Phosphatase Nuclear Targeting Subunit Research Articles

Abstract 5295: Targeting Rb phosphatase activity to circumvent CDK4/6 inhibitor resistance

Abstract The Retinoblastoma (Rb) tumor suppressor protein plays a role in the control of proliferation, apoptosis, differentiation, invasion and the maintenance of genome stability. Its activity is regulated by phosphorylation on several serine/threonine sites, which is thought to inactivate its tumor suppressive function. Cyclin dependent kinases (CDKs) are responsible for most of the Rb phosphorylation that occurs in cells. It is well established that alterations in the Rb pathway (consisting of CDK4/6, D-type cyclins, the CDK inhibitor p16INK4a, and Rb itself) that lead to excessive phosphorylation of Rb have been observed in almost all cancer types. Alterations that lead to increased CDK activity toward Rb are more common than mutations to the Rb gene itself, a finding that prompted investigations into the development of clinical treatments that inhibit the phosphorylation of Rb by CDKs. The CDK4/6 inhibitors palbociclib and abemaciclib were recently clinically approved for use in breast cancer. However, acquired resistance to CDK4/6 inhibition has been shown to limit clinical efficiency. Targeting Rb phosphorylation by CDK4/6 inhibition has been shown to activate alternate pro-survival pathways such as the AKT pathway. In our studies we developed a method of siRNA-mediated activation of Rb phosphatase that targets Rb phosphorylation in attempt to circumvent the development of resistance. Phosphatase Nuclear Targeting Subunit (PNUTS) associates with and regulates PP1 activity toward Rb. In several cancer cell types, such as HCT116 colon cancer, MIA PaCa-2 pancreatic cancer and MCF7 breast cancer, PNUTS depletion causes dephosphorylation of Rb without AKT activation. Furthermore, in Panc1 pancreatic cancer cells, activation of AKT is observed after 1-hour palbociclib (500nM) treatment, whereas PNUTS depletion does not induce AKT. In MD-MBA-231 breast cancer cells, 1-hour treatment of palbociclib or abemaciclib (20nM) induces the AKT pathway as well as the STAT pathways, however PNUTS depletion in these cells does not induce AKT activation. It has been shown that CDK4/6 inhibitors activate AKT by causing loss of Rb interaction with SIN1, a component the mTORC2 complex. Our studies reveal that PNUTS depletion causes dephosphorylation of Rb, without disrupting Rb-mediated inhibition of mTORC2. Thus, the stimulation of Rb phosphatase activity may target Rb phosphorylation without the development of resistance. Citation Format: Kathleen H. DiSalvo, Brian C. Velez, Rebecca L. Kravtsov, Nancy A. Krucher. Targeting Rb phosphatase activity to circumvent CDK4/6 inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5295.

Dephosphorylation of the Retinoblastoma protein (Rb) inhibits cancer cell EMT via Zeb

ABSTRACTThe tumor suppressor Retinoblastoma (Rb) protein is highly phosphorylated in cancer cells largely due to the overexpression of cyclins or the loss of expression of cyclin dependent kinase inhibitors (cdki). Hyperphosphorylation of Rb promotes proliferation, and plays a role in the regulation of apoptosis. Recently, inhibition of cyclin dependent activity toward Rb has been identified as a strategy that has shown clinical efficacy. We utilized a method to induce phosphatase activity toward Rb in cells by shRNA silencing of PNUTS (Phosphatase Nuclear Targeting Subunit) that regulates PP1-mediated dephosphorylation of Rb. In this study, the effect of Rb dephosphorylation on the epithelial to mesenchymal transition (EMT) was determined. The EMT transition is observed in cancer cells that have acquired invasive characteristics. In breast cancer cells grown in 3D Matrigel cultures, MCF7 cells undergo apoptosis in response to Rb dephosphorylation, whereas MDA-MB-231 and Hs578T cells exhibit a reduction in the EMT. Cells devoid of phosphorylated Rb (nontransformed MCF10A and Rb-null MDA-MB-468) lacked any response to PNUTS depletion, showing the effect is Rb-dependent. In addition, these studies showed that Rb dephosphorylation in 3D Matrigel cultures of highly invasive HT1080 cells led to the inhibition of the EMT. Furthermore we observed association between dephosphorylated Rb with ZEB1, a zinc-finger E-box-binding transcription factor that regulates expression of E- and N-cadherins. Finally Rb dephosphorylation led to inhibition of ZEB1 transcriptional activity, this data supports the notion that Rb dephosphorylation modulates the EMT. These studies suggest targeting Rb phosphorylation in mesenchymal cancer cells may decrease invasiveness.

Abstract 1675: Activation of phosphatase toward the retinoblastoma protein inhibits invasion in MDA-MB-231 breast cancer cells

Abstract Excessive phosphorylation of the Retinoblastoma protein (Rb) is found in most cancer tumor types. Several cyclin dependent kinase (cdk) inhibitors are in development or in clinical trials at the present time. Our previous studies have focused on the regulation of Rb phosphorylation by the phosphatase, PP1. In proliferating cells, PP1 is regulated by a protein called PNUTS (Phosphatase Nuclear Targeting Subunit). PNUTS is a competitive inhibitor of Rb binding to PP1 and siRNA mediated knockdown of PNUTS in several cancer cell types (breast, colon, ovarian) leads to an increase in Rb directed PP1 activity, Rb dephosphorylation on several sites, and a dramatic induction of apoptosis. In this study we have used MDA-MB-231 breast cancer cells grown in Matrigel. By lentiviral-mediated doxycycline inducible expression of PNUTS siRNA, expression of PNUTS protein is blocked and phosphorylation of Rb cells is reduced. Cells become less invasive and expression of mesenchymal markers N-cadherin, Zeb and Slug is lost. A reversion to the epithelial phenotype is suggested by an increase in E-cadherin expression. PNUTS knockdown has no effect on cells that do not contain highly phosphorylated Rb such as non-transformed MCF-10A cells or Rb-negative MDA-MB-468 cells. Further experiments using highly invasive HT-1080 cells show that reduction of Rb phosphorylation appears to reduce invasiveness in this cell model, supporting the idea that Rb phosphorylation plays a critical role in the regulation of invasion. Citation Format: Jacklynn Egger, Maria Lane, Brixhilda Dedi, Nancy A. Krucher. Activation of phosphatase toward the retinoblastoma protein inhibits invasion in MDA-MB-231 breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1675.

Abstract 4942: Activation of Phosphatase toward the Retinoblastoma protein in breast and colorectal cancer cell spheroids

Abstract Excessive phosphorylation of the Retinoblastoma protein is found in most cancer tumor types. Several cyclin dependent kinase (cdk) inhibitors are in development or in clinical trials at the present time. Our previous studies have focused on the regulation of Rb phosphorylation by the phosphatase, PP1. Specificity toward substrates is imparted onto PP1 by many different interacting proteins. In proliferating cells, PP1 is associated with a regulatory protein called PNUTS (Phosphatase Nuclear Targeting Subunit). Our previous experiments have shown that PNUTS inhibits PP1 activity toward specific Rb phosphorylation sites. Further we have demonstrated that siRNA mediated knockdown of PNUTS in several cancer cell types (colon, breast, ovarian) leads to an increase in Rb specific PP1 activity, Rb dephosphorylation on several sites, and a dramatic induction of apoptosis. Furthermore, the ability of PNUTS siRNA to induce apoptosis was shown to be dependent on Rb expression. Interestingly, further studies showed that PNUTS knockdown had no effect on non-transformed breast and colorectal cells, in which Rb is not hyperphosphorylated. Thus we have developed a method to activate the Rb phosphatase in cells by knockdown of the PP1 binding protein, PNUTS. The current study was undertaken to analyze the effect of PNUTS knockdown in cancer cells grown in three-dimensional spheroids grown by incubation of cells in hanging drop plates. The effect of PNUTS knockdown on Rb phosphorylation, cell proliferation and cell viability using this model system will be presented. Citation Format: Jacklynn Egger, Lisa Antonucci, Maria Lane, Nancy A. Krucher. Activation of Phosphatase toward the Retinoblastoma protein in breast and colorectal cancer cell spheroids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4942. doi:10.1158/1538-7445.AM2015-4942

Abstract 593: Rb association with Bax is regulated by phosphorylation

Abstract The Retinoblastoma protein (Rb) is important in the control of cell proliferation and apoptosis. Its activity is controlled by reversible phosphorylation on several serine and threonine residues that mediate binding to various cellular proteins. The role of Rb phosphorylation in the control of apoptosis is largely unknown, although several apoptotic stimuli result in dephosphorylation of Rb. Our previous work identified Rb (threonine-821) dephosphorylation as required for induction of apoptosis caused by UV light and inhibitions of cyclin dependent kinases. These studies led to the identification of proteins that bind to Rb under apoptotic conditions. One such Rb binding protein is the pro-apoptotic protein Bax. Bax, a member of the Bcl-2 family, undergoes a conformational change during apoptosis that is associated with the permeabilization of the outer mitochondrial membrane. In this study, we examined the role of Rb phosphorylation in the regulation of binding to Bax. In MCF7 cells, by activating the phosphatase responsible for dephosphorylating Rb, PP1, apoptotic death is triggered. We utilize siRNA to block the expression of PNUTS (Phosphatase Nuclear Targeting Subunit) which causes Rb dephosphorylation, and cell death. We have found that Bax binds to Rb under normal cell conditions, but when Rb becomes dephosphorylated on specific amino acid sites, Bax dissociates from Rb. Our cell fractionation studies have shown that phosphorylated Rb co-localizes with the mitochondrial fraction. Further examination of the specific sites of Rb phosphorylation required for binding to Bax is underway. Citation Format: Lisa Antonucci, Jacklynn V. Egger, Nancy A. Krucher. Rb association with Bax is regulated by phosphorylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 593. doi:10.1158/1538-7445.AM2014-593

Abstract 77: MicroRNA-34a Accelerates Aging in Vascular Smooth Muscle Cells (SMCs) Isolated from Transgenic Mice Overexpressing Nox1 in SMCs

Background: Aging increases cardiovascular morbidity. Aging in vascular smooth muscle cells (VSMC) induces cell senescence, leading to impaired vasodilation with decreased vascular compliance, increased inflammation and oxidative stress. MicroRNA-34a (miR-34a) has been reported as a biomarker of aging. Methods and Results: After screening for a group of miRs, there was a > 2 fold of miR-34a in aortic SMCs isolated from transgenic mice overexpressing Nox1 in SMCs (Nox-1 SMCs). Therefore, we hypothesized that miR34 contributes to VSMC aging. TaqMan miR assay confirmed an increased miR-34a by 2.1 fold in young NOX-1 SMCs (passage 3-7) with an increase by 5.25 fold in activity of senescence-associated β-galactosidase (SA-β-Gal ) compared to WT. miR-34a in old NOX-1 and WT SMCs (passage 14-16) was increased by 3.8 fold and 2.1 fold, respectively, compared to young SMCs (both P< 0.01 ). There was 1.8 fold more miR-34a detected in old NOX-1 vs. old WT SMCs. SA-β-Gal positive cells accounted for 0.8±1% and 4±1.3% in young WT and NOX-1 SMCs (P<0.01), and 5±1.5% and 32±5% in old WT and NOX-1 SMCs (P <0.005). After transfection with miR-34a inhibitor, mature miR-34a was decreased by 75% and SA-β-Gal activity by 58% in old NOX-1 cells with parallel changes in the protein expression for Gamma-H2AX( a DNA damage marker) and p21( an activator for cell senescence). Moreover, phosphatase nuclear targeting subunit (PNUTS, a recently identified anti-age protein and direct miR-34a target) was not changed in young NOX-1 SMCs but reduced by 78% in old NOX-1 SMCs. Interestingly, nuclear factor (erythroid-derived 2)-like 2 (Nrf2, a key transcription factor for antioxidant genes) mRNA and protein were decreased by 55% and 32% in old NOX-1 SMCs (P<0.01 and 0.05). Nrf2 activator tert-butylhydroquinone (tBHQ) reduced miR-34a from 2.9±0.4 to 1.8±0.2 fold in those cells. Conclusion: Increased NOX-1 in SMCs induces miR-34a that accelerates SMC senescence. The pathway may involve downregulation of Nrf2 and increased P21 through P53 pathway. This provides potential targets for protecting against vascular aging.

Abstract 5058: Phosphatase Nuclear Targeting Subunit (PNUTS) is phosphorylated by AKT kinase

Abstract Phosphorylation of the tumor suppressor protein, Retinoblastoma (Rb) is associated with a highly proliferative state of the cell and cancer. Rb phosphorylation state is partly controlled by the activity of Protein Phosphatase 1 (PP1) which is regulated by Phosphatase Nuclear Targeting Subunit (PNUTS). It is likely that PNUTS plays a role in cell survival and/or apoptosis because siRNA mediated knockdown of PNUTS causes apoptosis in breast and colon cancer cells. This occurs through activation of PP1 phosphatase activity toward Rb which leads to Rb dephosphorylation which can trigger apoptosis. Here we show that PNUTS may be regulated by the PI3K-AKT pathway which has been shown to be involved in many cellular functions including proliferation, growth and survival. In addition, signaling in this pathway has been shown to be disrupted in several cancers. In response to a sub-lethal dose of UV radiation of HCT116 colon cancer cells, activation of the AKT kinase occurs, with phosphorylation of known AKT substrates GSK-3 and mdm2 observed. Phosphorylation of Ser473 of AKT further verifies AKT activation. Under these conditions PNUTS is phosphorylated by the AKT kinase. Inhibition of PNUTS phosphorylation occurs when cells are treated with the LY294002 PI3K-AKT pathway inhibitor. Thus PNUTS is a putative substrate of AKT, and may play an important role in the survival of cells in response to stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5058.

PNUTS knockdown potentiates the apoptotic effect of Roscovitine in breast and colon cancer cells

The phosphorylation state of Retinoblastoma protein (Rb) plays a role in cell proliferation and apoptosis. Within cells, cyclin dependent kinases (cdks) phosphorylate Rb in response to growth stimulatory signals, whereas protein phosphatase 1 (PP1) dephosphorylates Rb when cells stop proliferating or undergo apoptosis in response to anti-proliferative or stress signals. Stimulation of PP1 activity via siRNA mediated knockdown of its interacting protein PNUTS (Phosphatase Nuclear Targeting Subunit) leads to Rb dephosphorylation and apoptosis in cancer cells. We utilized two separate methods to modulate the phosphorylation state of Rb in cancer cells. Kinase activity toward Rb is inhibited by the clinically relevant cdk inhibitor, Roscovitine. In addition, siRNA mediated PNUTS knockdown stimulates phosphatase activity toward Rb. Either of these treatments in cancer cells causes a 2-fold stimulation of apoptosis. When activation of phosphatase activity is combined with inhibition of cdk activity toward Rb, however, cells exhibit a 4-fold increase in apoptosis. The mechanism by which PNUTS knockdown mediated PP1 activation leads to apoptosis was determined to be dependent on the activity of the transcription factor E2F1. The Rb phosphorylation profiles resulting from each treatment were analyzed and found to be similar but not identical. In addition, the two treatments differentially effect the expression of bcl-2 family proteins. Thus inhibition of cdk activity and activation of PP1 activity toward pRb are functionally distinct processes that together increase the apoptotic effect in cells.

TRF2 functions as a protein hub and regulates telomere maintenance by recognizing specific peptide motifs

In mammalian cells, the telomeric repeat binding factor (TRF) homology (TRFH) domain-containing telomeric proteins TRF1 and TRF2 associate with a collection of molecules necessary for telomere maintenance and cell-cycle progression. However, the specificity and the mechanisms by which TRF2 communicates with different signaling pathways remain largely unknown. Using oriented peptide libraries, we demonstrate that the TRFH domain of human TRF2 recognizes [Y/F]XL peptides with the consensus motif YYHKYRLSPL. Disrupting the interactions between the TRF2 TRFH domain and its targets resulted in telomeric DNA-damage responses. Furthermore, our genome-wide target analysis revealed phosphatase nuclear targeting subunit (PNUTS) and microcephalin 1 (MCPH1) as previously unreported telomere-associated proteins that directly interact with TRF2 via the [Y/F]XL motif. PNUTS and MCPH1 can regulate telomere length and the telomeric DNA-damage response, respectively. Our findings indicate that an array of TRF2 molecules functions as a protein hub and regulates telomeres by recruiting different signaling molecules via a linear sequence code.

Reduced expression of PNUTS leads to activation of Rb-phosphatase and caspase-mediated apoptosis

There is abundant evidence that Retinoblastoma (Rb) activity is important in the control of cell proliferation and apoptosis. Reversible phosphorylation of the Rb protein that is carried out by cyclin dependent kinases and Protein phosphatase 1 (PP1) regulates its functions. A PP1 interacting protein, PNUTS (Phosphatase Nuclear Targeting Subunit) is proposed to be a regulator of Rb phosphorylation. In this study, PNUTS knockdown in MCF7, SKA and HCT116 cancer cells causes a reduction in viability due to increased apoptosis. However, normal cells (MCF10A breast and CCD-18Co colon) do not exhibit reduced viability when PNUTS expression is diminished. PNUTS knockdown has no effect in Rb-null Saos-2 cells. However, when Rb is stably expressed in Saos-2 cells, PNUTS knockdown reduces cell number. Knockdown of PNUTS in p53-/- HCT116 cells indicates that p53 is dispensable for the induction of apoptosis. Loss of PNUTS expression results in increased Rb-phosphatase activity and Rb dephosphorylation. E2F1 dissociates from Rb in cells depleted of PNUTS and the resulting apoptosis is dependent on caspase-8. These results indicate that Rb phosphorylation state can be manipulated by targeting Rb phosphatase activity and suggest that PNUTS may be a potential target for therapeutic pro-apoptotic strategies.

Dephosphorylation of Rb (Thr-821) in response to cell stress

The retinoblastoma tumor suppressor Rb is regulated by reversible phosphorylation that is dependent upon cyclin-dependent kinase (CDK) and protein phosphatase type 1 (PP1) activity in replicating cells. Hyperphosphorylated Rb allows cells to proliferate, whereas the hypophosphorylated isoform of Rb inhibits proliferation. Of the many phosphorylation sites of Rb, there is functional information available for a very few. In this report, we show that threonine-821 (Thr-821) of Rb is dephosphorylated earlier than other phosphorylation sites when cells are grown under hypoxic conditions which leads to Rb activation and G 1 arrest. This finding is interesting because Thr-821 of Rb remains phosphorylated throughout the cell division cycle in replicating cells. We hypothesized that the phosphorylation state of Thr-821 of Rb may depend on cellular stress. We report in this study that, when nontransformed CV1 epithelial cells and Hs578T breast cancer cells are treated with the chemotherapeutic agent cytosine arabinoside (Ara-C), Thr-821 of Rb is rapidly dephosphorylated concomitant with dissociation of the PP1 regulatory subunit PNUTS (phosphatase nuclear targeting subunit) from PP1 enzyme. These data are consistent with the concept that differential regulation of Rb-directed phosphatase activity exists when cells are progressing through the cell cycle compared to that observed when cells are under stress.

PNUTS (phosphatase nuclear targeting subunit) inhibits retinoblastoma-directed PP1 activity

Protein phosphatase type 1 catalytic subunit (PP1c) is a serine/threonine phosphatase involved in the dephosphorylation of many proteins in eukaryotic cells. It associates with several known targeting or regulatory subunits that directly regulate PP1c activity toward specific substrates. The recently identified Phosphatase Nuclear Targeting Subunit (PNUTS) binds to PP1c and inhibits PP1 activity toward phosphorylase a. One of the substrates of PP1c has been shown to be the cell cycle regulatory protein, Retinoblastoma (pRb). In this study, we show that PNUTS dissociates from PP1c under mildly hypoxic cell growth conditions that lead to an increase of PP1c activity toward pRb. We developed an assay that measures pRb-directed PP1c activity and show that a GST-PNUTS fusion protein inhibits phosphatase activity toward pRb when using PP1c from cell lysates, GST-PP1c, or purified PP1c. These studies suggest that PNUTS is involved in the regulation of PP1c activity toward pRb.