , Jiang Lingxiang , Chang Chawnshang , Tang Dean , Nastiuk Kent L. , Krolewski John J. 
Published: February 2019

Abstract
Purpose: The dramatic responses tumors display to targeted therapies are frequently limited by acquired or pre-existing mechanisms of therapy resistance. In prostate cancer (PCa), we found that targeted androgen receptor signaling blockade by the androgen antagonist enzalutamide (Enz) paradoxically enhanced metastasis. In mouse models, these prometastatic effects are mediated by the chemoattractant CCL2. We have previously demonstrated that androgen blockade-dependent induction of CCL2 is regulated by TNF signaling. Thus, we asked whether TNF mediates the prometastatic effects of Enz. Further, since androgen-deprivation therapy (ADT) selectively increases stem/progenitor (S/P) cell populations in PCa, we asked whether the increase in TNF expression following ADT is due to selection for a TNF-expressing prostate cancer S/P cell population.
Methods: PCa and microenvironment-derived cell lines were employed as in vitro model systems. These include the human (LNCaP, C4-2) and murine (P8, CaP8) PCa cell lines and their castration-resistant prostate cancer (CRPC) and immortalized macrophage-like (THP1) and the stromal myofibroblast (WPMY1) cell lines. Following Enz and anti-TNF treatment, cytokine mRNA levels and protein secretion from CRPC and microenvironment cells was measured by Q-RT-PCR and ELISA, respectively. PCa expression of S/P cell markers following Enz treatment was analyzed by immunofluorescence. S/P cells were sorted by fluorescence-activated cell sorting (FACS) and captured. Cytokine secretion from S/P and “bulk” (more differentiated) tumor cell populations were measured by ELISA. S/P cell phenotype was validated by soft agar colony assay. CRPC and macrophage migration/invasion were measured in transwells. Cytokines in PTEN-null tumors at various times post-castration were measured by ELISA. Macrophage infiltration into these tumors was analyzed by immunohistochemistry using an F4/80 antibody.
Results: Androgen withdrawal or Enz induced TNF mRNA and protein secretion in human CRPC (C4-2) cells. Enz induced autocrine CCL2 expression in C4-2 as well as a murine CRPC cell line PTEN-CaP8, while exogenous TNF induced CCL2 in THP1 and WPMY1 cells. TNF was most potent in myofibroblast cultures, suggesting that ADT induces CCL2 via paracrine interactions within the tumor microenvironment. A soluble TNF receptor (etanercept) blocked Enz-induced CCL2 secretion and mRNA expression in CRPC cells, implying dependence on secreted TNF. Etanercept inhibited Enz-induced migration and invasion of C4-2. Oncomine and GEO analysis of gene expression in primary PCa and metastatic CRPC datasets showed coordinate upregulation of both TNF and an S/P cell marker (integrin a6) in CRPC, relative to castration-sensitive primary PCa. Consistent with the Oncomine data, we found Enz increased the fraction of S/P marker (CD166 and integrin a6) expressing cells in C4-2 and LNCaP, with time. In parallel, TNF and CCL2 were also induced in these cultures, suggesting that the increase in TNF expression that occurs following ADT may be due to selection for a TNF-expressing S/P cells. Indeed, TNF and CCL2 secretion from sorted CD166+ C4-2 cells was significantly higher than from CD166- cells. Coculturing CD166+ cells with WPMY1 cells led to higher CCL2 secretion and more C4-2 and THP1 cell migration relative to CD166- cells. Consistent with this in vitro data, TNF and CCL2 expression, along with macrophage infiltration, was significantly elevated in PTEN-null tumors 35-day post-castration.
Conclusions: ADT induces TNF production by selecting for a TNF-expressing S/P cell-like population. TNF signaling then mediates ADT induction of CRPC metastasis via CCL2. Since anti-TNF therapies are safe and FDA approved, combining ADT with anti-TNF therapy might reduce resistance (metastasis) while allowing continued tumor control.
Citation Format: Kai Sha, Lingxiang Jiang, Chawnshang Chang, Dean Tang, Kent L. Nastiuk, John J. Krolewski. Cancer stem cell selection drives adverse response to androgen-deprivation therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B043.