HIF1 and ID1 Interplay Confers Adaptive Survival to HIF1α-Inhibition.

Hypoxia is a universal pathological feature of solid tumors. Hypoxic tumor cells acquire metastatic and lethal phenotypes primarily through the activities of hypoxia-inducible factor 1 alpha (HIF1α). Therefore, HIF1α is considered as a promising therapeutic target. However, HIF inhibitors have not proven to be effective in clinical testing. The underlying mechanism is unclear. We report that oncogenic protein ID1 is upregulated in hypoxia by HIF1α shRNA or pharmacological inhibitors. In turn, ID1 supports tumor growth in hypoxia in vitro and in xenografts in vivo, conferring adaptive survival response and resistance. Mechanistically, ID1 proteins interfere HIF1-mediated gene transcription activation, thus ID1 protein degradation is accelerated by HIF1α-dependent mechanisms in hypoxia. Inhibitions of HIF1α rescues ID1, which compensates the loss of HIF1α by the upregulation of GLS2 and glutamine metabolism, thereby switching the metabolic dependency of HIF1α -inhibited cells from glucose to glutamine.

A Multifunctional Therapy Approach for Cancer: Targeting Raf1- Mediated Inhibition of Cell Motility, Growth, and Interaction with the Microenvironment.

Prostate cancer cells move from their primary site of origin, interact with a distant microenvironment, grow, and thereby cause death. It had heretofore not been possible to selectively inhibit cancer cell motility. Our group has recently shown that inhibition of intracellular activation of Raf1 with the small-molecule therapeutic KBU2046 permits, for the first time, selective inhibition of cell motility. We hypothesized that simultaneous disruption of multiple distinct functions that drive progression of prostate cancer to induce death would result in advanced disease control. Using a murine orthotopic implantation model of human prostate cancer metastasis, we demonstrate that combined treatment with KBU2046 and docetaxel retains docetaxel's antitumor action, but provides improved inhibition of metastasis, compared with monotherapy. KBU2046 does not interfere with hormone therapy, inclusive of enzalutamide-mediated inhibition of androgen receptor (AR) function and cell growth inhibition, and inclusive of the ability of castration to inhibit LNCaP-AR cell outgrowth in mice. Cell movement is necessary for osteoclast-mediated bone degradation. KBU2046 inhibits Raf1 and its downstream activation of MEK1/2 and ERK1/2 in osteoclasts, inhibiting cytoskeleton rearrangement, resorptive cavity formation, and bone destruction in vitro, with improved effects observed when the bone microenvironment is chemically modified by pretreatment with zoledronic acid. Using a murine cardiac injection model of human prostate cancer bone destruction quantified by CT, KBU2046 plus zoledronic exhibit improved inhibitory efficacy, compared with monotherapy. The combined disruption of pathways that drive cell movement, interaction with bone, and growth constitutes a multifunctional targeting strategy that provides advanced disease control.

Loss of dihydrotestosterone-inactivation activity promotes prostate cancer castration resistance detectable by functional imaging.

Androgens such as testosterone and dihydrotestosterone are a critical driver of prostate cancer progression. Cancer resistance to androgen deprivation therapies ensues when tumors engage metabolic processes that produce sustained androgen levels in the tissue. However, the molecular mechanisms involved in this resistance process are unclear, and functional imaging modalities that predict impending resistance are lacking. Here, using the human LNCaP and C4-2 cell line models of prostate cancer, we show that castration treatment-sensitive prostate cancer cells that normally have an intact glucuronidation pathway that rapidly conjugates and inactivates dihydrotestosterone and thereby limits androgen signaling, become glucuronidation deficient and resistant to androgen deprivation. Mechanistically, using CRISPR/Cas9-mediated gene ablation, we found that loss of UDP glucuronosyltransferase family 2 member B15 (UGT2B15) and UGT2B17 is sufficient to restore free dihydrotestosterone, sustained androgen signaling, and development of castration resistance. Furthermore, loss of glucuronidation enzymatic activity was also detectable with a nonsteroid glucuronidation substrate. Of note, glucuronidation-incompetent cells and the resultant loss of intracellular conjugated dihydrotestosterone were detectable in vivo by 18F-dihydrotestosterone PET. Together, these findings couple a mechanism with a functional imaging modality to identify impending castration resistance in prostate cancers.

Loss of an Androgen-Inactivating and Isoform-Specific HSD17B4 Splice Form Enables Emergence of Castration-Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) requires tumors to engage metabolic mechanisms that allow sustained testosterone and/or dihydrotestosterone to stimulate progression. 17ß-Hydroxysteroid dehydrogenase type 4 (17ßHSD4), encoded by HSD17B4, is thought to inactivate testosterone and dihydrotestosterone by converting them to their respective inert 17-keto steroids. Counterintuitively, HSD17B4 expression increases in CRPC and predicts poor prognosis. Here, we show that, of five alternative splice forms, only isoform 2 encodes an enzyme capable of testosterone and dihydrotestosterone inactivation. In contrast with other transcripts, functional expression of isoform 2 is specifically suppressed in development of CRPC in patients. Genetically silencing isoform 2 shifts the metabolic balance toward 17ß-OH androgens (testosterone and dihydrotestosterone), stimulating androgen receptor (AR) and CRPC development. Our studies specifically implicate HSD17B4 isoform 2 loss in lethal prostate cancer.

Suppression of chemotaxis by SSeCKS via scaffolding of phosphoinositol phosphates and the recruitment of the Cdc42 GEF, Frabin, to the leading edge.

Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS-null MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS-null leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS-null MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS-null MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.

A genome-wide RNAi screen identifies FOXO4 as a metastasis-suppressor through counteracting PI3K/AKT signal pathway in prostate cancer.

Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness.

A transgenic mouse model for early prostate metastasis to lymph nodes.

The emergence of recurrent, metastatic prostate cancer following the failure of androgen-deprivation therapy represents the lethal phenotype of this disease. However, little is known regarding the genes and pathways that regulate this metastatic process, and moreover, it is unclear whether metastasis is an early or late event. The individual genetic loss of the metastasis suppressor, SSeCKS/Gravin/AKAP12 or Rb, genes that are downregulated or deleted in human prostate cancer, results in prostatic hyperplasia. Here, we show that the combined loss of Akap12 and Rb results in prostatic intraepithelial neoplasia (PIN) that fails to progress to malignancy after 18 months. Strikingly, 83% of mice with PIN lesions exhibited metastases to draining lymph nodes, marked by relatively differentiated tumor cells expressing markers of basal (p63, cytokeratin 14) and luminal (cytokeratin 8 and androgen receptor) epithelial cells, although none expressed the basal marker, cytokeratin 5. The finding that PIN lesions contain increased numbers of p63/AR-positive, cytokeratin 5-negative basal cells compared with WT or Akap12-/- prostate lobes suggests that these transitional cells may be the source of the lymph node metastases. Taken together, these data suggest that in the context of Rb loss, Akap12 suppresses the oncogenic proliferation and early metastatic spread of basal-luminal prostate tumor cells.