Retinoblastoma Phosphorylation Research Articles

Antiproliferative effects of the CDK6 inhibitor PD0332991 and its effect on signaling networks in gastric cancer cells.

PD0332991 (palbociclib/Ibrance®) is a cyclin-dependent kinase (CDK)4/6 inhibitor that has recently been approved for the treatment of estrogen receptor-positive advanced breast cancer. The present study investigated the antiproliferative effects of PD0332991 on gastric cancer (GC) cells and the underlying molecular mechanisms. The activity of PD0332991 was tested in several GC cell lines, including AGS, KATO-III, NCI-N87 and HS746T. Growth inhibitory activity of PD0332991, alone or in combination with fluorouracil (5-FU), was measured by MTT assay. The effects of PD0332991 on cell cycle progression were analyzed by flow cytometry and western blotting. Protein pathway array and Ingenuity Pathway Analysis were used to identify signaling pathways that may mediate the antiproliferative effects of PD0332991. PD0332991 inhibited proliferation in a dose-dependent manner and enhanced the activity of 5-FU in all GC cell lines tested. Cells treated with PD0332991 exhibited cell cycle arrest in G1 phase of the cell cycle, whereas the number of cells in G2/M phase was decreased. PD0332991 also inhibited CDK6-specific phosphorylation of retinoblastoma on Ser780, reduced the expression of cyclin D1, and induced expression of p53 and p27. Furthermore, 31 proteins were identified, the expression of which was significantly altered following treatment with PD0332991 in at least three cell lines. Pathway analysis indicated that the altered proteins were frequently associated with cell death, cell cycle and the molecular mechanism of cancer. The results of the present study indicated that PD0332991 may inhibit cell proliferation via modulation of the cell cycle, and may affect numerous oncogenic signaling pathways. Therefore, PD0332991 may be considered effective for the treatment of GC.

Phase II trial of palbociclib in patients with advanced esophageal or gastric cancer.

68 Background: Dysregulation of the cell cycle is a hallmark of cancer. Progression through the G1/S transition requires phosphorylation of retinoblastoma (RB) by cyclin-dependent kinases 4 and 6 (CDK4/6), which are regulated by cyclins D and E. A positive feedback loop between apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP kinase pathway, and cyclin D1 has been shown to drive cell proliferation in gastric cancer. In addition, amplification of cyclin D loci and/or activating mutations in CDKs are frequent molecular aberrations in gastroesophageal malignancies. We hypothesized that palbociclib, a potent inhibitor of CDK4/6 would disrupt proliferative signaling, and arrest the growth of gastric cancer. We conducted a phase II trial of palbociclib in gastric and esophageal cancers as an initial test of efficacy. Methods: We screened 38 subjects with gastric, GE junction, or esophageal cancer for RB nuclear expression by immunohistochemistry, and 38/38 (100%) were positive. We enrolled 21 subjects, of whom 5 had gastric adenocarcinoma, 3 had GE junction adenocarcinoma, 8 had esophageal adenocarcinoma, and 5 had esophageal squamous cell carcinoma. Four of 19 subjects tested positive for CCND1 overexpression by FISH. Patients received 125mg daily of palbociclib for days 1-21 of 28-day cycles. Results: Subjects remained on treatment for a median of 1.7 months. By the initial 2-month assessment, 5 of 21 subjects had stable disease, and 16 subjects had progressive disease by imaging and/or clinical progression. No objective responses were seen. The maximum duration of therapy was 5.5 months in two subjects. One of these subjects had progressing HER2-amplified gastric adenocarcinoma, and continued concurrent trastuzumab with palbociclib, while the other had squamous cell carcinoma of the esophagus. Grade 3 or 4 cytopenias occurred in 9 of 21 subjects (43%), with neutropenia in 8 (38%), anemia in 4 (19%), and thrombocytopenia in 1 (5%). One subject discontinued therapy due to grade 4 thrombocytopenia with GI bleed. All other subjects discontinued therapy due to disease progression. Conclusions: Palbociclib has modest single-agent activity in gastroesophageal tumors despite universal RB expression. Clinical trial information: NCT01037790.

MELK is a novel therapeutic target in high-risk neuroblastoma.

Maternal embryonic leucine zipper kinase (MELK) is known to modulate intracellular signaling and control cellular processes. However, the role of MELK in oncogenesis is not well defined. In this study, using two microarray datasets of neuroblastoma (NB) patients, we identified that MELK expression is significantly correlated to poor overall survival, unfavorable prognosis, and high-risk status. We found that MELK is a direct transcription target of MYCN and MYC in NB, and MYCN increases MELK expression via direct promoter binding. Interestingly, knockdown of MELK expression significantly reduced the phosphorylation of target protein Retinoblastoma (pRb) and inhibited NB cell growth. Furthermore, pharmacological inhibition of MELK activity by small-molecule inhibitor OTSSP167 significantly inhibited cell proliferation, anchorage-independent colony formation, blocked cell cycle progression, and induced apoptosis in different NB cell lines including a drug-resistant cell line. Additionally, OTSSP167 suppressed NB tumor growth in an orthotopic xenograft mouse model. Overall, our data suggest that MELK is a novel therapeutic target for NB and its inhibitor OTSSP167 is a promising drug for further clinical development.

Recent advances of cyclin-dependent kinases as potential therapeutic targets in HR+/HER2- metastatic breast cancer: a focus on ribociclib.

In normal cell cycle progression, transition of G0/G1 phase to synthesis (S) phase for breast and other cells is regulated by association of cyclin D and cyclin-dependent kinases 4 and 6 (CDK4/6) that leads to phosphorylation of retinoblastoma (Rb) protein. Imbalance of this cyclin D-CDK4/6-inhibitors of CDK4/6-Rb phosphorylation pathway is associated with tumorigenesis of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2−) breast cancers. Despite effective first-line endocrine therapy, HR+/HER2− metastatic breast cancers remain still incurable. Currently, advances in understanding of cell cycle checkpoints are evolving as promising strategy to target in treatment of various types of cancers including breast cancer. Therapies that target this cell cycle machinery in HR+/HER2− breast cancers are getting approval by the US Food and Drug administration (FDA) including ribociclib (LEE011). Ribociclib got the first FDA approval in March 13, 2017, as an initial therapy for HR+/HER2− advanced or metastatic breast cancer in combination with an aromatase inhibitor. This review, therefore, addresses the role of selective CDK4/6 inhibitors in advanced or metastatic breast cancer with a specific focus on ribociclib. Some findings of clinical trials involving ribociclib found pivotal benefits of ribociclib in HR+/HER2− metastatic breast cancer in terms of prolonging progression-free survival and objective response rates. Daily dosage range of the drug for such benefits is 50–900 mg with common daily doses of 400 or 600 mg and 600 mg in early and advanced breast cancer therapies, respectively. Along with its therapeutic benefits, however, more incident but manageable dose-limiting grade 3 or 4 toxicities, primarily hematologic adverse events, are common in patients treated with ribociclib. Generally, there are several active clinical trials undergoing to investigate the clinical efficacy and toxicity profile of the drug in various cancerous conditions other than breast cancer and will likely benefit patients with other cancer types.

CDKN2B deletion is essential for pancreatic cancer development instead of unmeaningful co-deletion due to juxtaposition to CDKN2A

Pancreatic cancer is among the deadliest malignancies; however, the genetic events that lead to pancreatic carcinogenesis in adults remain unclear. In vivo models in which these genetic alterations occur in adult animals may more accurately reflect the features of human cancer. In this study, we demonstrate that inactivation of Cdkn2b (p15ink4b) is necessary for induction of pancreatic cancer by oncogenic KRASG12D expression and inactivation of Tp53 and Cdkn2a in adult mouse pancreatic ductal cells (P60 or older). KRASG12D overexpression in these cells activated transforming growth factor-β signaling and expression of CDKN2B, which, along with CDKN2A, led to cellular senescence and protected cells from KRAS-mediated transformation via inhibition of retinoblastoma phosphorylation. These results show a critical role of CDKN2B inactivation in pancreatic carcinogenesis, and provide a useful adult animal model by genetic engineering via lentiviral delivery.

Phase 1 safety, pharmacokinetic and pharmacodynamic study of the cyclin-dependent kinase inhibitor dinaciclib administered every three weeks in patients with advanced malignancies

Background:Dinaciclib is a potent inhibitor of cell cycle and transcriptional cyclin-dependent kinases. This Phase 1 study evaluated the safety, tolerability and pharmacokinetics of various dosing schedules of dinaciclib in advanced solid tumour patients and assessed pharmacodynamic and preliminary anti-tumour activity.Methods:In part 1, patients were enrolled in escalating cohorts of 2-h infusions administered once every 3 weeks, utilising an accelerated titration design until a recommended phase 2 dose (RP2D) was defined. In part 2, 8- and 24-h infusions were evaluated. Pharmacokinetic parameters were determined for all schedules. Pharmacodynamic effects were assessed with an ex vivo stimulated lymphocyte proliferation assay performed in whole blood.Effects of dinaciclib on retinoblastoma (Rb) phosphorylation and other CDK targets were evaluated in skin and tumour biopsies. In addition to tumour size, metabolic response was evaluated by 18F-fluorodeoxyglucose-positron emission tomography.Results:Sixty-one patients were enrolled to parts 1 and 2. The RP2Ds were 50, 7.4 and 10.4 mg m−2 as 2- 8- and 24-hour infusions, respectively. Dose-limiting toxicities included pancytopenia, neutropenic fever, elevated transaminases, hyperuricemia and hypotension. Pharmacokinetics demonstrated rapid distribution and a short plasma half-life. Dinaciclib suppressed proliferation of stimulated lymphocytes. In skin and tumour biopsies, dinaciclib reduced Rb phosphorylation at CDK2 phospho-sites and modulated expression of cyclin D1 and p53, suggestive of CDK9 inhibition. Although there were no RECIST responses, eight patients had prolonged stable disease and received between 6 and 30 cycles. Early metabolic responses occurred.Conclusions:Dinaciclib is tolerable at doses demonstrating target engagement in surrogate and tumour tissue.

Abstract 4160: Impact of oral selective estrogen receptor degrader RAD1901 in preclinical models of endocrine sensitive/resistant breast cancer

Abstract Aromatase inhibitors (AI) are the mainstay for treatment of postmenopausal estrogen receptor positive (ER+) primary breast cancer (BC). However, many patients relapse whilst retaining a functional ER. The selective ER degrader (SERD) fulvestrant has been evaluated as a potential 2nd or 3rd line therapy for patients who relapse on AI treatment. Although exhibiting promising potential, its low bioavailability has limited its use to combination therapy in metastatic patients. Here, we assessed the efficacy of the orally available SERD, RAD1901, in a panel of BC cells with varying genetic backgrounds modelling patients both sensitive and resistant to AI therapy (LTED). Fulvestrant was used in parallel to allow relative responses to be compared. Cell proliferation assays in 2D and spheroids the presence of 0.01nM 17β-estradiol showed a concentration-dependent decrease in proliferation in response to RAD1901 and fulvestrant. GI50 values for RAD1901 in general were 10-fold higher than fulvestrant but equated to doses that are clinically achievable for RAD1901. Most importantly, RAD1901 effectively suppressed proliferation of two LTED models harbouring naturally occurring ESR1 mutations, MCF7 LTEDY537C (GI50 5nM) and SUM44-LTEDY537S (GI50 100nM). GI50 values of RAD1901 and fulvestrant showed similar reduction of ER, progesterone receptor (PGR) and cyclin D1 together with decreased phosphorylation of retinoblastoma (RB), concordant with cell cycle arrest. Furthermore, chromatin immunoprecipitation (ChIP) for ER in response to RAD1901 or fulvestrant showed a 70% reduction in recruitment of ER to TFF1, GREB1 and PGR promoters and concomitant reduction in mRNA expression of these genes. In MCF7-Arom cells, combination of letrozole with RAD1901 or fulvestrant showed enhanced antiproliferative effect compared to letrozole alone. The addition of RAD1901 to CDK4/6 inhibitor palbociclib or abemaciclib demonstrated additivity compared with monotherapy. In addition, RAD1901 effectively inhibited growth of palbociclib-resistant MCF7 LTED cells.These preclinical findings highlight the potential utility of RAD1901 as a potent drug in the treatment of ER+ BC. Combined with its bioavailability profile, RAD1901 warrants clinical testing versus fulvestrant after relapse on an AI, either alone or in combination with a CDK4/6 inhibitor. Citation Format: Sunil Pancholi, Joanna Nikitorowicz-Buniak, Ricardo Ribas, Nikiana Simigdala, Fiona Garner, Teeru Bihani, Stephen R. Johnston, Mitch Dowsett, Lesley-Ann Martin. Impact of oral selective estrogen receptor degrader RAD1901 in preclinical models of endocrine sensitive/resistant breast cancer [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 4160. doi:10.1158/1538-7445.AM2017-4160

Preclinical in vitro and in vivo evaluation of antitumor activity of UD-017, a novel selective and orally available CDK7 inhibitor, in colorectal cancer.

e14085 Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for cancer drugs due to its dual roles, cell cycle regulation and gene transcription/RNA procession. We synthesized UD-017, a small molecule, highly selective CDK7 inhibitor with a novel chemotype. In this study, we characterize antitumor activity of UD-017 in vitro and in vivo, and try to elucidate underlying mechanisms by which CDK7 inhibition contributes to the antitumor efficacy in colorectal cancer cells. Methods: We examined CDK7 selectivity of UD-017 against the other CDKs and kinases. We evaluated the antitumor activity in a variety of human cancer cell lines including colorectal cancer cells. We investigated the mechanism of antitumor activity of UD-017 using a human colorectal cancer cell line, HCT-116. In vivo antitumor efficacy of UD-017 was assessed in HCT-116 xenograft models and patient derived xenograft (PDX) model. Results:UD-017 inhibited CDK7 enzyme (IC50 = 16 nM), which is at least 300-fold more selective against other CDKs. In a panel of 313 kinases assay, UD-017 inhibited CDK7 almost mono-specifically. UD-017 potently inhibited the growth of human cancer cells including the patient-derived colorectal cancer cells.In the mechanism study, UD-017 inhibited phosphorylation of CDK1, 2 and retinoblastoma, which was followed by cell-cycle arrest. Inhibition of both RNA polymerase II phosphorylation and c-Myc expression was observed followed by apoptosis induction. HCT-116 xenograft model and PDX model demonstrated the potent antitumor activity of UD-017 with dose-dependence by daily oral administration for 2 weeks. Furthermore, UD-017 showed the combination effect with 5-FU without further affecting the side effects of the chemotherapy. Conclusions:UD-017 is a potent and highly selective CDK7 inhibitor, and significantly inhibits the growth of human cancer cell lines with cell-cycle arrest and apoptosis induction. UD-017 showed potent and dose-dependent antitumor response in the HCT-116 xenograft model and PDX model. Further investigation towards clinical development is warranted.

The efficacy of the cyclin-dependent kinase 4/6 inhibitor in endometrial cancer.

BackgroundPD-0332991, the selective cyclin-dependent kinase 4/6 inhibitor palbociclib, causes cell cycle arrest by inhibiting phosphorylation of retinoblastoma (Rb) protein. The aim of this study was to evaluate the therapeutic potential of PD-0332991 in endometrial cancer.Methods and findingsFour human endometrial cancer cell lines, ECC, HEC1A, HEC108 and TEN, were treated with PD-0332991 and their function was evaluated. In vivo, the therapeutic efficacy was evaluated in a model of subcutaneous endometrial cancer. An immunohistochemical analysis was performed in 337 endometrial cancer specimens. A proliferation assay revealed that 2 of the 4 cell lines that expressed Rb were sensitive to PD-0332991 with an IC50 of 0.65 μM (HEC1A) and 0.58 μM (HEC108), respectively. Both cell lines had G0/G1 cell cycle arrest after treatment with PD-0332991 according to flow cytometry. In vivo, PD-0332991 had antitumoral efficacy with a reduction in the activity of Ki67 and phosphorylation of Rb. Immunohistochemical analyses revealed that the positive rate of Rb was 67.7%, however, there was no significant relationship between the expression levels of Rb and the tumor grade.ConclusionsPD-0332991 had therapeutic potential against endometrial cancer cell lines expressing Rb protein. Our immunohistochemical analysis revealed that approximately 70% of patients with endometrial cancer might have therapeutic indications for PD-0332991. Of note, the tumor grade had no impact on the indications for treatment.

Potential role of dietary fat‐ and obesity‐sensitive adipose PKCbeta signaling in pathophysiology of adipose dysfunction

Excess delivery of nutrients to adipose tissue in obesity results in an increase in adipose tissue mass, followed by an increase in stress signals, and thereby an altered production of proinflammatory adipokines, ultimately leading to the progression of chronic inflammation and development of insulin‐resistance. Although the features of chronic inflammation in obese adipose tissue are clearly defined, the signals and mechanisms that trigger chronic inflammation (origin or mechanism of initiation of inflammatory signaling) and maintenance are not well understood and remains a critical area of research. There is clear evidence that the inflammatory trigger is metabolic and a strong correlation between adipocyte size and macrophage infiltration has also been reported. Importantly, specialized metabolic cells (such as adipocytes) are the cells that sustain the insult and whose expansion begins the inflammatory program. Accordingly, adipocyte hypertrophy and local hypoxia are implicated in immune cell infiltration, cytokine secretion and disruption of different signaling cascades. Our recent study suggested that protein kinase C beta (PKC beta) might be a pivotal protein regulating triglyceride storage and could be a target for the pathogenesis of adipose dysfunction in response to nutritional changes. Accordingly, a systemic PKC beta deficiency protects mice from diet‐induced adiposity and development of insulin‐resistance. To determine how PKC beta deficiency exerts its effect, we determined high‐fat diet‐PKC beta interaction and the impact of PKC beta deficiency on relevant adipose signaling pathways and metabolism. Here, we report that dietary fat strongly increased adipose PKC beta expression in a time‐dependent manner. The induction was observed at 2–6 weeks, peaked at 10 weeks, and remained elevated for at least 16 weeks (the last time point measured). We and others have reported involvement of PKC beta in regulating phosphorylation of retinoblastoma, I kappa b kinase, insulin‐receptor substrate, and p66shc, as well as dictates autophagy levels, we therefore compared changes in phosphorylation of these proteins and autophagy levels in different adipose tissues of above mice. The time‐dependent changes were also compared using adipose tissues from PKC beta‐deficient mice fed same high‐fat diet for identical periods. The available results support the possibility that adipocyte PKC beta is a key sensor to detect dangerous dietary signal, whose activation initiates adipose inflammation by sequential events consisting of PKC beta‐induced changes, thus mediating the interface between metabolic input and inflammatory output in the expanding adipose tissue. This study opens the possibility that modulation of PKC beta activity in the adipose tissue may be an effective approach to attenuate adipose dysfunction.Support or Funding InformationHL079091

Dietary fat/cholesterol‐sensitive PKCbeta‐RB signaling: Potential role in diet‐induced NASH/HCC axis

Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis. Recent studies suggest that abnormal lipid metabolism may play an essential role in nonalcoholic steatohepatitis (NASH) development and oncogenesis of HCC. Excess dietary cholesterol intake has been shown to cause development of experimental NASH in different animal models, especially in the setting of a concurrent high‐fat diet. Despite knowledge that Western‐style high‐fat and high‐cholesterol diet is a risk factor in the development of NASH and subsequent progression to HCC, diet‐induced signaling changes implicated in NASH/HCC axis are not well understood. Understanding molecular mechanisms altered by diet is crucial for developing preventive and therapeutic strategies. We previously assessed whether metabolic adaptation to high‐fat/cholesterol diet is associated with changes in hepatic PKCβ expression and also studied the consequence of PKCβ deficiency on diet‐induced hepatic cholesterol homeostasis. We reported that diets enriched with high‐fat and high‐cholesterol (with or without bile acid), shown to produce NASH and HCC, induce hepatic PKCβ expression, and a systemic loss of PKCβ promotes hepatic cholesterol accumulation in response to such diets. These results support the possibility that PKCβ is a physiological transducer of dietary fat/cholesterol and plays a critical role in hepatic adaptation to such diets. Here, we sought to determine how PKCβ and diet functionally interact during tumorigenesis. We examined effects of dietary fat/cholesterol on the PKCβ phosphorylation of retinoblastoma (RB) protein and family members, as well as compared PKCβ expression in human HCC versus normal tissue. We show that diet‐induced hepatic PKCβ expression is accompanied by increased phosphorylation of specific residues of RB protein in a time‐dependent manner. Intriguingly, PKCβ−/− livers exhibited reduced phospho‐RB and RB protein levels, which ultimately resulted in increased transcription of RB gene. It is also accompanied by reduced RBL‐1 with no significant effect on RBL‐2 protein level. To investigate the potential effect of PKCβ deficiency on DNA content of preneoplastic livers, flow cytometric analysis was conducted on hepatocytes obtained from high‐fat/cholesterol diet‐fed WT and PKCβ−/− mice. We also report reduced expression of PKCβ in HCC compared to an adjacent non‐tumorous liver in human patients. These results raise an interesting possibility that diet‐induced PKCβ activation represents an important mediator in the functional wiring of cholesterol metabolism and tumorigenesis through modulating stability of cell cycle‐associated proteins. Identification of PKCβ as a cellular relay in diet‐mediated change in RB functions can define the signaling events that could participate in diet‐induced oncogenesis.Support or Funding InformationThe Ohio State University College of Medicine Research Funds

Feasibility of cotargeting cell cycle and androgen signaling in metastatic hormone-sensitive prostate cancer (mHSPC).

151 Background: Most patients (pts) with mHSPC treated with androgen deprivation therapy (ADT) will progress to castration resistance. Androgens drive PC cell proliferation via upregulation of cyclin D which complexes with the cyclin-dependent protein kinases 4/6 (CDK4/6) resulting in phosphorylation of retinoblastoma (Rb) tumor suppressor protein and G1/S progression. Alterations in this pathway contribute to progression to castration-resistance (CR). In preclinical PC models, palbociclib, a novel specific CDK4/ 6 inhibitor, blocked proliferation and promoted G1 arrest in an Rb and Cyclin D dependent manner. Rb is lost in <10% of localized PC, but >30% of CR disease. Rb loss in mHSPC is estimated to be 10-20%. Based on this preclinical data, we designed a randomized phase II clinical trial (NCT02059213) to test the hypothesis that palbociclib + ADT in newly metastatic Rb-positive PC pts will significantly increase ADT efficacy. Methods: mHSPC pts with Rb intact tumors based on metastatic biopsy are stratified by disease extent and randomized (1:2) to ADT or ADT+ palbociclib. Primary endpoint is confirmed prostate specific antigen (PSA) response (≤ 4 ng/mL) after 28 weeks of therapy. With 20 patients randomized to ADT and 40 to ADT + palbociclib there will be a 64.2% power to detect a 20% difference in proportions with a one-sided type I error of 0.10 using the mid p-value method of the Fisher’s exact test. Secondary endpoints: safety and tolerability, rate of undetectable PSA (≤ 0.2ng/mL), biochemical and clinical PFS, overall PSA and radiographic RR, therapeutic biomarkers (circulating DNA and tumor cells, tumor protein and transcriptome) and to establish a repository of mHSPC samples. Results: Since July 2014, 68 patients have been registered and underwent tumor metastatic disease biopsy (36 soft tissue, 32 bone). 60/68 (88%) had adequate tissue for Rb assessment. 58/60 (97%) tumors were Rb positive (IHC). To date, 58/60 patients have been randomized to ADT (19 patients) or ADT + palbociclib (39 patients). Conclusions: Biopsy driven studies are feasible in the mHSPC population. Rb expression was present in 58/60 (97%) patients. This study is ongoing. Support: Movember-PCF Challenge Award, Pfizer. Clinical trial information: NCT02059213.

Molecular targeting of VEGF/VEGFR signaling by the anti-VEGF monoclonal antibody BD0801 inhibits the growth and induces apoptosis of human hepatocellular carcinoma cells in vitro and in vivo

ABSTRACTHepatocellular carcinoma (HCC) is the third-leading cause of cancer-related deaths with 750,000 newly diagnosed cases each year. Surgery, radiotherapy, and chemotherapy constitute the main treatment modalities for HCC, but liver cirrhosis and damage often occur. Molecular targeted drugs have been recently developed to treat HCC. Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) autocrine signaling is closely related to the growth, progression, and metastasis of HCC, making the VEGF/VEGFR axis an ideal target for the development of molecular targeted agents. Here, we report the effects of the novel anti-VEGF humanized monoclonal antibody BD0801 on the growth of HCC cells in vitro and in vivo as well as the underlying mechanisms. BD0801 significantly inhibited the proliferation of HepG2, SMMC-7721, and Bel7402 cells in vitro, accompanied with an induction of apoptosis and cell cycle arrest at the G1 phase. BD0801 potently suppressed AKT, Erk1/2, and retinoblastoma (Rb) phosphorylation, while increasing p21 and decreasing cyclin D1 protein levels. BD0801 significantly inhibited growth in mouse tumor xenografts and induced cell apoptosis of HepG2 and SMMC-7721 tumor xenografts. Furthermore, BD0801 effectively reduced the vascular density and tumor tissue microvessel density (MVD). Similarly, BD0801 decreased AKT, Erk1/2, and Rb phosphorylation and cyclin D1 expression whereas it increased p21 protein expression in mouse HCC tumor xenografts. Importantly, BD0801 showed a better effect than Bevacizumab (Bev) on the inhibition of cell growth and induction of apoptosis in HCC cells in vitro and in vivo. These findings suggest that BD0801 is a potent anti-VEGF monoclonal antibody for the treatment of HCC.

Anticancer Effects of Sinocrassulosides VI/VII from Silene viscidula on HeLa Cells.

Natural products are becoming increasingly important in chemoprevention and for cancer therapy. Silene viscidula (S. viscidula), a traditional Chinese herb, has long been used as an anti-inflammatory and neuroleptic agent. However, the anticancer activity of S. viscidula has remained unclear. In this study, 16 compounds were extracted from S. viscidula. Among those compounds, sinocrassulosides VI/VII, an inseparable isomer mixture, possess the strongest inhibitory activity on HeLa cells with the IC50 value of 2.37 μM. Mechanism studies found that sinocrassulosides VI/VII downregulated the expression of cyclin D1 and decreased retinoblastoma (Rb) phosphorylation, which arrested HeLa cells in the G1 phase. Also, sinocrassulosides VI/VII could induce senescence via the upregulation of p16 and a significant increase of β-galactosidase (β-gal) staining. Our results suggest that sinocrassulosides VI/VII may be a new therapeutic potential agent for cervical cancer. In addition, we explored the structure-activity relationships of three groups of the configurational isomer with similar chemical structure from S. viscidula. We first demonstrated that the length of the ester chains linked to the carboxyl group of the glucuronic acid residue could affect the potent cytotoxicity. This finding will open new avenues for developing effective anticancer compounds by modifying the components derived from plants in nature.

Inhibition of Skp2 sensitizes lung cancer cells to paclitaxel.

S-phase kinase-associated protein 2 (Skp2) is an E3 ubiquitin ligase and plays an important role in the control of cell cycle progression. Skp2 is upregulated in several cancers, including lung cancers, but the role of Skp2 in the tumorigenesis and anticancer drug resistance in human lung cancer remains to be determined. We report here that Skp2 positively regulated mitotic arrest deficient 2 (MAD2) expression and that inhibition of Skp2 sensitizes human lung cancer cells to paclitaxel. Knockdown of Skp2 by small interfering RNA (siRNA) decreased Mad2 messenger RNA (mRNA) and protein levels in A549 and NCI-H1975 cells, accompanied with upregulation of p27 but decrease of the phosphorylation of retinoblastoma (Rb). In contrast, ectopic overexpression of Skp2 increased Mad2 mRNA and protein levels and phosphorylation of Rb, while it decreased p27. Pharmacological inhibition of CDK1/2 by flavopiridol or E2F1 with HLM006474 led to downregulation of Mad2 expression and prevented the increase of Mad2 expression by Skp2. Most importantly, pharmacological inhibition of Skp2 sensitized A549 and NCI-H1299 cells to paclitaxel. Our results demonstrated that SKP2 positively regulates the gene expression of MAD2 through p27-CDKs-E2F1 signaling pathway and that inhibition of Skp2 sensitizes A549 and NCI-H1299 cells to paclitaxel, suggesting that small molecule inhibitors of Skp2 are potential agents for the treatment of lung cancer with upregulation of Skp2.

An RB insensitive to CDK regulation

ABSTRACTThe N-term phosphorylation of Retinoblastoma (RB) by the p38 stress-activated protein kinase (SAPK) makes RB insensitive to cyclin-dependent kinase (CDK)-Cyclin inhibition, which enhances the transcriptional repression of E2F-driven promoters and delays tumor cell growth. This novel mechanism of RB regulation opens up a window for developing new cancer drug treatments for tumors harboring high CDK-Cyclin activity and a wild-type RB gene.

Multitarget Antileukemic Effects of Metformin in Myeloproliferative Neoplasm Cells: Inhibition of JAK2/STAT Signaling and Mitochondrial Activity

Background: The recurrent JAK2V617F gain-of-function mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). The lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying novel therapeutic strategies. Metformin is a biguanide that inhibits the mitochondrial complex I and metformin antineoplastic activity has been described in leukemia, lymphoma and multiple myeloma. More importantly, metformin antineoplastic effects appear to act selectively in cancer cells, since cytotoxicity was not observed for normal hematopoeitic cells. Considering that metformin has been widely used in clinical practice, preclinical studies in an attempt to determine its effects, alone or in combination with ruxolitinib, in MPN, may be of interest, since these findings have a translational potential. Objectives: To investigate the effects of metformin and/or ruxolitinib on cell signaling and cellular functions of JAK2V617F cells. Metformin and/or ruxolitinib effects were also evaluated on spontaneous erythroid colony formation from polycythemia vera (PV) patients. Materials and methods: HEL, SET2 (JAK2V617F) and Ba/F3 JAK2V617F cell lines treated or not with metformin (5 and 10 mM) and/or ruxolitinib (300 nM) were submitted for cell viability (MTT), clonogenicity (colony formation), cell cycle (flow cytometry), cell proliferation (Ki-67 staining), apoptosis (annexin-V/PI and caspase 3/PARP1 cleavage), PCR array for PI3K/AKT-related genes and oxygen consumption (high-resolution respirometry). For spontaneous erythroid colony formation assays, mononuclear cells from peripheral blood or bone marrow of PV patients (n=3) were cultured on methylcellulose supplied with hematopoietic growth factors except EPO, in the presence or not of metfomin (2.5 mM) and/or ruxolitnib (50 nM). Gene and protein expressions were evaluated by qPCR and Western blot. Statistical analyses were performed by ANOVA test or Student’s t-test. Results: In HEL, SET2 and Ba/F3 JAK2V617F cells, metformin or ruxoltinib treatment significantly reduced cell viability and clonogenicity (p<0.05). Ruxolitinib plus metformin significantly decreased cell viability and induced apoptosis when compared with monotherapy (p<0.05). Metformin and/or ruxolitinib delay cell cycle progression (p<0.05). In HEL cells, PCR-array identified 11, 14 and 17 genes modulated by metformin, ruxolitinib, and combined treatment, respectively. Reduced cyclin D1 (CCND1) and upregulated p27 (CDKN1B) were validated in HEL and SET2 cells (p<0.05). We also observed downregulation of retinoblastoma (RB) phosphorylation, a cyclin D1 target and a key cell cycle progression-related protein, in both JAK2V617F cell lines upon all treatment conditions. Ruxolitinib treatment reduced STAT3, STAT5, ERK1/2, 4EBP1 and P70S6K phosphorylation. Metformin also reduced activation of same proteins, but combined treatment presented more intense inhibition of ERK1/2 and P70S6K activation compared to metformin or ruxolitinib monotherapy. A strong reduction of cellular oxygen consumption supported by exogenous substrates in culture media (ROUTINE state), at the non-phosphorylating state (LEAK) and at the maximum respiratory capacity (ETS state) was observed in metformin- and metformin plus ruxolitinib-treated HEL, SET2 and Ba/F3 JAK2V617F cells (p<0.05). Ruxolitinib significantly reduced oxygen consumption at routine, leak and ETS states only in SET2 and Ba/F3 JAK2V617F cells and at LEAK state in HEL cells. In primary cells from PV patients, a reduction of 71±21%, 94±4%, 98±1% was observed on spontaneous erythroid colony formation upon metformin, ruxolitinib and metformin plus ruxolitinib treatment, respectively. Conclusion: Metformin exerts an anticancer activity and downregulates JAK2/STAT and MAPK signaling in JAK2V617F cells. PCR-array identified cyclin D1 and p27 as target of metformin and ruxolitinib, corroborating cell cycle and proliferation findings. Our exploratory study establishes novel molecular mechanisms of metformin and ruxolitinib action on JAK2V617F aberrant signaling and provides insights for development of alternative/complementary therapeutic strategies for MPN. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic methylation status of P16, MGMT and SPOCK2 in diffuse Large B cell lymphoma

Introduction: Epigenetic methylation has been implicated in the pathogenesis of diffuse large B cell lymphoma (DLBCL). This study investigated the methylation status of p16, MGMT and SPOCK2. Aberrantly methylated p16 and MGMT have been linked to DLBCL, but not SPOCK2. p16 inhibits cyclin-dependent kinase, which results in retinoblastoma phosphorylation and blockage of cell cycle at G1 phase. MGMT removes alkyl adduct at O 6 - guanine, thus preventing lethal cross-links. SPOCK2, an extracellular chondroitin and heparin sulfate proteoglycans, abolishes the inhibition of membrane-type 1-matrix metalloproteinase which might enhance the angiogenesis. The absence of SPOCK2 methylation was therefore hypothesized in the majority of cases in this study. Methods: Extracted DNA from 88 formalin-fixed paraffin-embedded (FFPE) tissues of DLBCL were subjected to bisulfite conversion followed by methylation-specific PCR (MSP) analysis for p16, MGMT and SPOCK2 methylation. p16 methylation was also quantified in 16 samples through pyrosequencing assay. Results: p16 methylation was observed in 65/88 (74%) samples by MSP. Pyrosequencing detected p16 methylation in all 16 samples ranging from 18% to 81%. MGMT methylation was detected in all 88 (100%) cases. Methylated SPOCK2 was found in 83 (94.3%) samples. There was a significant association between p16 methylation status with patients above 50 years of age (p= 0.04). Conclusions: These preliminary discoveries may serve as a good platform in order to gain a comprehensive overview on the epigenetics contribution in the pathogenesis of DLBCL. Pyrosequencing is a robust tool in detecting and quantifying methylation.

Abstract IA06: Co-Targeting cell cycle and androgen signaling to personalize therapy for hormone dependent prostate cancer

Abstract Prostate cancer (PC) is an androgen driven and dependent disease and androgen deprivation therapy (ADT) is the standard for patients with metastatic hormone sensitive disease. Despite a high response rate, most patients will progress to castration resistance. The expanding molecular knowledge highlights the biologic heterogeneity and adaptive capacity of PC, and provides the rationale for a preemptive multi-targeted therapeutic strategy. Compelling support for the latter strategy is provided by recent data from two randomized trials demonstrating an unprecedented impact on survival with the addition of docetaxel to ADT. Androgens drive proliferation of PC cells via upregulation of cyclin D which complexes with the cyclin-dependent protein kinases 4/6 (CDK4/6) resulting in phosphorylation of retinoblastoma (Rb) tumor suppressor protein and G1/S progression. Alterations in this pathway contribute to progression to castration-resistance. Palbociclib is a novel specific CDK4/ 6 inhibitor which was recently approved in combination with letrozole for postmenopausal women with ER +, HER2 negative advanced breast cancer. In preclinical prostate cancer models, palbociclib inhibited proliferation and promoted G1 arrest in an Rb and Cyclin D dependent manner. Current data indicate that wild-type Rb expression is present in in 95% vs 75% of patients with newly diagnosed vs metastatic castration resistant PC respectively. We estimate that 80-90% of early metastatic hormone-sensitive PC will harbor intact Rb. Based on the biologic and preclinical data, we developed a multicenter randomized phase II clinical trial (NCT02059213) to test the hypothesis that adding palbociclib to ADT in patients with newly metastatic Rb-positive PC will significantly increase the efficacy of ADT. Patients (n = 60) with new metastatic hormone sensitive PC and Rb intact tumors based on metastatic biopsy are stratified by disease extent and randomized (1:2) to ADT or ADT+ palbociclib. Primary endpoint is confirmed prostate specific antigen (PSA) response (&amp;lt; 4 ng/mL) after 28 weeks of therapy. With 20 patients randomized to ADT and 40 randomized to ADT plus palbociclib there will be a 64.2% power to detect a 20% difference in proportions with a one-sided type I error of 0.10 using the mid p-value method of the Fisher's exact test. Secondary endpoints: safety and tolerability of ADT + palbociclib, rate of undetectable PSA (&amp;lt; 0.2ng/mL), biochemical and clinical progression-free survival, overall PSA and radiographic response rates, assessment of biomarkers which predict therapy response (circulating DNA and tumor cells, tumor protein and transcriptome analysis) and to establish a repository of metastatic hormone sensitive prostate tumor samples. To date 39 patients have been registered and underwent tumor metastatic disease biopsy (soft tissue 26, bone 13). 2 patients were Rb negative and 5 had inadequate tissue. 32 patients have been randomized to ADT (11 patients) or ADT + palbociclib (21 patients). The study is in progress. Support: Movember-PCF Challenge Award, Pfizer. Citation Format: Maha Hussain, Phillip L. Palmbos1, Scott A. Tomlins1, Stephanie Daignault-Newton, Neeraj Agarwal, Przemyslaw Twardowski, Alicia K. Morgans, Emmanuel S. Antonarakis, Karen E. Knudsen, Felix Y. Feng. Co-Targeting cell cycle and androgen signaling to personalize therapy for hormone dependent prostate cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA06.

Cell-specific Kaiso (ZBTB33) Regulation of Cell Cycle through Cyclin D1 and Cyclin E1

The correlation between aberrant DNA methylation with cancer promotion and progression has prompted an interest in discerning the associated regulatory mechanisms. Kaiso (ZBTB33) is a specialized transcription factor that selectively recognizes methylated CpG-containing sites as well as a sequence-specific DNA target. Increasing reports link ZBTB33 overexpression and transcriptional activities with metastatic potential and poor prognosis in cancer, although there is little mechanistic insight into how cells harness ZBTB33 transcriptional capabilities to promote and progress disease. Here we report mechanistic details for how ZBTB33 mediates cell-specific cell cycle regulation. By utilizing ZBTB33 depletion and overexpression studies, it was determined that in HeLa cells ZBTB33 directly occupies the promoters of cyclin D1 and cyclin E1, inducing proliferation by promoting retinoblastoma phosphorylation and allowing for E2F transcriptional activity that accelerates G1- to S-phase transition. Conversely, in HEK293 cells ZBTB33 indirectly regulates cyclin E abundance resulting in reduced retinoblastoma phosphorylation, decreased E2F activity, and decelerated G1 transition. Thus, we identified a novel mechanism by which ZBTB33 mediates the cyclin D1/cyclin E1/RB1/E2F pathway, controlling passage through the G1 restriction point and accelerating cancer cell proliferation.