Integrated stress response plasticity governs normal cell adaptation to chronic stress via the PP2A-TFE3-ATF4 pathway.

The integrated stress response (ISR) regulates cell fate during conditions of stress by leveraging the cell's capacity to endure sustainable and efficient adaptive stress responses. Protein phosphatase 2A (PP2A) activity modulation has been shown to be successful in achieving both therapeutic efficacy and safety across various cancer models; however, the molecular mechanisms driving its selective antitumor effects remain unclear. Here, we show for the first time that ISR plasticity relies on PP2A activation to regulate drug response and dictate cellular fate under conditions of chronic stress. We demonstrate that genetic and chemical modulation of the PP2A leads to chronic proteolytic stress and triggers an ISR to dictate cell fate. More specifically, we uncovered that the PP2A-TFE3-ATF4 pathway governs ISR cell plasticity during endoplasmic reticular and cellular stress independent of the unfolded protein response. We further show that normal cells reprogram their genetic signatures to undergo ISR-mediated adaptation and homeostatic recovery thereby successfully avoiding toxicity following PP2A-mediated stress. Conversely, oncogenic specific cytotoxicity induced by chemical modulation of PP2A is achieved by activating chronic and irreversible ISR in cancer cells. Our findings propose that a differential response to chemical modulation of PP2A is determined by intrinsic ISR plasticity, providing a novel biological vulnerability to selectively induce cancer cell death and improve targeted therapeutic efficacy.

Small-Molecule-Mediated Stabilization of PP2A Modulates the Homologous Recombination Pathway and Potentiates DNA Damage-Induced Cell Death.

High-grade serous carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP inhibitors (PARPi) have become the mainstay of HGSC-targeted therapy, given that these tumors are driven by a high degree of genomic instability (GI) and homologous recombination (HR) defects. Nonetheless, approximately 30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and an increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a small-molecule activator of protein phosphatase 2A (PP2A; SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in patient-derived HGSC cells and xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for cellular transformation (B56α, B56γ, and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061-induced stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and HR-deficient HGSC cells and PDX models. Our studies identify PP2A as a novel regulator of HR and indicate PP2A modulators as a therapeutic therapy for HGSC. In summary, our findings further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 presents benefits in overcoming PARPi resistance driven by BRCA1/2 mutation reversions.

miR-181a initiates and perpetuates oncogenic transformation through the regulation of innate immune signaling.

Genomic instability (GI) predisposes cells to malignant transformation, however the molecular mechanisms that allow for the propagation of cells with a high degree of genomic instability remain unclear. Here we report that miR-181a is able to transform fallopian tube secretory epithelial cells through the inhibition of RB1 and stimulator-of-interferon-genes (STING) to propagate cells with a high degree of GI. MiR-181a targeting of RB1 leads to profound nuclear defects and GI generating aberrant cytoplasmic DNA, however simultaneous miR-181a mediated inhibition of STING allows cells to bypass interferon mediated cell death. We also found that high miR-181a is associated with decreased IFNγ response and lymphocyte infiltration in patient tumors. DNA oncoviruses are the only known inhibitors of STING that allow for cellular transformation, thus, our findings are the first to identify a miRNA that can downregulate STING expression to suppress activation of intrinsic interferon signaling. This study introduces miR-181a as a putative biomarker and identifies the miR-181a-STING axis as a promising target for therapeutic exploitation.

Inactivation of PP2A by a recurrent mutation drives resistance to MEK inhibitors.

The serine/threonine Protein Phosphatase 2A (PP2A) functions as a tumor suppressor by negatively regulating multiple oncogenic signaling pathways. The canonical PP2A holoenzyme comprises a scaffolding subunit (PP2A Aα/ß), which serves as the platform for binding of both the catalytic C subunit and one regulatory B subunit. Somatic heterozygous missense mutations in PPP2R1A, the gene encoding the PP2A Aα scaffolding subunit, have been identified across multiple cancer types, but the effects of the most commonly mutated residue, Arg-183, on PP2A function have yet to be fully elucidated. In this study, we used a series of cellular and in vivo models and discovered that the most frequent Aα R183W mutation formed alternative holoenzymes by binding of different PP2A regulatory subunits compared with wild-type Aα, suggesting a rededication of PP2A functions. Unlike wild-type Aα, which suppressed tumorigenesis, the R183W mutant failed to suppress tumor growth in vivo through activation of the MAPK pathway in RAS-mutant transformed cells. Furthermore, cells expressing R183W were less sensitive to MEK inhibitors. Taken together, our results demonstrate that the R183W mutation in PP2A Aα scaffold abrogates the tumor suppressive actions of PP2A, thereby potentiating oncogenic signaling and reducing drug sensitivity of RAS-mutant cells.

Protein phosphatase 2A Aα regulates Aß protein expression and stability.

Protein phosphatase 2A (PP2A) represses many oncogenic signaling pathways and is an important tumor suppressor. PP2A comprises three distinct subunits and forms through a highly regulated biogenesis process, with the scaffolding A subunit existing as two highly related isoforms, Aα and Aß. PP2A's tumor-suppressive functions have been intensely studied, and PP2A inactivation has been shown to be a prerequisite for tumor formation. Interestingly, although partial loss of the Aα isoform is growth promoting, complete Aα loss has no transformative properties. Additionally, in cancer patients, Aα is found to be inactivated in a haploinsufficient manner. Using both cellular and in vivo systems, colorectal and endometrial cancer cell lines, and biochemical and cellular assays, here we examined why the complete loss of Aα does not promote tumorigenesis. CRISPR/Cas9-mediated homozygous Aα deletion resulted in decreased colony formation and tumor growth across multiple cell lines. Protein expression analysis of PP2A family members revealed that the Aα deletion markedly up-regulates Aß protein expression by increasing Aß protein stability. Aß knockdown in control and Aα knockout cell lines indicated that Aß is necessary for cell survival in the Aα knockout cells. In the setting of Aα deficiency, co-immunoprecipitation analysis revealed increased binding of specific PP2A regulatory subunits to Aß, and knockdown of these regulatory subunits restored colony-forming ability. Taken together, our results uncover a mechanism by which PP2A Aα regulates Aß protein stability and activity and suggests why homozygous loss of Aα is rarely seen in cancer patients.

Small-Molecule Activators of Protein Phosphatase 2A for the Treatment of Castration-Resistant Prostate Cancer.

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065-80. ©2018 AACR.

Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth.

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.