AKT activation not sufficient to induce prostatic adenocarcinoma. The loss of PTEN is one of the most prevalent changes in primary PC disease [54] (Fig. 1A). PTEN loss is a likely marker of cancer initiation because biallelic deletion of Pten in transgenic mouse models is sufficient to induce HG-PIN in less oncogenic backgrounds such as C57BL/6 [55]. The loss of PTEN lipid phosphatase activity is thought to activate oncogenic AKT by allowing it to bind through its intrinsic PH domain to PI3K-generated PIP3 at the plasma membrane [56, 57]. Indeed, PTEN loss correlates with increased AKT activation levels in more advanced cases of human PC, based on increased relative levels of AKTpoS473 staining [58, 59], serving as strong predictor of biochemical recurrence [60]. Progression to prostatic adenocarcinoma and distal metastases requires additional losses in tumor suppressors such as Rb or Smad4 [40, 61], mimicking their frequent losses in primary PC (Fig. 1A). The notion that PTEN, RB1, or SMAD4 play important roles in regulating metastatic PC (mPC) is evidenced by gene losses that are more frequent in mPC than in primary lesions (Figs. 1A&B; Supplementary Fig. S1A). Consistent with AKAP12’s known metastasis suppressor function [10], AKAP12 loss is 3.5-fold more frequent in mPC than in primary PC (Figs. 1A&B).
Although the loss of AKAP12 is less frequent than PTEN loss in primary PC (10% vs. 31% in the TCGA dataset), PTEN or AKAP12 losses statistically co-occur with RB1 loss (Supplementary Table S1), and these combinations show statistical significance in predicting disease-free survival (DFS) using TCGA datasets (Fig. 1C). AKAP12 is thought to attenuate oncogenic Src signaling by scaffolding pools of Src to lipid rafts, away from integrin/FAK/growth factor receptor-rich plasma membrane sites [13]. Indeed, the inducible upregulation of Akap12 in MLL[TetOFF-SSeCKS/Akap12] PC cells [7] decreased relative phosphotyrosyl-p85α and AKTpoS473 levels without affecting total AKT, p85α or PTEN levels (Fig. 1D). This correlates with a statistical co-occurrence between AKAP12 loss and either increased levels of the SFK member, LYN (copy number gain, transcriptional upregulation) or SrcpoY416, a shared marker of SFK activation [62] (Fig. 1E). This is consistent with the notion that LYN promotes PC progression and mPC formation [16, 63–65], whereas FYN, whose levels trend towards mutual exclusivity with AKAP12 loss (Fig. 1E), is thought to promote progression of neuroendocrine PC [66]. Yet, whereas both Pten/Rb- and Akap12/Rb-null prostate lesions exhibit Akt activation (Fig. 2A) [3, 40], their mPC progression profiles differ (Table 1): Pten/Rb-null mice develop aggressive prostatic adenocarcinomas associated with systemic metastases, whereas Akap12/Rb-null mice develop HG-PIN plus local, indolent lymph node metastases. This suggests that PTEN or AKAP12 control divergent AKT oncogenic progression pathways, with the latter likely depending more on SFK roles. Indeed, the majority of primary PC cases with PTEN loss are distinct from those with SRC or LYN gain (Supplementary Fig. S1A). Thus, “AKT activation” in the context of RB loss is not sufficient for progression to adenocarcinoma.
Table 1
Pathology of Prostate Lesions in Transgenic CaP Models
Genotype
|
Prostate Lesion
|
Metastasis
|
AKTpoS473
|
WT
|
none
|
no
|
-
|
Pb4*-Cre:Rbfl/fl
|
hyperplasia
|
no
|
-
|
Akap12−/−
|
hyperplasia
|
no
|
+
|
Pb4-Cre:Ptenfl/fl
|
PIN
|
rare
|
+
|
Akap12−/−;Pb4-Cre:Rbfl/fl
|
HG-PIN
|
yes (local LN)
|
+
|
Pb4-Cre:Ptenfl/fl;Rbfl/fl
|
adenocarcinoma
|
yes (lung, liver, LN, bone)
|
+
|
*, Pb, probasin; PIN, prostatic intraepithelial neoplasia; HG, high-grade; LN, lymph node |
Preferential dependence on AKT2 in PTEN-deficient PC cells. We addressed whether differential Akt isoform usage/dependence might account for the varying mPC progression profiles of the two transgenic models. AKT isoforms exert different effects on the survival of PC cell lines [32, 38, 39, 67, 68], and importantly, Chin et al. [38] showed that PTEN-deficient human LNCaP cells show a greater reliance on AKT2 for maintenance and survival in anchorage-independent growth conditions. Analysis of the three AKT isoforms, AKT1, AKT2, and AKT3, identified a statistically-significant increase in only AKT2 in mPC in three human Oncomine datasets [14, 25, 69] (Supplemental Fig. S1B), suggesting a more important role for AKT2 in mPC progression. IB analysis of prostate lysates from 12 week-old WT, Akap12/Rb-, or Pten/Rb-null mice indicated that relative AktpoS473 levels were increased in Akap12/Rb- and Pten/Rb-null lesions compared to levels in WT prostates (Fig. 2A). Ser473 is phosphorylated by mechanistic target-of-rapamycin complex 2 (mTORC2) [70] and PI3K [71], and is required to potentiate AKT serine/threonine kinase activity [30]. In addition, the relative increase of the pan-AKT substrate, PRAS40poT246, suggests similar levels of overall Akt activation in Akap12/Rb- and Pten/Rb-null compared to WT prostates (Fig. 2A). In contrast, the relative level of AktpoT308, a PDK1-mediated phosphorylation required for AKT activity [72], was elevated only in Pten/Rb-null prostates, when normalized to β-actin as a loading control. However, we found no change in relative PDK1 protein or activation levels in WT, Akap12/Rb-, or Pten/Rb-null prostates (Fig. 2B). As we showed previously [19], the loss of Akap12 alone was sufficient to induce activated Akt in all four prostatic lobes (Fig. 2C) using an Ab that recognizes poS473/474 shared by AKT1/2. Compared to WT prostates, higher Akt1/2poS473/474 levels were also detected in Akap12/Rb- and Pten/Rb-null prostates (Fig. 2C). There was increased nuclear signal in Pten/Rb-null adenocarcinomas (Fig. 2C; bottom), consistent with a previous report of localization of AKT1 in the cytoplasm and AKT2 in the nucleus of PC-3 cells [39]. Moreover, whereas total Akt1 protein levels were similar in all three prostate genotypes, the relative levels of Akt2 and Akt3 were increased in Pten/Rb-null prostates (Fig. 2A).
RNA-seq analysis showed no overall changes in Akt1 and Akt3 levels between Akap12/Rb- and Pten/Rb-null prostates, compared to an upregulation of Akt2 RNA in the Pten/Rb-null prostates (Fig. 2D). In order to assess the relative activation levels of AKT isoforms, AKT isoform proteins were immunoprecipitated using isoform-specific Abs and the pull-downs probed for AktpoS473 (Fig. 2E). The relative activation level of Akt1 was similar in Akap12/Rb- and Pten/Rb-null prostates whereas Pten/Rb-null prostates showed increases in Akt2 and Akt3 activation levels, correlating with increased protein levels (Fig. 2A).
Next, we probed these tissue samples with an Ab that detects AKT canonical substrates based on the shared phosphorylated motif, RXXSpo/Tpo (Fig. 2F). Whereas the Akap12/Rb-null lysates had quantitative differences in the levels of several substrates compared to WT lysates, the Pten/Rb-null lysates also had qualitative differences, suggesting the targeting of unique substrates. This finding is consistent with the notion that different AKT isoforms may predominate in the two Tg PC models.
We then addressed how PTEN status controls PC oncogenic growth by re-expressing PTEN-GFP (vs. GFP alone in controls) in LNCaP or T402 cells (Table 2, Fig. 3A), the latter derived from a murine Pten/Rb-null adenocarcinoma [40]. As well, we produced an isogenic pair of PTEN-positive 22Rv1 cells expressing shPTEN or scrambled (control) shRNA (Supplementary Fig. S2D). PTEN re-expression in T402 cells neither changed protein levels of Akt isoforms or Ar (Fig. 3A), the relative expression of an Ar-regulated 19-gene panel (Fig. 3B), nor proliferation in 2D conditions with androgen-containing media (Supplementary Figs. S2A-C). Consistent with its role as a tumor suppressor, PTEN re-expression decreased relative PC invasiveness (Fig. 3C), clonogenicity (Fig. 3E), chemotaxis (Figs. 4A&C) and tumor formation (Fig. 6F), whereas PTEN knockdown in isogenic 22Rv1 cells increased clonogenicity (Fig. 3E).
Table 2: CaP Cell Line Models
Model
|
Species
|
PTEN
|
AR
|
*AKTpoSer473
|
T402 (Pten, Rb-negative)
|
mouse
|
deleted
|
+
|
++
|
T402[PTEN]
|
mouse
|
+
|
+
|
low (in 3D)
|
LNCaP
|
human
|
del/mut**
|
+
|
++
|
LNCaP[PTEN]
|
human
|
+
|
+
|
low (in 3D)
|
22Rv1
|
human
|
+
|
H874Y
|
low (in 3D)
|
22Rv1[shPTEN]
|
human
|
low
|
H874Y
|
+
|
*, based on IB, relative to total AKT1 protein levels
|
**, one copy deleted, one copy with a truncation mutation
|
Based on the increase in Akt2 expression and activation in the more metastatic Pten/Rb-null model (Figs. 2A&E), we asked if the knockdown of Akt2 would inhibit in vitro parameters of metastatic growth, using transwell assays for chemotaxis or Matrigel invasion, or by assaying for survival using either clonogenic or anoikis assays. AKT knockdowns in T402 and LNCaP cells were isoform-specific (Fig. 3D, lower panel; Supplementary Fig. S3E). The knockdown of Akt2, but not Akt1, in the Pten-negative T402 PC cells (Supplementary Fig. S3E) decreased invasiveness (Fig. 3C). In contrast, the re-expression of PTEN, which decreased the invasiveness of control T402 cells, switched dependence from Akt2 to Akt1. Similarly, the knockdown of either AKT2 or AKT3, but not AKT1, inhibited LNCaP invasiveness (Fig. 3D). PTEN re-expression decreased the invasiveness of LNCaP cells to the limits of detection (< 20 cells/field), thereby making it impossible to assess the effects of AKT isoform knockdown. Although AKT3 levels were quite low in LNCaP cells (requiring IP from 0.5mg of lysate protein followed by IB), knockdown caused a decrease in invasiveness (Fig. 3D), strongly suggesting that AKT3 promotes invasiveness in these cells. Taken together, these data identify critical roles for AKT2 and AKT3 in the invasiveness of PTEN-deficient PC, and that upon PTEN re-expression, reliance on AKT1 increases.
Role of PI3K-p110 and AKT isoforms in PTEN-regulated survival and chemotaxis. Because PTEN-negative tumor cells have been reported to depend more on p110β for oncogenic growth [33, 56], we next assessed how PTEN expression affected clonogenic survival of the PC isogenic pairs, and if PTEN affected sensitivity to small molecule inhibitors of PI3K-p110 and AKT isoforms, or after knockdown of p110/AKT isoforms. PTEN re-expression decreased the relative clonogenic survival of LNCaP or T402 cells, whereas PTEN knockdown increased survival of 22Rv1 cells (Fig. 3E). To address the role of PI3K and AKT isoforms in controlling clonogenic survival, we first identified concentrations of p110α, p110β, AKT1 and AKT2 inhibitors that had minimal effect on the 2D proliferation of PC lines but were significantly above the IC50's reported for each drug (examples in Supplementary Figs. S2E & S4D). Importantly, we sought to show that the effects of the isoform-specific inhibitory drugs mimicked what we found with isoform-specific AKT and/or p110 si/shRNAs. Only the combination of p110βi and Akt2i significantly reduced the number of colonies in T402, whereas in T402[PTEN] cells, sensitivity changed to a combination of Akt1i and p110αi (Fig. 3F). Similar results were seen in LNCaP (Fig. 3G) and with other PTEN-positive or -negative human PC cells lines (Supplementary Fig. S3A&B), or when combining knockdown of p110 (Supplementary Fig. S3F) and AKT isoforms (Supplementary Figs. S3C&D). Thus, these data suggest a plasticity with which PTEN directs survival dependency through both p110α and AKT1, whereas PTEN-deficient cells depend on both p110β and Akt2. Akt3 was not included because of the lack of Akt3-specific inhibitors.
We then analyzed the PC isogenic cell panel for the effects of PTEN on chemotaxis. 22Rv1, which are very poor at chemotaxis even if PTEN is knocked down, were omitted. The reintroduction of PTEN significantly reduced chemotaxis in LNCaP and T402 (Fig. 4A). We next asked if the differential PI3K and AKT drug sensitivities observed in the clonogenic assays also affected chemotaxis. Chemotaxis in T402 was inhibited by the combination of AKT2 and p110β inhibitors (Fig. 4B; Supplementary Fig. 4B). PTEN re-expression abrogated most of the combined effect of AKT2i plus p110βi. As was observed in the clonogenic assays, T402 chemotaxis was not inhibited by p110αi and/or AKT1i, whereas in T402[PTEN], chemotaxis was sensitive to the combination of p110αi and Akt1i. Similar results were observed in LNCaP using isoform-inhibitory drugs (Fig. 4C) or RNAi (Supplementary Fig. S4A), noting that 0.3 µM AKT2i was insufficient to inhibit LNCaP 2D proliferation (Supplementary Fig. S2E) but sufficient to inhibit chemotaxis (Fig. 4D).
Zhang et al. [37] showed that the dependence of PTEN-deficient BT549 (breast) and PC3 (prostate) cancer cells on p110β was likely due to the selective binding by CRKL to p110β, facilitated by Src-phosphorylated p130Cas. This correlated with increased suppression of PTEN-deficient tumor growth by combining p110β and Src inhibitors. We recapitulated these findings in LNCaP and T402 cells using both out p110βi and the Src inhibitor, Saracatinib (Supplementary Fig. S4C). Additionally, Crkl RNA levels are roughly 4.2-fold higher in Pten/Rb-null than in Akap12/Rb-null tumors (Supplementary Fig. S4D). These data strengthen the notion that the p110β/AKT2 pathway activated in the absence of PTEN is separate from the Src/p110α/AKT1 pathway activated in the absence of AKAP12 (Fig. 1D).
We then attempted gain-of-function experiments using constitutively-active (CA) AKT1S473D or AKT2S474D. In a previous study, CA-AKT1S473D, but not CA-AKT2S474D, rescued the phosphorylation of the mTORC2-dependent substrate, ATP-citrate lyase, in brown adipocytes [41]. The stable expression of AKT1S473D or AKT2S474D in T402 cells equally increased the number and abundance of phospho-AKT substrates irrespective of PTEN status (Supplementary Fig. S4E), validating the notion that they encode CA kinase variants. However, there was no distinction in the ability of CA-AKT isoform to increase chemotaxis in either T402 or T402[PTEN] cells (Supplementary Fig. S4F). We found similar results using AKT1 or AKT2 constructs fused to an N-terminal myristylation domain known to potentiate associated kinase activity [73, 74], namely, no distinction in the ability to induce phospho-AKT substrates and chemotaxis (data not shown). Thus, it is likely that in LNCaP or T402 cells, the CA mutants cannot differentiate AKT1- vs. AKT2-specific functions.
SMAD4 loss as a marker of p110β/AKT2 dependence in PTEN-deficient PC cells. Previous data showed that Smad4 loss potentiates PC metastasis formation in PtenPE:−/− mice [61]. We analyzed whether Smad4 might serve as a marker of metastatic progression that could differentiate the aggressive Pten/Rb-null PC model from the indolent Akap12/Rb-null HG-PIN model. SMAD4 RNA levels inversely correlated with AKT2, but not AKT1, RNA levels in human PC cell lines (Fig. 5A) and when comparing Akap12/Rb- vs. Pten/Rb-null prostate lesions (Fig. 5B). This corresponded to lower Smad4 protein levels in the more metastatic Pten/Rb-null tumors (Fig. 5C) and in human metastatic PC (Supplementary Fig. S5). Smad4 protein levels were increased 2- to 2.5-fold by the knockdown of Akt2 (Fig. 5D) but not by the knockdown of Akt1 or Akt3 (Fig. 5E). In LNCaP and 22Rv1 cells, the knockdown of SMAD4 using two different siRNAs led to a significant increase in chemotaxis (Fig. 5F). Treatment with AKT2i induced SMAD4 expression in LNCaP (Figs. 5G&H) but not in LNCaP[PTEN] cells (Fig. 5H), and this correlated with decreased abundance of po-AKT substrates but not total AKT1 or AKT2 (Fig. 5H), confirming the efficacy of AKT2i. In contrast, concentrations of AKT1i or p110αi that did not inhibit LNCaP 2D proliferation or survival (Supplementary Figs. S2E & S3A) also failed to decrease LNCaP chemotaxis (Fig. 4C), strengthening the role of AKT2 in controlling chemotactic motility of PTEN-negative PC cells.
We next compared how PTEN re-expression affected AKT signaling in 2D vs. 3D growth. This is because we previously showed that activated Src had a maximal ability to activate AKT in 3D growth conditions [75], and because the effect of AKT2 on LNCaP survival was manifest in 3D, but not in 2D growth [38]. Interestingly, the re-expression of PTEN did not significantly reduce activated AKT (relative AKTpoSer473 or AKT2poSer474 levels, the latter using an Ab specific for activated AKT2) in T402 grown in 2D (Fig. 6A). In contrast, PTEN-mediated reduction in relative AKTpoT308 levels were observed in cells grown in 3D (suspension in methylcellulose) (Fig. 6B). We then analyzed how PTEN affected the ability of serum to induce AKT activation in 2D vs. 3D conditions. While the overnight growth in 3D with serum had minimal to no ability to activate AKT, a 30 min treatment of FBS (“3D + FBS Stim.”) to serum-starved (“3D + 0.5%FBS”) LNCaP or T402 cells induced more relative AKTpoSer473/474 and AKTpoT308 in PTEN-negative cells than in PTEN re-expressing cells (Figs. 6C&D). We next determined if PTEN controlled survival under anoikis conditions through a greater dependence on AKT1 or AKT2. LNCaP and LNCaP[PTEN] cells were grown in non-adherent conditions (48 h on agarose-coated plates) while being treated with DMSO, AKT1i or AKT2i, followed by quantification of cell viability. LNCaP viability was more dependent on AKT2, whereas viability of LNCaP[PTEN] cells was more dependent on AKT1 (Fig. 6E). Taken together, these data strongly suggest that PTEN suppression of AKT activation is potentiated under 3D conditions, exemplified here by increased survival under anchorage-independent conditions but also by other 3D conditions shown earlier such as invasiveness and chemotaxis.
Therapeutic targeting of PTEN-negative PC requires combining PI3K-p110β and AKT2 inhibitors. We addressed how targeting p110 and/or AKT isoforms affected the progression of primary orthotopic tumors and the establishment of spontaneous metastases in SCID male mice. Consistent with PTEN’s tumor suppressor function, the orthotopic injection of T402[PTEN] cells (prostatic anterior lobe) failed to yield growing tumors after 80 days (Fig. 6F). Individual inhibitors for p110α, p110β, AKT1 or AKT2, or for the p110α/AKT1 combination, slightly decreased tumor growth from days 12–18 of drug treatment in comparison to vehicle controls, however, none of these translated to statistically significant effects at day 35. In contrast, treatment with the combination of p110β/AKT2 inhibitors showed statistically significant tumor suppression over controls. Moreover, the p110β/AKT2 inhibitor combination resulted in a statistically significant decrease in the metastatic colonization by LNCaP-C4-2B[luc/GFP] cells, as assessed by Alu-specific qPCR as described previously [52] (Fig. 6G).