Dephosphorylation Of Rb Research Articles

Palbociclib inhibits the progression of head and neck cancer and improves the Cetuximab response under specific condition in vitro and in vivo.

In head and neck squamous cell carcinoma (HNSCC) with human papillomavirus (HPV)-negative, deregulation of cell cycle is partly due to inactivation of p16INK4 and overexpression of cyclin D1. Here we investigated the in vitro and in vivo effects of the CDK4/6 inhibitor Palbociclib alone or combined with EGFR inhibitor Cetuximab in HNSCC. CCK-8, soft agar assay, colony formation assay, wound healing assay and transwell assay, β-galactosidase assay, western blotting, and cell-derived xenografts were used to investigated the in vitro and in vivo activity of drugs. Cell viability and colony formation decreased in a dose-dependent manner in Tu686, AMC-HN8, and Fadu cells under Palbociclib treatment. Palbociclib remarkably inhibited migration, invasion and the expression MMP-9 in HNSCC cells. Palbociclib also induced senescence. Palbociclib caused the dephosphorylation of RB but increased the cyclin D1 level in a dose-dependent manner. Moreover, combination with Cetuximab could significantly prevent the induction of cyclin D1 and activation of EGFR signals from Palbociclib treatment. Nevertheless, only within the range of certain concentrations, a synergistic inhibitory effect on cell growth was observed when combined with Palbociclib and Cetuximab. Although the synergistic effect in Palbociclib/Cetuximab combined therapy was comparable to that in traditional chemotherapeutic regimens (cisplatin/Cetuximab) in Fadu tumor xenograft, the combination therapy was less active than Cetuximab monotherapy in Tu686 tumor xenograft. In HPV-negative HNSCC, CDK4/6 inhibitor shows promising anti-tumor effects, which exhibits a synergistic effect when combined with EGFR inhibitor only in specific drug concentration and mouse model.

CDK4/6 signaling attenuates the effect of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) mutations, such as exon 19 deletion and exon 21 L858R, are driver oncogenes of non-small cell lung cancer (NSCLC), with EGFR tyrosine kinase inhibitors (TKIs) being effective against EGFR-mutant NSCLC. However, the efficacy of EGFR-TKIs is transient and eventually leads to acquired resistance. Herein, we focused on the significance of cell cycle factors as a mechanism to attenuate the effect of EGFR-TKIs in EGFR-mutant NSCLC before the emergence of acquired resistance. Using several EGFR-mutant cell lines, we investigated the significance of cell cycle factors to attenuate the effect of EGFR-TKIs in EGFR-mutant NSCLC. In several EGFR-mutant cell lines, certain cancer cells continued to proliferate without EGFR signaling, and the cell cycle regulator retinoblastoma protein (RB) was not completely dephosphorylated. Further inhibition of phosphorylated RB with cyclin-dependent kinase (CDK) 4/6 inhibitors, combined with the EGFR-TKI osimertinib, enhanced G0/G1 cell cycle accumulation and growth inhibition of the EGFR-mutant NSCLC in both in vitro and in vivo models. Furthermore, residual RB phosphorylation without EGFR signaling was maintained by extracellular signal-regulated kinase (ERK) signaling, and the ERK inhibition pathway showed further RB dephosphorylation. Our study demonstrated that the CDK4/6-RB signal axis, maintained by the MAPK pathway, attenuates the efficacy of EGFR-TKIs in EGFR-mutant NSCLC, and targeting CDK4/6 enhances this efficacy. Thus, combining CDK4/6 inhibitors and EGFR-TKI could be a novel treatment strategy for TKI-naïve EGFR-mutant NSCLC.

Identification of c-Met as a novel target of γ-glutamylcyclotransferase.

γ-Glutamylcyclotransferase (GGCT) is highly expressed in multiple types of cancer tissues and its knockdown suppresses the growth of cancer cells in vitro and in vivo. Although GGCT is a promising target for cancer therapy, the mechanisms underlying the antitumor effects remain unclear. The knockdown of GGCT inhibited the MEK-ERK pathway, and activated the tumor suppressor retinoblastoma gene (RB) at the protein level in cancer cell lines. c-Met was down-regulated by the knockdown of GGCT in cancer cells and its overexpression attenuated the dephosphorylation of RB and cell cycle arrest induced by the knockdown of GGCT in lung cancer A549 cells. STAT3 is a transcription factor that induces c-Met expression. STAT3 phosphorylation and its nuclear expression level were decreased in GGCT-depleted A549 and prostate cancer PC3 cells. The simultaneous knockdown of AMPK and GGCT restored the down-regulated expression of c-Met, and attenuated the dephosphorylation of STAT3 and MEK-ERK-RB induced by the knockdown of GGCT in PC3 cells. An intraperitoneal injection of a GGCT inhibitor decreased c-Met protein expression in a mouse xenograft model of PC3 cells. These results suggest that the knockdown of GGCT activates the RB protein by inhibiting the STAT3-c-Met-MEK-ERK pathway via AMPK activation.

Estrogen receptor alpha mediates 17β-estradiol, up-regulates autophagy and alleviates hydrogen peroxide-induced vascular senescence.

Atherosclerosis threatens human health by developing cardiovascular diseases, the deadliest disease world widely. The major mechanism contributing to the formation of atherosclerosis is mainly due to vascular endothelial cell (VECs) senescence. We have shown that 17β-estradiol (17β-E2) may protect VECs from senescence by upregulating autophagy. However, little is known about how 17β-E2 activates the autophagy pathway to alleviate cellular senescence. Therefore, the aim of this study is to determine the role of estrogen receptor (ER) α and β in the effects of 17β-E2 on vascular autophagy and aging through in vitro and in vivo models. Hydrogen peroxide (H2O2) was used to establish Human Umbilical Vein Endothelial Cells (HUVECs) senescence. Autophagy activity was measured through immunofluorescence and immunohistochemistry staining of light chain 3 (LC3) expression. Inhibition of ER activity was established using shRNA gene silencing and ER antagonist. Compared with ER-β knockdown, we found that knockdown of ER-α resulted in a significant increase in the extent of HUVEC senescence and senescence-associated secretory phenotype (SASP) secretion. ER-α-specific shRNA was found to reduce 17β-E2-induced autophagy, promote HUVEC senescence, disrupt the morphology of HUVECs, and increase the expression of Rb dephosphorylation and SASP. These in vitro findings were found consistent with the in vivo results. In conclusion, our data suggest that 17β-E2 activates the activity of ER-α and then increases the formation of autophagosomes (LC3 high expression) and decreases the fusion of lysosomes with autophagic vesicles (P62 low expression), which in turn serves to decrease the secretion of SASP caused by H2O2 and consequently inhibit H2O2-induced senescence in HUVEC cells.

Dual PPARγ/ɑ agonist oroxyloside suppresses cell cycle progression by glycolipid metabolism switch-mediated increase of reactive oxygen species levels

Cancer cells prefers to rely on aerobic glycolysis than pyruvate oxidation to meet the high demand of energy for rapidly proliferation. Peroxisome proliferator-activated receptors (PPARs) are a kind of important ligand-inducible transcription factors and play crucial roles in glucose and lipid metabolism. Careful designing of novel agonists for PPARs, may show improvement with the side effects and also increase the therapeutic value for cancer and other metabolic disorder diseases. Compared with normal human liver cells, lower expression or acitivity of PPARs is observed in hepatocellular carcinoma (HCC). In this study, we show that oroxyloside (OAG) is a new dual agonist of PPARγ/ɑ, and inhibits cell proliferation of HCC based on metabolic switch. Via both PPAR-dependent and PPAR-independent regulations on glycolipid metabolic enzymes, OAG shuts down the catabolism of glucose and promotes fatty acids oxidation to generate acetyl-CoA for TCA cycle and oxidative phosphorylation. The metabolic switch induced by OAG results in a marked increase of reactive oxygen species (ROS) levels, leading to rapid dephosphorylation of RB and cell-cycle arrest in G1 phase. Pyruvate dehydrogenase kinase 4 (PDK4) and β-Oxidation are required for the suppression of cell cycle progression by OAG. Together, our findings provide a new drug candidate and a viable therapeutic strategy for HCC based on metabolic reprogram.

Dual Targeting of CDK4/6 and BCL2 Pathways Augments Tumor Response in Estrogen Receptor-Positive Breast Cancer.

Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor-positive (ER+) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor. BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER+ breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER+ mouse mammary tumor model was used to study the effect of combination therapy on the immune system. Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G1-S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib-induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER+ mammary tumor model and extended tumor response when combined with anti-PD1 therapy. This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER+ breast cancer.

Tumor suppressor properties of the small C-terminal domain phosphatases in non-small cell lung cancer.

Non-Small Cell Lung Cancer (NSCLC) is responsible for the majority of deaths caused by cancer. Small C-terminal domain (CTD) phosphatases (SCP), CTDSP1, CTDSP2 and CTDSPL (CTDSPs) belong to SCP/CTDSP subfamily and are involved in many vital cellular processes and tumorigenesis. High similarity of their structures suggests similar functions. However their role in NSCLC remains insufficiently understood. For the first time we revealed the suppressor function of CTDSPs leading to a significant growth slowdown and senescence of A549 lung adenocarcinoma (ADC) cells in vitro. Their tumor-suppressive activity can be realized through increasing the proportion of the active form of Rb protein dephosphorylated at Ser807/811, Ser780, and Ser795 (P<0.05) thereby negatively regulating cancer cell proliferation. Moreover, we observed that a frequent (84%, 39/46) and highly concordant (Spearman’s rank correlation coefficient (rs) = 0.53–0.62, P≤0.01) down-regulation of CTDSPs and RB1 is characteristic of primary NSCLC samples (n=46). A clear difference in their mRNA levels was found between lung ADCs with and without lymph node metastases, but not in squamous cell carcinomas (SCCs) (P≤0.05). Based on The Cancer Genome Atlas (TCGA) data and the results obtained using the CrossHub tool, we suggest that the well-known oncogenic cluster miR-96/182/183 could be a common expression regulator of CTDSPs. Indeed, according to our qPCR, the expression of CTDSPs negatively correlates with these miRs, but positively correlates with their intronic miR-26a/b. Our results reflect functional association of CTDSP1, CTDSP2, and CTDSPL, expand knowledge about their suppressor properties through Rb dephosphorylation and provide new insights into the regulation of NSCLC growth.

RBTT-05. A SINGLE-ARM, OPEN-LABEL, PHASE II STUDY EVALUATING THE EFFICACY AND SAFETY OF ABEMACICLIB IN PATIENTS WITH RECURRENT OLIGODENDROGLIOMA: TRIALS IN PROGRESS

Abstract BACKGROUND Oligodendroglioma is a rare primary brain tumor characterized by mutation in the IDH1/IDH2 genes and codeletion of chromosomes 1p and 19q. Although median overall survival extends beyond 10 years for patients with oligodendroglioma who receive adjuvant radiation and alkylating chemotherapy, the disease uniformly relapses and has no other effective treatments. The CIC gene, which encodes a high-mobility group box transcriptional repressor, is mutated in about 70% of oligodendrogliomas and is associated with worse prognosis and overexpression of cyclin D1 (CCND1). Cyclin D1 assembles with the cyclin-dependent kinases CDK4 and CDK6 to cause phosphorylation of Rb protein and subsequent cell cycle progression from the G1 to S phase. Therapeutic inhibition of CDK4/6 activity, which leads to Rb dephosphorylation and cell cycle arrest, has revolutionized the care of breast cancer. The current phase 2 study will investigate the efficacy and safety of abemaciclib, a selective and potent small molecule inhibitor of CDK4 and CDK6 with significant brain-brain barrier penetration, in patients with 1p/19q codeleted oligodendroglioma that has recurred after standard radiation and alkylating chemotherapy. METHODS This protocol (NCT03969706) has a single-arm, open-label, single-stage phase 2 design. Subjects will be treated with abemaciclib 200mg tablet twice daily on 28-day cycles until disease progression or unacceptable toxicity. Tumor response will be assessed according to modified Response Assessment in Neuro-Oncology (mRANO) criteria. The primary objective of the study is to determine the efficacy of abemaciclib for recurrent oligodendroglioma. The primary endpoint is the progression-free survival status of the subject at 6 months after enrollment (PFS-6). The alternative hypothesis is that the proportion of subjects without an event at 6 months will be ³85%. We will test this hypothesis against a null hypothesis of PFS-6 = 50%. With enrollment of 10 planned subjects, the power to detect this difference is 82% (one-sided alpha = 0.05).

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.

Palbociclib, a selective CDK4/6 inhibitor, restricts cell survival and epithelial-mesenchymal transition in Panc-1 and MiaPaCa-2 pancreatic cancer cells.

The mortality rate of pancreatic cancer has close parallels to its incidence rate because of limited therapeutics and lack of effective prognosis. Despite various novel chemotherapeutics combinations, the 5-year survival rate is still under 5%. In the current study, we aimed to modulate the aberrantly activated PI3K/AKT pathway and epithelial-mesenchymal transition (EMT) signaling with the treatment of CDK4/6 inhibitor PD-0332991 (palbociclib) in Panc-1 and MiaPaCa-2 pancreatic cancer cells. It was found that PD-0332991 effectively reduced cell viability and proliferation dose-dependently within 24 hours. In addition, PD-0332991 induced cell cycle arrest at the G1 phase by downregulation of aberrant expression of CDK4/6 through the dephosphorylation of Rb in each cell lines. Although PD-0332991 treatment increased epithelial markers and decreased mesenchymal markers, the nuclear translocation of β-catenin was not prevented by PD-0332991 treatment, especially in MiaPaCa-2 cells. Effects of PD-0332991 on the regulation of PI3K/AKT signaling and its downstream targets such as GSK-3 were cell type-dependent. Although the activity of AKT was inhibited in both cell lines, the phosphorylation of GSK-3β at Ser9 increased only in Panc-1. In conclusion, PD-0332991 induced cell cycle arrest and reduced the cell viability of Panc-1 and MiaPaCa-2 cells. However, PD-0332991 differentially affects the regulation of the PI3K/AKT pathway and EMT process in cells due to its distinct influence on Rb and GSK-3/β-catenin signaling. Understanding the effect of PD-0332991 on the aberrantly activated signaling axis may put forward a new therapeutic strategy to reduce the cell viability and metastatic process of pancreatic cancer.

Mutations in the RAS pathway as potential precision medicine targets in treatment of rhabdomyosarcoma

Precision medicine strategies for treating rhabdomyosarcoma (RMS), a childhood malignancy, have not been developed. We examined the effect of CH5126766, a potent selective dual RAF/MEK inhibitor, on RMS cell lines. Among the eleven cell lines studied, one NRAS and two HRAS mutated cell lines were detected. CH5126766 inhibited the proliferation and growth in all of the RAS-mutated RMS cell lines, while it induced G1 cell cycle arrest in two of them. G1 cell cycle arrest was accompanied by p21 up-regulation and RB dephosphorylation. CH5126766 also suppressed the in vivo growth of RAS-mutated RMS tumor, and the mice showed improved survival. Thus, our results demonstrate that CH5126766 is an effective RAF/MEK inhibitor in RAS-mutated RMS. This study not only shows that in RMS, mutations in the RAS pathway can be a target for precision medicine, but also demonstrates that the evaluation of the gene mutation status is important in childhood malignancies.

17β-estradiol inhibits human umbilical vascular endothelial cell senescence by regulating autophagy via p53.

Vascular endothelial cell (VEC) senescence is an initiating factor in numerous cardiovascular diseases. Recent studies showed that 17β-estradiol (17β-E2), an estrogen with numerous biological activities such as inhibition of atherosclerosis, protects VECs from senescence. However, the effects of 17β-E2 on human umbilical VECs (HUVECs) remain unknown. This study investigated the anti-senescent effect of 17β-E2 on HUVECs and explored the underlying mechanism with respect to autophagy and p53 activity. First, rapamycin and 3-methyladenine were used to clarify the relationship between autophagy and senescence in HUVECs, and an inverse relationship was demonstrated. Next, the effect of 17β-E2 on H2O2-induced senescence of HUVECs was examined. Increased autophagy induced by 17β-E2 inhibited H2O2-induced senescence of HUVECs, increased cell viability, and maintained HUVEC morphology. 17β-E2 pre-treatment also decreased cell cycle arrest, decreased the dephosphorylation of Rb, decreased the production of ET-1, and increased the production of NO. Most importantly, 17β-E2 pre-treatment increased autophagy by activating p53 and its downstream effector p53-upregulated modulator of apoptosis (PUMA). Overall, our data indicate the critical role of autophagy in the anti-senescent effect of 17β-E2 on HUVECs.

Abstract 314: Evaluation of effective drug combinations of sonidegib and ribociclib for treatment of Sonic hedgehog medulloblastoma using mathematical models of the cell cycle and Hedgehog pathways

Abstract Sonic hedgehog (SHH) tumors account for roughly 25% of all pediatric medulloblastoma. Aberrant signaling of the SHH pathway, typically via Patched-1 (PTCH) mutation, results in cell proliferation and subsequent tumor formation. Smoothened (SMO) inhibitors such as sonidegib have been used clinically to combat tumor progression and have shown initial promise. However, many tumors acquire resistance to therapy. Combination therapy with an additional agent targeting another node in pathways affecting cell-cycle progression such as ribociclib, a cyclin D-dependent kinase (CDK) 4/6 inhibitor, may improve therapy and reduce tumor burden by delaying or preventing the development of resistance. Identification and evaluation of effective anti-cancer drug combinations, along with their schedule, sequence, and dosage are a major challenge due to the large number of possible combinations in addition to the cost and time involved in experimentally testing each possible permutation. Mathematical models can aid in the discovery of optimal drug combinations. A mathematical model of the SHH and Cyclin D / CDK / Rb pathways was designed to evaluate effective sequences and schedules of sonidegib and ribociclib for the treatment of SHH medulloblastoma. The model was parameterized using in vitro data from studies treating the NIH3T3 cell line with sonidegib and ribociclib singly or in combination. Flow cytometry analysis was used to quantify response to treatment by obtaining cell-cycle phase distributions for each drug as a single agent or in combination. Uncertainty and sensitivity analysis of the model parameters were evaluated via Latin Hypercube Sampling and Partial Rank Correlation Coefficient analysis to interpret the effects of amplification or deletion of nodes in the pathway, evaluate potential targetable nodes, and identify sensitive parameters that need robust quantification. Sensitivity analysis indicated that the formation of Cyclin D/CDK4/6 complexes and the dephosphorylation of Rb were significant nodes that should be included in the model. Uncertainty analysis indicated that the majority of the variability lies in the distribution of cells in G0/G1 and S phase. Simulations of single agent treatment predict a similar response between sonidegib and ribociclib, with co-treatment indicative of an additive effect. Optimal control theory methods are being applied to this model to help determine effective sequence, schedule, and dose of sonidegib and ribociclib to improve efficacy and reduce drug resistance in the treatment of SHH medulloblastoma. Citation Format: Zack Jones, Jessica Roberts, Haley Houke, Martine Roussel, Clinton Stewart, Carl Panetta. Evaluation of effective drug combinations of sonidegib and ribociclib for treatment of Sonic hedgehog medulloblastoma using mathematical models of the cell cycle and Hedgehog pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 314. doi:10.1158/1538-7445.AM2017-314

Enantiomerically pure β-dipeptide derivative induces anticancer activity against human hormone-refractory prostate cancer through both PI3K/Akt-dependent and -independent pathways.

The use of peptides that target cancer cells and induce anticancer activities through various mechanisms is developing as a potential anticancer strategy. KUD983, an enantiomerically pure β-dipeptide derivative, displays potent activity against hormone-refractory prostate cancer (HRPC) PC-3 and DU145 cells with submicromolar IC50. KUD983 induced G1 arrest of the cell cycle and subsequent apoptosis associated with down-regulation of several related proteins including cyclin D1, cyclin E and Cdk4, and the de-phosphorylation of RB. The levels of nuclear and total c-Myc protein, which could increase the expression of both cyclin D1 and cyclin E, were profoundly inhibited by KUD983. Furthermore, it inhibited PI3K/Akt and mTOR/p70S6K/4E-BP1 pathways, the key signaling in multiple cellular functions. The transient transfection of constitutively active myristylated Akt (myr-Akt) cDNA significantly rescued KUD983-induced caspase activation but did not blunt the inhibition of mTOR/p70S6K/4E-BP1 signaling cascade suggesting the presence of both Akt-dependent and -independent pathways. Moreover, KUD983-induced effect was enhanced with the down-regulation of anti-apoptotic Bcl-2 members (e.g., Bcl-2, and Mcl-1) and IAP family members (e.g., survivin). Notably, KUD983 induced autophagic cell death using confocal microscopic examination, tracking the level of conversion of LC3-I to LC3-II and flow cytometric detection of acidic vesicular organelles-positive cells. In conclusion, the data suggest that KUD983 is an anticancer β-dipeptide against HRPCs through the inhibition of cell proliferation and induction of apoptotic and autophagic cell death. The suppression of signaling pathways regulated by c-Myc, PI3K/Akt and mTOR/p70S6K/4E-BP1 and the collaboration with down-regulation of Mcl-1 and survivin may explain KUD983-induced anti-HRPC mechanism.

The Novel Cyclin-Dependent Kinase 4/6 Inhibitor Ribociclib (LEE011) Alone and in Dual-Targeting Approaches Demonstrates Antitumoral Efficacy in Neuroendocrine Tumors in vitro

Background/Aim: Cyclin-dependent kinases (CDKs) are crucial for cell cycle regulation, and alterations in the cell cycle are often observed in human cancer. CDK4/6 in particular orchestrates G1 phase progression and the G1/S transition. Here, we investigated the in vitro effects of the CDK4/6 inhibitor LEE011 in human neuroendocrine tumor cells. Methods: The human neuroendocrine tumor cell lines BON1, QGP1, NCI-H727 and GOT1 were treated with different concentrations of LEE011 alone and in combination with 5-fluorouracil and everolimus. Results: Cell viability decreased in a time- and dose-dependent manner in BON1, QGP1, and NCI-H727 cells upon LEE011 treatment, whereas GOT1 cells were treatment resistant. Treatment sensitivity towards LEE011 was associated with the high expression of cyclin D1 and Rb. LEE011 caused the dephosphorylation of Rb and a subsequent G1 phase cell cycle arrest. Combined treatment with LEE011 and 5-fluorouracil or everolimus showed a significant enhancement in the inhibition of cell viability when compared to single-substance treatments due to PI3K-Akt-mTOR and Ras-Raf-MEK-ERK pathway downregulation and cooperative downregulation of cell cycle components. However, LEE011 also exhibited antagonizing effects with 5-fluorouracil, protecting NET cells from DNA-damaging chemotherapy by blocking PARP cleavage and caspase-3/7 activity. Conclusions: Our data demonstrate that the CDK 4/6 inhibitor LEE011 exhibits promising anti-tumoral properties alone and in combination treatment approaches with 5-fluorouracil or everolimus in human neuroendocrine tumor cell lines.

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

Pin1 inhibits PP2A-mediated Rb dephosphorylation in regulation of cell cycle and S-phase DNA damage.

Inactivation of the retinoblastoma protein (Rb) has a key role in tumorigenesis. It is well established that Rb function is largely regulated by a dynamic balance of phosphorylation and dephosphorylation. Although much research has been done to understand the mechanisms and function of RB phosphorylation, the regulation of Rb dephosphorylation is still not well understood. In this study, we demonstrate that Pin1 has an important role in the regulation of Rb function in cell cycle progression and S-phase checkpoint upon DNA damage. We show that the Rb C-pocket directly binds to the Pin1 WW domain in vitro and in vivo, and that the phosphorylation of Rb C-pocket by G1/S Cyclin/Cyclin-dependent kinase complexes is critical for mediating this interaction. We further show that Rb-mediated cell cycle arrest and Rb-induced premature cellular senescence are effectively inhibited by Pin1 expression. In addition, DNA damage induces Rb dephosphorylation in a PP2A-dependent manner, and this process is inhibited by Pin1. Furthermore, the overexpression of Pin1 promotes Rb hyperphosphorylation upon S-phase DNA damage. Importantly, both the Pin1 WW domain and isomerase activity are required for its effect on S-phase checkpoint. Moreover, the overexpression of Pin1 is correlated with Rb hyperphosphorylation in breast cancer biopsies. These results indicate that Pin1 has a critical role in the modulation of Rb function by the regulation of Rb dephosphorylation, which may have an important pathological role in cancer development.

Abstract A06: NORE1A is a double-barreled Ras senescence effector linking Ras to p53 and Rb

Abstract In addition to driving growth and transformation, activated forms of Ras are potent inducers of oncogene induced senescence (OIS). OIS appears to be a major barrier that must be overcome to permit Ras driven tumorigenesis. The signaling pathways utilized by Ras to induce senescence, and how they are subverted during tumor development remain poorly characterized. NORE1A is a member of the RASSF family of tumor suppressors. It binds directly to activated Ras via the Ras effector domain and acts as a Ras death effector. Frequent loss of NORE1A expression is observed in many tumor types and hereditary genetic defects in NORE1A predispose carriers to cancer. We have found that NORE1A can connect Ras to the induction of p21CIP1 and cell cycle arrest. Therefore, we sought to determine if NORE1A might play a role in Ras induced senescence. We now show that NORE1A is a potent mediator of Ras induced senescence. Knockdown of NORE1A suppresses the ability of Ras to induce senescence in multiple cell systems and enhances Ras driven transformation. We have identified two novel signaling pathways activated by Ras/NORE1A. First, NORE1A forms a Ras regulated, endogenous complex with the kinase HIPK2. HIPK2 can phosphorylate p53 to induce apoptosis or recruit acetyltransferases to acetylate p53 to induce senescence signaling. NORE1A suppresses HIPK2 apoptotic post-translational modifications of p53 but activates pro-senescence post-translational modifications. NORE1A also binds and destabilizes mdm2 to enhance the stability of nuclear p53. Primary human tumors show a close correlation between the expression levels of NORE1A and acetylated p53. In addition to p53, we show that NORE1A also links Ras to the regulation of Retinoblastoma (Rb) protein. NORE1A promotes a potent, Ras dependent stabilization of the Rb protein. It also promotes dephosphorylation of RB, an activating event. Thus, NORE1A is a double-barreled Ras senescence effector which connects Ras to two of the most important senescence regulating tumor suppressors in the cell. Loss of NORE1A activity in tumors is usually due to epigenetic inactivation or aberrant protein degradation by calpains. Both of these mechanisms, in principal, may be subject to clinical intervention to restore NORE1A function. This may provide a novel approach to antagonizing Ras driven tumors. Citation Format: Howard Donninger, Diego Calvisi, Thibaut Barnoud, M. Lee Schmidt, Geoffrey J. Clark. NORE1A is a double-barreled Ras senescence effector linking Ras to p53 and Rb. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A06. doi: 10.1158/1557-3125.RASONC14-A06