PI3K isoform dependence of PTEN-deficient tumors can be altered by the genetic context.

There has been increasing interest in the use of isoform-selective inhibitors of phosphatidylinositide-3-kinase (PI3K) in cancer therapy. Using conditional deletion of the p110 catalytic isoforms of PI3K to predict sensitivity of cancer types to such inhibitors, we and others have demonstrated that tumors deficient of the phosphatase and tensin homolog (PTEN) are often dependent on the p110ß isoform of PI3K. Because human cancers usually arise due to multiple genetic events, determining whether other genetic alterations might alter the p110 isoform requirements of PTEN-null tumors becomes a critical question. To investigate further the roles of p110 isoforms in PTEN-deficient tumors, we used a mouse model of ovarian endometrioid adenocarcinoma driven by concomitant activation of the rat sarcoma protein Kras, which is known to activate p110α, and loss of PTEN. In this model, ablation of p110ß had no effect on tumor growth, whereas p110α ablation blocked tumor formation. Because ablation of PTEN alone is often p110ß dependent, we wondered if the same held true in the ovary. Because PTEN loss alone in the ovary did not result in tumor formation, we tested PI3K isoform dependence in ovarian surface epithelium (OSE) cells deficient in both PTEN and p53. These cells were indeed p110ß dependent, whereas OSEs expressing activated Kras with or without PTEN loss were p110α dependent. Furthermore, isoform-selective inhibitors showed a similar pattern of the isoform dependence in established Kras(G12D)/PTEN-deficient tumors. Taken together, our data suggest that, whereas in some tissues PTEN-null tumors appear to inherently depend on p110ß, the p110 isoform reliance of PTEN-deficient tumors may be altered by concurrent mutations that activate p110α.

The phosphatidylinositol 3-kinase (PI3K) isoform dependence of tumor formation is determined by the genetic mode of PI3K pathway activation rather than by tissue type.

UNLABELLED: Previous work has shown that prostate cancer in a Pten-null murine model is dependent on the p110ß isoform of phosphatidylinositol 3-kinase (PI3K), while breast cancer driven by either polyoma middle T antigen (MT) or HER2 is p110α dependent. Whether these differences in isoform dependence arise from tissue specificity or from the nature of the oncogenic signal activating the PI3K pathway is important, given increasing interest in using isoform-specific PI3K inhibitors in cancer therapy. To approach this question, we studied the PI3K isoform dependence of our recently constructed prostate cancer model driven by MT. Since MT activates a number of signaling pathways, we first confirmed that the MT-driven prostate cancer model was actually dependent on PI3K. A newly generated transgenic prostate line expressing an MT allele (Y315F) known to be defective for PI3K binding displayed a markedly reduced ability to drive tumor formation. We next selectively ablated expression of either p110α or p110ß in mice in which wild-type MT was expressed in the prostate. We found that tumor formation driven by MT was significantly delayed by the loss of p110α expression, while ablation of p110ß had no effect. Since the tumor formation driven by MT is p110α dependent in the prostate as well as in the mammary gland, our data suggest that PI3K isoform dependence is driven by the mode of PI3K pathway activation rather than by tissue type. IMPORTANCE: Middle T antigen (MT), the oncogene of polyomavirus, can drive tumor formation in a variety of cell types and tissues. Interestingly, MT has no intrinsic enzymatic activity but instead functions by binding and activating cellular signaling proteins. One of the most important of these is the lipid kinase PI3K, which was first studied in MT immunoprecipitates. Ubiquitously expressed PI3K comes in two major isoforms: p110α and p110ß. Previous work in animal models showed that p110α was the key isoform in breast tumors driven by oncogenes, including MT and HER2, while p110ß was key in prostate tumors driven by Pten loss. We asked the simple question of whether a prostate tumor driven by MT depends on p110α, which would suggest that the mode of activation determines p110 isoform dependence, or p110ß, which would suggest that tissue type determines isoform dependence. The clear answer is that MT depends on p110α in both the prostate and breast.

Blocking PI3K p110ß Attenuates Development of PTEN-Deficient Castration-Resistant Prostate Cancer.

A common outcome of androgen deprivation in prostate cancer therapy is disease relapse and progression to castration-resistant prostate cancer (CRPC) via multiple mechanisms. To gain insight into the recent clinical findings that highlighted genomic alterations leading to hyperactivation of PI3K, we examined the roles of the commonly expressed p110 catalytic isoforms of PI3K in a murine model of Pten-null invasive CRPC. While blocking p110α had negligible effects in the development of Pten-null invasive CRPC, either genetic or pharmacologic perturbation of p110ß dramatically slowed CRPC initiation and progression. Once fully established, CRPC tumors became partially resistant to p110ß inhibition, indicating the acquisition of new dependencies. Driven by our genomic analyses highlighting potential roles for the p110ß/RAC/PAK1 and ß-catenin pathways in CRPC, we found that combining p110ß with RAC/PAK1 or tankyrase inhibitors significantly reduced the growth of murine and human CRPC organoids in vitro and in vivo. Because p110ß activity is dispensable for most physiologic processes, our studies support novel therapeutic strategies both for preventing disease progression into CRPC and for treating CRPC. IMPLICATIONS: This work establishes p110ß as a promising target for preventing the progression of primary PTEN-deficient prostate tumors to CRPC, and for treating established CRPC in combination with RAC/PAK1 or tankyrase inhibitors.

CRKL Mediates p110ß-Dependent PI3K Signaling in PTEN-Deficient Cancer Cells.

The p110ß isoform of PI3K is preferentially activated in many tumors deficient in the phosphatase and tensin homolog (PTEN). However, the mechanism(s) linking PTEN loss to p110ß activation remain(s) mysterious. Here, we identify CRKL as a member of the class of PI3Kß-interacting proteins. Silencing CRKL expression in PTEN-null human cancer cells leads to a decrease in p110ß-dependent PI3K signaling and cell proliferation. In contrast, CRKL depletion does not impair p110α-mediated signaling. Further study showed that CRKL binds to tyrosine-phosphorylated p130Cas in PTEN-null cancer cells. Since Src family kinases are known both to be regulated by PTEN and to phosphorylate and activate p130Cas, we tested and found that Src inhibition cooperated with p110ß inhibition to suppress the growth of PTEN-null cells. These data suggest both a potential mechanism linking PTEN loss to p110ß activation and the possible benefit of dual inhibition of Src and PI3K for PTEN-null tumors.

LIN54 is an essential core subunit of the DREAM/LINC complex that binds to the cdc2 promoter in a sequence-specific manner.

Recently, the conserved human LINC/DREAM complex has been described as an important regulator of cell cycle genes. LINC consists of a core module that dynamically associates with E2F transcription factors, p130 and the B-MYB transcription factor in a cell cycle-dependent manner. In this study, we analyzed the evolutionary conserved LIN54 subunit of LINC. We found that LIN54 is required for cell cycle progression. Protein interaction studies demonstrated that a predicted helix-coil-helix motif is required for the interaction of LIN54 with p130 and B-MYB. In addition, we found that the cysteine-rich CXC domain of LIN54 is a novel DNA-binding domain that binds to the cdc2 promoter in a sequence-specific manner. We identified two binding sites for LIN54 in the cdc2 promoter, one of which overlaps with the cell cycle homology region at the transcriptional start site. Gel shift assays suggested that, in quiescent cells, the binding of LIN54 at the cell cycle homology region is stabilized by the binding of E2F4 to the adjacent cell cycle-dependent element. Our data demonstrate that LIN54 is an important and integral subunit of LINC.

The human synMuv-like protein LIN-9 is required for transcription of G2/M genes and for entry into mitosis.

Regulated gene expression is critical for the proper timing of cell cycle transitions. Here we report that human LIN-9 has an important function in transcriptional regulation of G2/M genes. Depletion of LIN-9 by RNAi in human fibroblasts strongly impairs proliferation and delays progression from G2 to M. We identify a cluster of G2/M genes as direct targets of LIN-9. Activation of these genes is linked to an association between LIN-9 and B-MYB. Chromatin immunoprecipitation assays revealed binding of both LIN-9 and B-MYB to the promoters of G2/M regulated genes. Depletion of B-MYB recapitulated the biological outcome of LIN-9 knockdown, including impaired proliferation and reduced expression of G2/M genes. These data suggest a critical role for human LIN-9, together with B-MYB, in the activation of genes that are essential for progression into mitosis.

LINC, a human complex that is related to pRB-containing complexes in invertebrates regulates the expression of G2/M genes.

Here we report the identification of the LIN complex (LINC), a human multiprotein complex that is required for transcriptional activation of G2/M genes. LINC is related to the recently identified dREAM and DRM complexes of Drosophila and C. elegans that contain homologs of the mammalian retinoblastoma tumor suppressor protein. The LINC core complex consists of at least five subunits including the chromatin-associated LIN-9 and RbAp48 proteins. LINC dynamically associates with pocket proteins, E2F and B-MYB during the cell cycle. In quiescent cells, LINC binds to p130 and E2F4. During cell cycle entry, E2F4 and p130 dissociate and LINC switches to B-MYB and p107. Chromatin Immunoprecipitation experiments demonstrate that LINC associates with a large number of E2F-regulated promoters in quiescent cells. However, RNAi experiments reveal that LINC is not required for repression. In S-phase, LINC selectively binds to the promoters of G2/M genes whose products are required for mitosis and plays an important role in their cell cycle dependent activation.