The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through multiple phases in the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase to anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity. With that aim and considering the limitations of conventional 2D cell culture in mimicking the tumor environment and predicting drug responses in animal models and humans, we also established 3D organoid cultures of PCa cells and immortalized human prostate epithelial cells (hPrEC) in Matrigel. This allowed us to explore cell to extracellular matrix (ECM) interactions. PC3 cells initially form round organoids in Matrigel, followed by an invasive switch to where cell protrusions invade the surrounding ECM. Strikingly, B55α reconstitution, dramatically suppressed rupture of the basement lamina and ECM invasion, while proliferation appeared not affected. Tracking organoid growth at defined time points or using live imaging, shows that protrusions in PC3 organoids are very dynamic and resemble invadopodia. Interestingly, reconstitution of B55α in PC3 organoids just prior the invasive switch results in reduction of invasive leads and those protrusions that appear to initiate keep forming and collapsing, with most organoids remain round. Our previous phosphoproteomics data in 2D culture suggests that cell-to-ECM signaling is likely altered with B55α reconstitution, identifying potential B55α/PP2A substrates among key mediators of integrin signaling. In sum, reconstitution of B55α suppresses invasion in PC3 organoids, possibly by regulating potential B55α substrates in focal adhesion signaling, such as Paxillin and/or Talin. Alternatively, centrosomal defects due to dephosphorylation of B55α substrates (e.g. HAUS6, NEDD1) may cause microtubule defects that preclude invasion. Further studies are required to identify the mechanism. Moreover, because our studies presented above are based on prostate cancer cell lines with undefined genetic alterations, we have immortalized primary human PrEC by a novel approach to generate a cell model to study cooperation of PPP2R2A loss with step-wise introduction of specific oncogenes and/or tumor suppressor gene alterations in transformation, tumorigenicity and invasion. Our newly develop method combines expression of hTERT, which appears insufficient for immortalization of hPrEC with CDKN2A knockout, which we predicted will prevent stress-induced replicative senescence. We have obtained two independent immortalized clones (hPrEC-T-ΔN2A) using this method and confirmed their identity using PCR and western blot analyses. Although cytogenetic analysis showed these two clones are of mixed population with minor alterations in karyotype, 4 out of 11 cells examined in clone 1 appear completely normal. We also find that the clones exit the cell cycle upon contact inhibition and induce p53 expression when treated with flavopiridol, further supporting that hPrEC-T-ΔN2A clones exhibit the features of normal cells. Characterization in 3D culture reveals that the clones are likely of basal epithelial origin. Finally, soft agar and clonogenic assays show hPrEC-T-ΔN2A clones are highly proliferative but not transformed. We are using these cell models to dissect the role of PPP2R2A depletion in step-wise transformation of immortalized PrEC and hope to develop a defined 3D organoid system to study invasion, which could also be suitable for drug screens. Altogether our work has very significantly advanced our understanding of B55α in suppressing transformation in prostate cancer cells and has developed novel tools for further mechanistic characterization of PPP2R2A haploinsufficiency and the development of potential pharmacologic therapeutic agents. / Biomedical Sciences
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/4095 |
| Date | January 2020 |
| Creators | Zhao, Ziran |
| Contributors | Graña-Amat, Xavier, Shore, Scott K., Haines, Dale, Zaidi, M. Raza, Gligorijevic, Bojana |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 192 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/4077, Theses and Dissertations |
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