Prostate cancer initiation, progression and advancement are associated with proto-oncogene activation and inhibition of tumor suppressor genes involved in PI3K/AKT, RAS/MAPK and STAT3 signaling. Abnormalities in the prostate epithelium result in pre-neoplastic lesions called prostatic intraepithelial neoplasia (PIN), which feature luminal epithelial hyperplasia and a reduction in the number of basal cells. PIN lesions progress to invasive adenocarcinoma (luminal phenotype) with loss of the basal cell layer and basement membrane resulting in various tumor grades, beginning with indolent to more aggressive forms of PCa, and subsequent development of metastasis and castration resistance. Studies involving murine PCa models provide support for the role of proteins involved in PI3K/AKT, RAS/MAPK and STAT3 signaling at various stages of PCa development. Murine PCa models discussed in the text are illustrated, with specific promoter-driven gene knockout or transgene overexpression indicated in brackets. Figure is adapted from ref. [5]. BM bone marrow; c-Akt constitutive-active Akt; CR castration-resistant; Cre Cre-recombinase; CreER/CreERT2 TAM-inducible Cre; LN lymph node; OT orthotopic; PB probasin promoter; TAM tamoxifen. 

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... to propagate PCSCs derived from human PCa cell lines including suspension or low adherence culture [67, 68], and media cultures containing either fetal bovine serum [60, 61, 66] or serum-free media supplemented with epidermal growth factor [75, 76] and basic fibroblast growth factor [67, 69, 70, 72]. However, the formation of free-floating spheres under serum-free conditions is routinely used as an in vitro approach towards propagating and evaluating the stem-like properties of putative CSCs derived from solid tumors [62], including those derived from PCa cells [69, 70, 72]. Despite its limitations [77], the sphere formation assay allows for the clonality and self-renewal capacity of stem-like cells to be evaluated upon culturing individual cells, reducing the impact of progenitor-like cells which display limited self-renewal potential [78]. The self-renewal capacity of sphere-derived human PCSCs has been evaluated [70, 76], which display enhanced tumor formation in vivo [69, 70, 72, 76]. While purification of human PCa cells based on the expression of surface markers isolates stem-like cells with tumor-initiating ability (Table 1), flow cytometry can also enrich for prospective PCSCs based on their ability to efflux the Hoechst 33342 dye. These cells, also known as side population (SP) cells [79], isolated from androgen-sensitive LAPC9 cells are capable of in vivo tumor formation unlike the non-SP cell population [80]. The SP phenotype is associated with the expression of the ATP-binding cassette membrane transporter family of proteins, particularly ABCG2 [81], which are involved in drug efflux and confer multi-drug resistance [82]. However, ABCG2 + and ABCG2 - PCa cells displayed similar tumorigenic potential in vivo [80], implicating the expression of additional ABC transporter proteins and/or other means of Hoechst 33342 dye efflux by these PCa cells. An alternative strategy towards purifying PCSCs involves measuring their aldehyde dehydrogenase (ALDH) activity. Elevated ALDH activity has been associated with CSCs in various solid tumors [83] and is required to maintain the drug-tolerant cancer cell subpopulation [84]. ALDH1A1, which is expressed by a minor basal cell population in human prostates [85], was observed to be heterogeneously expressed in prostate adenocarcinomas, with elevated ALDH1A1 expression being positively correlated with the severity (Gleason score and pathologic stage) of the disease and inversely correlated with patient survival [85, 86]. Primary and metastatic PCa also express ALDH7A1 protein [87], with knockdown of ALDH7A1 reducing the tumor-propagating and metastatic abilities of androgen-independent PCa cells [88]. Using a functional (AldefluorTM) assay validated to assess ALDH1 isoform activities [89, 90], PCa cells demonstrating high ALDH activity (ALDH hi ) were isolated and displayed increased tumorigenicity in vivo [87]. In addition, as few as 10 1 ALDH hi CD44 + α 2 β 1+ cells isolated from LAPC9 (low PSA- expressing; PSA low ) tumor xenografts generated secondary tumors in castrated mice whereas 10 4 ALDH low CD44 - α 2 β 1- cells were required for tumor formation, indicating that the ALDH hi CD44 + α 2 β 1+ phenotype in a PSA low cell population is enriched for castration-resistant PCSCs [91]. While 19 ALDH isoforms have been identified in humans [83], other ALDH isoforms have yet to be shown to be functionally- active in PCSCs or required for tumor formation in vivo . As no individual method isolates bona-fide human PCSCs exclusively, a combination of methods (sphere culture, and isolation based on cell surface marker expression and/or functional activity) can be used to enrich for and characterize human PCSCs, with subsequent implantation in immunocompromised mice to evaluate their tumor- propagating activity and serial transplantation to assess their long-term self-renewal capability in vivo [62]. A number of cell signaling pathways have been implicated in PCa progression towards an androgen- resistant state including receptor tyrosine kinases, like epidermal growth factor receptor (EGFR) [92-94], and developmental pathways including Wnt, Notch and Hedgehog signaling [95-97]. These developmental pathways have also been implicated in PCSC maintenance [98-100]. Human PSCs and PCSCs display low levels or lack AR expression [45, 64, 66, 72], with evidence to suggest that murine PSCs and PCSCs are capable of surviving in an androgen-independent state [26, 32, 101]. While not to undermine the importance of these developmental pathways and AR signaling in PCa, we will focus on the involvement of intracellular PI3K/AKT, RAS/MAPK and STAT3 pathways in maintaining PCSCs. Given that details of these signaling pathways and their ability to regulate various hallmarks of PCa have been reviewed thoroughly elsewhere [102-104], we highlight key proteins within these pathways that regulate PCSC activity (Figure 2), and the animal models that support their role in PCa development (Figure 3). Dysregulation of PI3K/AKT signaling has been implicated in PCa given the discovery that its negative regulator, PTEN, is mutated and frequently lost in PCa. Loss of PTEN function, as a result of mutation, deletion or reduced expression, occurs at a frequency of ~40% in PCa [105, 106]. Integrative analysis of mRNA expression, copy number and exon sequencing for somatic mutations conducted on a cohort of PCa patients revealed that the PI3K/AKT pathway was altered in 42% of primary cancers and 100% of metastases, while the RAS/MAPK pathway was altered in 43% of primary specimens and 90% of metastases [106]. Both PTEN loss and AKT activation, as measured by Ser 473 phosphorylation, are associated with biochemical relapse following radical prostatectomy [107, 108], while PTEN deletion (along with allelic c-MYC gain) is associated with biochemical relapse following local therapy [59]. Intense staining patterns for activated AKT are correlated with higher Gleason grade PCa [109] and tumor progression [110], while PTEN loss is associated with PCa progression and predicts a shorter time for metastasis-free survival in patients [111]. The role of PI3K/AKT signaling in PCa has also been examined in animal models. Pten +/- mice were first reported to display hyperplastic lesions in the prostate, with benign PIN lesions being observed in some mice at a young age (≤14 weeks) [112, 113]. Pten deletion in prostate basal or luminal cells has also been carried out to promote PI3K/AKT signal activation in order to determine the prostate cell lineage responsible for subsequent tumor initiation [26, 32, 35], as previously discussed. Prior to these studies, probasin ( PB ) promoter-driven Pten deletion was conducted, resulting in PI3K/AKT signal activation specifically in both prostate basal and luminal cells [114]. By 6 weeks of age, these mice formed PIN lesions, developed adenocarcinoma that invades stromal regions (9 weeks) and displays androgen-independent survival following castration, and generated metastases in the lymph nodes and lungs (12 weeks) [114]. Moreover, prostate epithelial cells lacking Pten require mammalian target of rapamycin (mTOR) complex 2 (mTORC2) for neoplastic transformation [115], thereby implicating downstream mTOR signaling in murine PCa development. Expression of a constitutively-active form of Akt1 (c-Akt) resulted in PIN formation when expression is driven by the PB promoter [116], or after reconstituting c-Akt- expressing prostate basal cells with UGSM cells and grafting under the kidney capsule of mice [49]. Therefore, the murine prostate epithelium has been shown to be vulnerable to Pten loss, with the propensity to undergo AKT-driven tumorigenesis. The role of PI3K/AKT signaling in the proliferation and maintenance of PCSCs has been examined in human PCa. PTEN knockdown in DU145 PCa cells resulted in an increased ability to enrich for CD133/AC141 + CD44 + stem-like cells [69]. Treatment with PI3K inhibitor LY294002 reduced sphere formation [69] while the dual PI3K/mTOR inhibitor NVP-BEZ235 reduced the CD133/ AC141 + CD44 + cell population in vivo and subsequently delayed tumor formation [117]. However, DU145 spheres display low levels of AKT activation while PTEN knockdown marginally increased AKT activation and did not affect sphere maintenance, suggesting that PTEN function may not be critical for PCSC self-renewal in vitro [70]. While PTEN may not be required to negatively- regulate PI3K/AKT-dependent PCSC maintenance, whether PTEN loss/inactivity and subsequent PI3K/AKT signal activation promotes PCSC differentiation into non- tumorigenic bulk tumor cells has not been thoroughly evaluated, despite PTEN knockdown increasing tumor formation in vivo [69]. Mitogen-activated protein kinases (MAPKs) are cytoplasmic Ser/threonine kinases that transduce signals by activating extracellular signal-regulated kinases (ERKs) through the RAS-RAF-MEK-ERK (RAS/MAPK) signal transduction cascade [118]. Similar to PI3K/AKT signaling, MAPK signal activation correlates with disease progression, with elevated staining for phosphorylated (active) forms of ERK1 and ERK2 (ERK1/2) being detected with increasing tumor stage of human PCa specimens [119]. Increased ERK1/2 activation in patients receiving neoadjuvant hormone therapy, and in recurrent CRPC patients, has been observed [120]. Mutations of all three RAS isoforms ( KRAS , HRAS and NRAS ) have been detected in human PCa specimens (3 – 30%), particularly among Japanese men [121-124], while the activating mutant B-RAF(V600E) has been observed in ~10% of Korean PCa patients [122]. These correlative studies reinforce that activated RAS/MAPK signaling contributes towards prostate tumorigenesis. Further evidence in support of a relationship between RAS/MAPK signaling and PCa progression towards an androgen-independent state has been demonstrated in the preclinical setting. Ectopic expression of HRAS(T35S) in androgen-sensitive LNCaP cells ...