Rb Protein Levels Research Articles

CDK4/6 inhibitor ribociclib induces cardiotoxicity in dynamic engineered heart tissues, mitigated by E2F1 overexpression

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council CDK4/6 inhibitor Ribociclib induces cardiotoxicity in dynamic engineered heart tissues, mitigated by E2F1 overexpression Background Ribociclib, a novel chemotherapeutic agent for metastatic breast cancer, functions as a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor1. Its mechanism of action involves the CDK4/6-Rb-E2F1 pathway. Ribociclib use has very recently been associated with development of heart failure (HF)2, yet the causality of its cardiac effects remains to be explored. Aims This study aims to investigate ribociclib's cardiotoxic effects in dynamic human engineered heart tissues (dyn-EHTs), focusing on the CDK4/6-Rb-E2F1 axis. Methods and Results Dyn-EHTs were generated using human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) and treated with repeated doses of ribociclib (7 µM) to mimic clinical drug administration. Post-treatment, dyn-EHTs exhibited a 17.0% (p<0.001) increase in tissue dilatation, an 8.9% (p<0.001) decrease in systolic force generation, and a 22.1% (p<0.001) increase in systolic stress, indicating significant cardiac dysfunction. Additionally, an increase in phosphorylation levels of Rb protein (Ser249 and Thr252) was observed (FC=0.445, p=0.009). Overexpression of E2F1 in iPSCs resulted in a threefold increase in E2F1 protein expression and successfully mitigated the ribociclib-induced tissue dilatation (p=0.007), decreased systolic force generation (p=0.005), and increase in systolic stress (p=0.004) making their performance comparable to healthy controls, as shown by linear regression analysis. Conclusions Our study demonstrates that ribociclib induces significant cardiotoxic effects in dyn-EHTs and iPSC-CMs through the CDK4/6-Rb-E2F1 pathway. The prevention of these effects by E2F1 overexpression highlights the pathway's involvement in ribociclib's cardiotoxic profile. These findings emphasize the need for careful cardiac monitoring in patients treated with CDK4/6 inhibitors and suggest further research into specific mechanisms of cardiotoxicity in cancer therapeutics.Graphical Abstract

Sequential activation of E2F via Rb degradation and c-Myc drives resistance to CDK4/6 inhibitors in breast cancer

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) are key therapeutic agents in the management of metastatic hormone-receptor-positive breast cancer. However, the emergence of drug resistance limits their long-term efficacy. Here, we show that breast cancer cells develop CDK4/6i resistance via a sequential two-step process of E2F activation. This process entails retinoblastoma (Rb)-protein degradation, followed by c-Myc-mediated amplification of E2F transcriptional activity. CDK4/6i treatment halts cell proliferation in an Rb-dependent manner but dramatically reduces Rb-protein levels. However, this reduction in Rb levels insufficiently induces E2F activity. To develop CDK4/6i resistance, upregulation or activating mutations in mitogenic or hormone signaling are required to stabilize c-Myc levels, thereby augmenting E2F activity. Our analysis of pre-treatment tumor samples reveals a strong correlation between c-Myc levels, rather than Rb levels, and poor therapeutic outcomes after CDK4/6i treatment. Moreover, we propose that proteasome inhibitors can potentially reverse CDK4/6i resistance by restoring Rb levels.

Abstract 91: Classification of cancer cells based on unexpected variation in levels and dynamics of pRb, pERK, and pAKT in response to cell synchronization from nutrient deprivation

Abstract The tumor suppressor Rb and the RAS-RAF-MEK-ERK and PI3K-AKT-mTOR signaling cascades are often dysregulated in cancer. As insufficient vasculature in the tumor microenvironment (TME) leads to nutrient deficiency, cellular responses to nutrient deprivation is relevant to consider in the treatment of solid tumors. Here, we demonstrate that levels of pRb, pERK, and pAKT after cell synchronization via nutrient deprivation and after release via serum stimulation vary unexpectedly across a panel of nine cancer and two normal cell lines. From these results, we describe a novel classification of tumor cells defined by their behavior in nutrient-limiting conditions. Four colorectal, four lung, and one breast cancer cell line, and two normal lung fibroblasts were included in the panel. A range of KRAS, NRAS, BRAF, and PIK3CA mutations as well as wild-type cells were represented. In 6-well plates, 1E5-5E5 cells were plated per well in media containing 10% serum, incubated for 16-24 hours, then were nutrient-deprived in media containing 0% or 1% serum for 2, 4, 8, 12, 24, 48, 72, or 96 hours. Synchronization was released by serum stimulation with 20% serum-containing media for 24-48 hours. Cells were imaged, counted, and harvested, then protein levels of Rb, pRb, ERK, pERK, AKT, and pAKT were measured in cell lysates using a western blot. Nutrient deprivation slowed the proliferation of each cell line. Surprisingly, phosphorylation of Rb at Ser795 and Ser807/811 after nutrient deprivation varied across cell lines. pRb remained at baseline in MCF7 cells, decreased in RKO and H1299 cells, and increased in HCT116 cells. Despite this, levels of the pRb upstream regulator CDK6 decreased consistently across these cell lines, suggesting possible alternate regulation of pRb in MCF7 and HCT116 cells. Unanticipated variation in pERK levels after nutrient deprivation was also observed, including a dramatic increase in pERK upon starvation in HCT116 cells but not in others. Similar notable findings were made as far as pAKT. pAKT levels changed dynamically across time points in some cell lines, but consistently increased in some (MCF7) and decreased in others (RKO). Cell synchronization release via serum stimulation caused varying levels of pRb and pERK restoration. Our novel findings reveal a broad new classification of tumor cells defined by their behavior nutrient-limiting conditions. Such classification may guide the use of nutrient deprivation as a predictive biomarker for CDK, MEK, or mTOR inhibitors in solid tumors with nutrient-poor TMEs, as we are currently exploring. We are also evaluating the impact of nutrient deprivation on immune cell killing and cytokine secretion of specific classes of cancer cells that we define here, and are working to establish real-time cell cycle markers to visualize differential effects of serum deprivation and stimulation. Citation Format: Lindsey Carlsen, Wafik S. El-Deiry. Classification of cancer cells based on unexpected variation in levels and dynamics of pRb, pERK, and pAKT in response to cell synchronization from nutrient deprivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 91.

Acer mono Extract Inhibits Invasive Activities and G1/S Transition of HT1080 Fibrosarcoma Cells.

Acer mono is known to contain bioactive substances that exhibit beneficial effects in osteoporosis, gastric ulcers, hepatic damage, and pathologic angiogenesis. The current study aimed to investigate the effects of Acer mono extract on the invasive activities and cell-cycle progression of human fibrosarcoma cells. Cytotoxicity of Acer mono extract was assessed by MTT assay, in-vitro invasiveness of HT1080 fibrosarcoma cells was measured using matrigel assay, expression of invasion- and cell-cycle-related proteins was analyzed by western blot analysis, and that of E2F target genes was quantified using qRT-PCR. Acer mono extract did not show distinct cytotoxicity in the experimental concentrations used. Invasiveness of HT1080 fibrosarcoma cells and expression of cyclin D1 and CDK4 in them were significantly reduced in a dose-dependent manner after treatment with Acer mono extract. Acer mono extract showed inhibitory effects on the G1/S transition during cell-cycle progression; the active phosphorylated Rb protein level was decreased, and expression of E2F target genes was downregulated by the Acer mono extract. Our data collectively demonstrated that Acer mono extract exerts inhibitory effects on the invasiveness and cell-cycle progression of HT1080 human fibrosarcoma cells.

An aryl hydrocarbon receptor agonist suppresses the growth of human umbilical vein endothelial cells in vitro: Potent effect with polyunsaturated fatty acids.

Human umbilical vein endothelial cells (HUVECs) are a pivotal component of the hematopoietic microenvironment linked to the modulation of the immune response, inflammation and carcinogenesis. HUVEC expresses the aryl hydrocarbon receptor (AHR), which regulates gene expression by binding to the xenobiotic-responsive element. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent agonist for AHR signalling. Treatment with TCDD (0.1-100nmol/L) was found to suppress the proliferation and to stimulate the death of HUVEC. TCDD's effects were abolished by culturing with CH223191, an inhibitor of AHR signalling. Mechanistically, TCDD treatment increased the protein levels of cell growth suppressors, including p53, Rb, p21 and regucalcin, and caspase-3 implicated in apoptotic cell death, and decreased the levels of Stat3, mitogen-activated protein kinase (MAPK/Erk1/2) and phospho-MAPK/Erk1/2. Treatment with polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid, eicosapentaenoic acid and arachidonic acid, suppressed the proliferation and stimulated the death of HUVEC in vitro, and decreased the levels of Stat3, MAPK/Erk1/2 and phospho-MAPK/Erk1/2 and increased caspase-3. Notably, the effects of TCDD in suppressing proliferation and stimulating death of HUVEC were modulated by coculturing with PUFAs. These effects were reversed by treatment with CH223191, an inhibitor of AHR. Treatment with both TCDD and PUFAs collaboratively enhanced the levels of AHR, CYP1A1, p53, p21, Rb and regucalcin. Moreover, TCDD suppressed migration with wound healing of HUVEC. Notably, the combination of TCDD and PUFAs revealed potent suppressive effects on angiogenesis of HUVEC, potentially related to disorders of the stromal microenvironment.

Abstract A48: YB-1-based oncolytic virotherapy in combination with CDK4/6-inhibitors against Ewing sarcoma

Abstract Oncolytic virotherapy is associated with immunogenic cell death and antitumor inflammatory immune responses. Thereby, a local as well as systemic immunosuppressive microenvironment might be abrogated, resulting in efficient phagocytic and antigen-presenting properties. Consecutively, priming of naive T cells against a multitude of de-repressed tumor and viral antigens is claimed to elicit strong antitumor immune responses accompanied by tumor-infiltrating T cells, especially in combination with immune checkpoint blockade. These features might be beneficial for patients with relapsed and metastatic Ewing sarcoma, who have poor prognosis and lack innovative therapy concepts, and whose tumor biopsies are characterized by low or absent T-cell infiltration. Here, we explore the efficacy of the oncolytic YB-1-selective adenovirus XVir-N-31 in combination with the CDK4/6-inhibitor abemaciclib (LY2835219) in established Ewing sarcoma cell lines. We demonstrate enhanced viral replication, particle formation, and oncolysis when combined with abemaciclib in vitro. Priming of tumor cells with abemaciclib before viral infection leads to cell cycle arrest in G1 phase, with consecutive decrease in Rb, pRb, and E2F1 protein levels. Interestingly, in the Rb-mutated hence CDK4/6-inhibitor-resistant cell line SK-N-MC, the combination with abemaciclib does not result in enhanced viral replication oor oncolysis. However, the same effects as for CDK4/6-inhibitor sensitive cell lines can be induced by siRNA-mediated E2F1 knockdown, indicating the crucial role of E2F1 as a repressor for initiation of viral replication. In summary, our results further support the hypothesis of E2F1 being a repressor of adenoviral replication (Holm et al., manuscript in preparation) and suggest the use of CDK4/6-inhibitors to boost oncolytic adenoviral efficacy. Further in vivo confirmation is planned in order to prepare a clinical phase I study of XVir-N-31 in combination with a CDK4/6-inhibitor and immune checkpoint blockade for treatment-refractory pediatric sarcoma patients. Citation Format: Sebastian J. Schober, Uwe Thiel, Melanie Thiede, Maximilian Ehrenfeld, Roman Nawroth, Guenther HS Richter, Stefan E.G. Burdach, Per Sonne Holm. YB-1-based oncolytic virotherapy in combination with CDK4/6-inhibitors against Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A48.

Caveolin-1 Scaffolding Domain Peptide Regulates Colon Endothelial Cell Survival through JNK Pathway.

It has been reported that pathological angiogenesis contributes to both experimental colitis and inflammatory bowel disease. Recently, we demonstrated that endothelial caveolin-1 plays a key role in the pathological angiogenesis of dextran sodium sulfate (DSS) colitis. However, the molecular mechanism of caveolin-1 regulation of endothelial function is unknown. In this study, we examined how the antennapedia- (AP-) conjugated caveolin-1 scaffolding domain (AP-Cav) modulates vascular endothelial growth factor- (VEGF-) dependent colon endothelial cell angiogenic responses, as seen during colitis. We used mouse colon endothelial cells and found that AP-Cav significantly inhibited VEGF-mediated bromodeoxyuridine (BrdU) incorporation into colon microvascular endothelial cells. AP-Cav significantly blunted VEGF-dependent extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation at 10 minutes and 2 hours after stimulation, compared with the AP control peptide. AP-Cav + VEGF-A treatment also significantly increased c-Jun N-terminal kinase (JNK) phosphorylation at 2 hours. AP-Cav + VEGF-A treatment significantly downregulated retinoblastoma (Rb) protein levels, upregulated cleaved caspase-3 protein levels at 4 hours, and induced apoptosis. Thus, our study suggests that disruption of endothelial caveolin-1 function via the AP-Cav diverts VEGF signaling responses away from endothelial cell proliferation and toward apoptosis through the inhibition of mitogen-activated protein (MAP) kinase signaling and the induction of JNK-associated apoptosis.

Abstract P6-03-09: Role of IL-6 in promoting endocrine therapy and palbociclib resistance estrogen receptor positive breast cancer cells

Abstract Background: CDK4/6 inhibitors combined with endocrine therapy (ET) are mainstay to treat metastatic estrogen receptor (ER) positive patients. Yet, almost 60% develop resistance to CDK4/6 inhibition within 2 years of initial treatment. An ongoing clinical challenge has thus been identifying biomarkers of response to predict patients that will either respond or not respond to palbociclib. Further, there is an unmet need to identify actionable targets for patients that have progressed on CDK4/6 blockade regimens. Currently, the only biomarker being used to identify patients for anti-CDK4/6 therapy is estrogen receptor (ER) by IHC. The goal of our study was thus to identify the therapeutic vulnerabilities of CDK4/6 inhibitor resistance cells and identify key markers that can longitudinally correlate with development of resistance. Methods: We have developed MCF7 and T47D resistant cells by treating them with increasing doses (1.2, 2.4, 3.6, and 4.8μΜ) of palbociclib over a 6-month period. At each dose, resistant cells were harvested to observe dose dependent changes that occur as the cells acquire resistance to palbociclib. At endpoint i.e. 4.8μM, multi-omics approach was used to compare sensitive vs. resistant cells. Results: Our multi-omics data indicates that palbociclib resistant cells show i) Upregulation of the STAT3-IL6 pathway ii) Loss of ERα and iii) Loss RB protein levels. Primarily, resistant cells adapt to palbociclib treatment by a dose dependent upregulation of IL6. Further, we observe that induction of IL-6 is an early event which also correlates with senescence-associated secretory phenotype (SASP) signature in resistant cells, predicted to be induced with CDK4/6 inhibition. Importantly, time-course studies using 1μM palbociclib indicates that IL-6 is needed in overcome SASP and promote proliferation in cells adapting and eventually developing resistance to CDK4/6 inhibition. Lastly, we subjected MCF7 and T47D parental cells, which are sensitive to endocrine therapy and palbociclib, with exogenous IL-6 and IL-6 receptor (IL-6R). Treatment with IL-6/IL-6R lead to the downregulation of ER and RB protein levels. IL-6 treated parental cells are 5-fold more resistant to tamoxifen and 7-fold more resistant to fulvestrant compared to parental cells not treated with IL-6. Additionally, dose response studies with palbociclib showed parental cells treated with IL-6 are 5-fold more resistant to palbociclib treatment compared to the cells cultured in the absence of IL-6. Conclusions: Collectively, our data suggests that resistance to palbociclib results in a cascade of events initiating with induction of senescence, leading to sustained upregulation of IL-6 which leads to the promotion of an aggressive tumor growth, accompanied by resistance to endocrine therapy. Our ongoing studies are geared towards delineating the role of each step of CDK4/6 resistance and determining if IL-6 is required to maintain CDK4/6 inhibition resistance. Our long-term goal is to correlate provide the pre-clinical rationale for using IL-6 as a predictive biomarker in patients receiving CDK4/6 inhibitor based therapies. Citation Format: Nicole M Kettner, Tuyen Bui, Xian Chen, Kelly K Hunt, Debu Tripathy, Khandan Keyomarsi. Role of IL-6 in promoting endocrine therapy and palbociclib resistance estrogen receptor positive breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-09.

Stress-Induced Premature Senescence Promotes Proliferation by Activating the SENEX and p16INK4a/Retinoblastoma (Rb) Pathway in Diffuse Large B-Cell Lymphoma

Objective:Cellular senescence has been thought to be an important barrier to tumor formation. Recent studies have shown that stress-induced premature senescence (SIPS) can promote partial tumor invasion, but how SIPS affects diffuse large B-cell lymphoma (DLBCL) remains inconclusive. This study aimed to address that issue.Materials and Methods:The immunophenotype of the LY8 cell line was measured with flow cytometry. SIPS induced by tert-butyl hydroperoxide (tBHP) was detected by senescence β-galactosidase staining. Cell proliferation was analyzed with CCK8 and expression levels of ARHGAP18 (SENEX gene-encoding protein), p16/p21, and Rb/pRb were measured with western blot. LY8 cells were transfected with SENEX-SiRNA/NC and verified by western blot.Results:Our results suggested that the immunophenotype of the LY8 cell line is CD19-, CD20-, and CD10-positive and the immunoglobulin light chain is the kappa type. The cellular senescence model of DLBCL could be successfully induced by 30 μM tBHP. ARHGAP18, p21, p16, and Rb protein levels were significantly increased but the level of pRb expression was decreased in the SIPS group compared with other groups. Meanwhile, the proliferation rate was increased in the SIPS group more than other tBHP groups. Furthermore, the expressions of p21 and p16 were significantly decreased in the SENEX-SiRNA group compared with the negative control group.Conclusion:SIPS formation activates ARHGAP18 and the p16/Rb pathway and promotes DLBCL cell proliferation. Furthermore, SENEX activates the p16 pathway in DLBCL. SIPS promotes proliferation by activating SENEX and the p16/Rb pathway in DLBCL. SENEX-related SIPS may serve as an important target for relapsed/refractory DLBCL therapy.

Abstract 4417: Synergy of cyclin dependentkinase 4/6 inhibitors with cytotoxic chemotherapy in cholangiocarcinoma

Abstract Purpose: Cholangiocarcinoma (CCA) is a highly lethal malignancy that often presents at advanced stages. Gemcitabine/cisplatin (GP) represents the standard-of-care but provides only a modest response rate and survival benefit. Loss of the CDK4/6 inhibitor CDKN2A is a prevalent genetic event in CCA, thus CDK4/6 inhibitors, FDA-approved in some breast cancer regimens, are hypothesized to have a role in treating CCA. Clinical challenges to target cell cycle dysregulation and exploit cytotoxic chemotherapy include predictive biomarkers for selecting patients most likely to respond. We hypothesized autophagy is involved in therapeutic resistance to CDK4/6 inhibitors, and plays a role in the activity of GP in CCA. Thus, we investigated CDK4/6 inhibitors as monotherapy, and combined with GP in CCA, measuring multiple readouts pertaining to drug synergy, putative pathway biomarkers and autophagy. Methods: The activity of 3 FDA approved CDK4/6 inhibitors, palbociclib, ribociclib and abemaciclib, as monotherapy (N=24), and combined with GP (N=10), was assessed in CCA cell lines by measuring relative cell number with Cell TiterGlo, and apoptosis with Annexin/PI and cleaved caspase3 quantification via flow cytometry. To characterize RB status for biomarker potential, protein levels of CCND1, CDK4, CDK6, RB, and CDKN2A were measured by western blot. Autophagy was measured using an autophagy flux assay, LysoTracker confocal staining, and by quantifying protein levels of LC3B, p62 and Beclin1A. Results: 22/24 (92%) of CCA cell lines were found to have at least one defect likely relevant to CDK4/6 inhibition: CDKN2A (92%), Rb1 (63%), CDK4 (23%) and cyclin D1 (32%). Protein levels of CDKN2A and RB1 showed moderate correlation with CDK4/6 inhibitor activity. Abemaciclib was the most potent CDK inhibitor in vitro (IC50≤0.5uM; palbociclib IC50>1uM; ribociclib IC50>5uM). Combined abemaciclib and GP showed higher cytotoxicity and synergy at lower doses compared to chemotherapy alone or single-agent abemaciclib. Compared to CDK4/6 inhibitors or chemotherapy alone, combination therapy enhanced growth inhibition, cell cycle arrest and caspase-dependent apoptotic cell death. Abemaciclib triggered autophagy mediated by significant increases in reactive oxygen species (ROS). Combination therapy reduced ROS production and thus autophagy, as seen by reduced autophagic flux, reduced LysoTracker mean fluorescence intensity, a decrease in the expression of LC3B-II, Beclin1A and an increase in p62. Conclusion: These results suggest CDK4/6 inhibitors interact synergistically with GP in CCA and mechanistically disrupt autophagy. This suggests potential to further explore CDKN2A and RB as biomarkers for preclinical sensitivity in CCA. Pre-clinical data supports ongoing clinical studies of this concept, as well as planned clinical studies of GP and alternative CDK4/6 inhibitors. Citation Format: Mansi Arora, James Bogenberger, Nagalo Bolni, Yumei Zhou, Latha Kannan, Jan Egan, Raquel Yokoda, Daniel H. Ahn, Mitesh J. Borad. Synergy of cyclin dependentkinase 4/6 inhibitors with cytotoxic chemotherapy in cholangiocarcinoma [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 4417.

Preclinical characterization of SHR6390, a novel CDK 4/6 inhibitor, in vitro and in human tumor xenograft models

Inhibition of the cyclin‐dependent kinase (CDK) 4/6‐retinoblastoma (RB) pathway is an effective therapeutic strategy against cancer. Here, we performed a preclinical investigation of the antitumor activity of SHR6390, a novel CDK4/6 inhibitor. SHR6390 exhibited potent antiproliferative activity against a wide range of human RB‐positive tumor cells in vitro, and exclusively induced G1 arrest as well as cellular senescence, with a concomitant reduction in the levels of Ser780‐phosphorylated RB protein. Compared with the well‐known CDK4/6 inhibitor palbociclib, orally administered SHR6390 led to equivalent or improved tumor efficacy against a panel of carcinoma xenografts, and produced marked tumor regression in some models, in association with sustained target inhibition in tumor tissues. Furthermore, SHR6390 overcame resistance to endocrine therapy and HER2‐targeting antibody in ER‐positive and HER2‐positive breast cancer, respectively. Moreover, SHR6390 combined with endocrine therapy exerted remarkable synergistic antitumor activity in ER‐positive breast cancer. Taken together, our findings indicate that SHR6390 is a novel CDK4/6 inhibitor with favorable pharmaceutical properties for use as an anticancer agent.

Stress activated p38 MAPK regulates cell cycle via AP-1 factors in areca extract exposed human lung epithelial cells.

Areca nut chewing habits are associated with several oral manifestations like leukoplakia, submucous fibrosis and oral squamous cell carcinoma. Although numerous evidence on areca toxicity is known but the mechanistic pathway of disease causation is to be studied. Aqueous areca nut extract treated A549 cells showed reduced cell viability by 48h with IC50 value of 0.50%. The toxic nature of areca nut induced the production of reactive oxygen species with decreased anti-oxidant glutathione S transferase levels lead to altered redox homeostasis. PCR studies showed decreased mRNA levels of Jun and Fos AP-1 subunits on extract treatment by 48h. The protein levels of PCNA, CDK4, RB, p53, c-Jun and c-Fos were found to be downregulated with upregulated CDK inhibitor p21 on extract treatment as compared to control. Results of FACS analysis further confirm G1/S phase cell cycle arrest on areca nut extract exposure. The regulation of downstream AP-1 subunits by MAPKs was studied by using specific inhibitors of ERK, JNK and p38 along with areca nut extract. Results showed the redox activation of MAP kinases down regulated the mRNA levels of AP-1 subunits in aqueous areca nut extract treated cells. Hence the present study aids in elucidating the role of MAP kinases in regulating the AP-1 subunits and their implications on target genes that are involved regulation of various cellular processes. Further, it would help in understanding the mechanistic aspects of the diseased state which may facilitate in designing of new therapeutic modalities that could help in cancer management.

Blockage of glutamine-dependent anaplerosis affects mTORC1/2 activity and ultimately leads to cellular senescence-like response.

ABSTRACTThe purpose of study was to explore the role of glutamine-dependent anaplerosis in cell fate determination (proliferation and senescence) and the potential associated mechanism by employing a pharmacological inhibitor of glutamine-dependent anaplerosis, amino-oxyacetate (AOA). Using the WI38 normal human embryonic fibroblast cell line, we found that exposure to AOA induced mTORC1 inactivation−mTORC2 activation (within day 1), cell cycle arrest (day 2–6) and cellular senescence (day 4–6). These AOA effects were blocked by concomitantly providing anaplerotic factors [α-ketoglutarate (αKG), pyruvate or oxaloacetate], and not affected by ROS scavenger N-acetyl-cysteine (NAC). Moreover, AOA-induced cellular senescence in WI38 cells is associated with elevated protein levels of p53, p21CIP1 and p16INK4A and decreased Rb protein level, which was blocked by αKG supplementation. In p16INK4A-deficient U2OS human osteosarcoma cells and p16INK4A-knockdown WI38 cells, AOA exposure also induced similar effects on cell proliferation, and protein level of P-Rb-S807/811 and Rb. Interestingly, no AOA induction of cellular senescence was observed in U2OS cells, yet was still seen in p16INK4A-knockdown WI38 cells accompanied by the presence of p16 antibody-reactive p12. In summary, we disclose that glutamine-dependent anaplerosis is essential to cell growth and closely associated with mTORC1 activation and mTORC2 inactivation, and impedes cellular senescence particularly associated with p16INK4A.

Overcoming palbociclib resistance by combined treatment with PI3K/AKT/mTOR inhibitors in mesothelioma cells

Carcinogenesis of malignant pleural mesothelioma (MPM) is strictly associated with chronic exposure of mesothelial cells from the pleura to asbestos fibers and MPM incidence is expected to peak in the next years. Extensive genome analyses of patient-derived MPM tumors revealed that the most frequent mutational event involves the inactivation of the CDKN2A gene that encodes for the cell cycle inhibitors p16$^{\rm INK4a}$ and p14$^{\rm ARF}$ with consequent constitutive activation of CDK4/6 - cyclin D complexes. Therefore, inhibition of the latter complexes may represent a new option for the treatment of MPM patients. However, despite the efficacy of the specific CDK4/6 inhibitor palbociclib in blocking MPM cell proliferation, acquired resistance inevitably occurs. Herein, palbocilib-resistant clones isolated after stepwise exposure of MSTO-211H cells to gradually increasing drug concentrations, showed a reduction in Rb protein levels as well as in the cell cycle inhibitor p21$^{\rm waf1}$, accompanied by increased phosphorylation of AKT and p70S6K. Simultaneous treatment of resistant clones with both palbociclib and specific PI3K/AKT/mTOR inhibitors produced an additive effect in terms of reduction in cell growth, without any signs of senescence but with increased cell death. In MSTO-211H sensitive cells, this combination drug treatment significantly delayed the adaptation to palbociclib, suggesting that this treatment approach may prevent or at least retard the emergence of palbociclib resistance. Collectively, these results suggest that the combination of palbociclib with PI3K/AKT/mTOR inhibitors may overcome the acquisition of resistance to palbociclib treatment and could represent a potential therapeutic approach to treat cancers with acquired resistance to CDK4/6 inhibitors in the presence of activation of the AKT/mTOR signaling pathway.

2,3,7,8‑Tetrachlorodibenzo‑p‑dioxin suppresses the growth of human liver cancer HepG2 cells in vitro: Involvement of cell signaling factors.

The aryl hydrocarbon receptor (AHR) is transcriptionally active in the form of a heterodimer with the AHR nuclear translocator, which then binds to the xenobiotic responsive element. AHR was originally discovered via its ligand, the polychlorinated hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we investigated whether TCDD regulates the growth of human liver cancer HepG2 cells in vitro. TCDD (0.1–100 nM) was found to exert suppressive effects on the colony formation and proliferation of HepG2 cells, and stimulatory effects on the death of HepG2 cells when the cells reached subconfluence. The effects of TCDD on the HepG2 cells were abolished by culture with CH223191, an inhibitor of AHR signaling. The effects of TCDD were dependent on the concentration of serum, which contains various signaling factors. The effects of TCDD were not potentiated by culture with tumor necrosis factor-α, which activates the signaling of nuclear factor-κB (NF-κB). The results of western blot analysis revealed that TCDD increased the protein levels of p53, Rb, p21, and regucalcin, which are suppressors of the growth of tumor cells. Moreover, TCDD enhanced the NF-κB p65, β-catenin, signal transducer and activator of transcription 3 (STAT3), Ras and Akt levels. Thus, the findings of this study indicate that TCDD may suppress liver cancer cell growth through various signaling pathways, mediated by AHR and its-related co-factors. Of note, the effects of TCDD were found to be potentiated by gemcitabine, which induces nuclear DNA damage in cancer cells, suggesting that their combined use may have potential as a suppressor of tumor cell growth.

Protein kinases orchestrate cell cycle regulators in differentiating BeWo choriocarcinoma cells.

Choriocarcinoma, a trophoblastic neoplasia, occurs in women as an incidence of abnormal pregnancy. BeWo choriocarcinoma cells derived from the abnormal placentation are a suitable model system to study the factors associated with differentiation, invasion and other cellular events as an alternative to clinical samples. Many protein kinases orchestrate the complex events of cell cycle and in case of malignancy such regulators are found to be mutated. In the present study, BeWo cells treated with forskolin (Fo) and phorbol 12-myristate 13-acetate (PMA) were used to study the role of PKA (protein kinase A) and PKC (protein kinase C), respectively, on the expression pattern of differentiation-related genes, membrane markers, PKC isoforms and cell cycle regulators. The effect of Fo and PMA on the cell proliferation was assessed. Progressive induction of alkaline phosphatase level and formation of multinucleated differentiated cells were observed in the cells treated with Fo. Exposure of cells to Fo and PMA induced the mRNA transcripts of α-hCG, β-hCG and endoglin and down-regulates E-cadherin at mRNA and protein levels. Synergistic levels of both up- and down-regulated genes/proteins were observed when cells were treated with the combination of Fo and PMA. The mRNA levels of cyclin D1, cyclin E1, p21, Rb, p53, caspase-3 and caspase-8 decreased gradually during differentiation. Fo significantly inhibited the protein levels of PCNA, Rb, PKC-α and PMA stimulated mRNA expression of PKC-ε and PKC-δ. Further, failure in the activation of essential components of the cell cycle machinery caused G2/M phase arrest in differentiating BeWo cells.

3-(2-Chloropropyl amide)-4-methoxy-N-phenylbenzamide inhibits expression of HPV oncogenes in human cervical cancer cell

BackgroundHuman papillomaviruses (HPVs) are the primary causative agents for cervical cancer, and HPV oncoproteins E6 and E7 are known to be the main reason for the onset and maintenance of the malignancies. Therefore, inhibition of viral E6 and E7 oncoproteins expression represents a viable strategy to cervical cancer therapies. This study is to evaluate the antiviral effect of a novel N-Phenylbenzamide derivative, 3-(2-Chloropropyl amide)-4-methoxy-N-phenylbenzamide (L17), against HPV16 in vitro and identify its associated mechanism of action in cervical cancer cells.MethodsThe cytotoxic effect of L17 was assessed by MTT assay. The mRNA and protein levels of E6 and E7 oncogenes were analyzed by quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot, respectively. p53 and Rb protein levels were also detected by Western blot. The effect of L17 on cell cycle was analyzed by flow cytometry.ResultsThe cytotoxic effect of L17 was greater in cervical carcinoma cells than in normal cells. L17 significantly reduced the expression of HPV16 E6 and E7 mRNA and protein, at least partly by enhancing degradation of HPV16 E6 and E7 mRNA. Moreover, reduced expression of E6 and E7 induced by L17 resulted in the up-regulation of p53 and Rb expression, which subsequently induced CaSki cells arrest at G0/G1 phase.ConclusionsL17 has antiviral activity through suppressing E6 and E7 oncogene expression and could inhibit CaSki cell proliferating by inducing cells arrest at G0/G1 phase at nontoxic concentration, implying that L17 might be exploited as a candidate agent for HPV-associated cervical cancer prevention and treatment.

SET/I2PP2A overexpression induces phenotypic, molecular, and metabolic alterations in an oral keratinocyte cell line.

The multifunctional SET/I2PP2A protein is known to be overexpressed in head and neck squamous cell carcinoma. However, SET has been reported to have apparently conflicting roles in promoting cancer cell survival under oxidative stress conditions and preventing invasion and metastasis, complicating efforts to understand the contribution of SET to carcinogenesis. In the present study, we overexpressed SETin a spontaneously immortalized oral keratinocyte cell line (NOK-SI SET) and demonstrated that SET upregulation alone was sufficient to transform cells. In comparison with NOK-SI cells, NOK-SI SET cells demonstrated increased levels of phosphorylated Akt, c-Myc and inactive/phosphorylated Rb, together with decreased total Rb protein levels. In addition, NOK-SI SET cells presented the following: (a) a spindle-cell shape morphology compared with the polygonal morphology of NOK-SI cells; (b) loss of mesenchymal stem cell markers CD44 and CD73, and epithelial cell markers CD71 and integrin α6/β4; (c) the ability to form xenograft tumors in nude mice; and (d) increased mitochondrial respiration accompanied by decreased ROSlevels. Overall, our results show that SEToverexpression promotes morphological and oncogenic cell transformation of an oral keratinocyte cell.

Abstract CT045: Ribociclib + letrozole for first-line treatment of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC): efficacy by baseline tumor markers

Abstract Background: Cyclin D-cyclin-dependent kinase (CDK) 4/6 complexes promote cell proliferation through phosphorylation of retinoblastoma protein (Rb). In breast cancer, cyclin D-CDK4/6 activity can be increased through cyclin D gene (CCND1) amplification or loss of the CDK4/6 negative regulator p16. Here we present efficacy data from the Phase III MONALEESA-2 study of ribociclib (CDK4/6 inhibitor) + letrozole vs. placebo + letrozole for first-line treatment of HR+, HER2- ABC, assessed in baseline tumors by protein levels of Rb, p16, the cell proliferation marker Ki67, and by gene expression levels of CDKN2A (p16) and CCND1. Methods: Postmenopausal women with HR+, HER2- ABC with no prior systemic therapy for advanced disease were randomized 1:1 to receive ribociclib or placebo (600 mg/day 3-weeks-on/1-week-off) + letrozole (2.5 mg/day continuous). The primary endpoint was investigator-assessed progression-free survival (PFS). Provision of a representative baseline tumor biopsy or archival tissue at screening was mandatory if available. Baseline tumor tissue was evaluated for protein biomarkers (immunohistochemistry) and gene expression (NanoString nCounter® Human Cancer Reference panel). Results: Of 668 patients randomized, 479 were evaluable for total Rb, and 416 (87%) displayed high levels (H-score ≥100). p16 protein levels were evaluable in 405 patients; 165 (41%) had low (H-score <50), 182 (45%) medium (H-score ≥50-149), and 58 (14%) had high (H-score ≥150) p16 levels. Ki67 was detected in ≤14% of tumor cells in 216 (47%) patients and in >14% of cells in 247 (53%) patients. The median messenger RNA expression level was used as the cut-off to define patients with low or high baseline CDKN2A and CCND1 gene expression. An improved PFS was observed by the addition of ribociclib to letrozole in all the above patient subgroups, with hazard ratios ranging from 0.40 (high p16 by H-score; 95% confidence interval [CI] 0.16-1.0; p=0.06) to 0.64 (≤14% Ki67-positive cells; 95% CI 0.39-1.0; p=0.07). Patients with less or greater than 14% Ki67-positive cells, lower or higher p16 levels, Rb levels, or CDKN2A or CCND1 gene expression benefitted from the addition of ribociclib to letrozole to a similar extent. Conclusions: A consistent benefit from ribociclib + letrozole vs. placebo + letrozole was observed irrespective of baseline Rb, p16, and Ki67 levels or CDKN2A and CCND1 gene expression levels. Hormone receptor positivity remains the only established biomarker of response to CDK4/6 inhibitors. Citation Format: Fabrice Andre, Salomon M. Stemmer, Mario Campone, Katarina Petrakova, Shani Paluch-Shimon, Yoon-Sim Yap, Norbert Marschner, Arlene Chan, Cristian Villanueva, Lowell L. Hart, Carlos L. Arteaga, Gabe S. Sonke, Eva-Maria Grischke, Emilio Alba, Arnd Nusch, Denise A. Yardley, Erik Jakobsen, Sibel Blau, Sara M. Tolaney, Faye Su, Wei He, Caroline Germa, Gabriel N. Hortobagyi. Ribociclib + letrozole for first-line treatment of hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC): efficacy by baseline tumor markers [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 CT045. doi:10.1158/1538-7445.AM2017-CT045

Abstract 4150: Anti-tumor activity of the PI3K/mTOR pathway inhibitors alpelisib (BYL719) and everolimus (RAD001) in xenograft models of acquired resistance to CDK-4/6 targeted therapy

Abstract The selective cyclin dependent kinases 4 and 6 (CDK-4/6) inhibitor, palbociclib, has recently been approved in combination with endocrine-based therapies for the treatment of advanced ER+/HER2- breast cancer (BC). Despite the improvements in progression-free and overall survivals, a significant number of patients on CDK-4/6 therapy will go on to develop progressive disease. As such, it is critical to identify the mechanisms by which tumor cells evade CDK-4/6 targeted therapy and to develop therapeutic strategies to overcome resistance. In this study, we evaluated the efficacy of a p110α-selective PI3K inhibitor, alpelisib (BYL719) and the mTORC1 specific inhibitor, everolimus (RAD001), in ER+/HER2- xenograft tumors conditioned in vitro and in vivo to acquire resistance to combined palbociclib + endocrine-based therapy. The EFM19 (ER+/HER2-, PIK3CA mt), MDA134 (ER+/HER2-, PIK3CA wt), and MDA361 (ER+/HER2+, PIK3CA mt) BC cell lines were conditioned to acquire palbociclib resistance through long-term culture in the presence of increasing concentrations of drug. Baseline total/phosphoprotein levels of over 280 cancer-associated analytes were measured in the parental and resistant cell lines by Reverse Phase Protein Array (RPPA). In vivo resistant models were developed from xenografts of the MCF7 and HCC1500 ER+ BC cell lines, treated until progression with palbociclib (75 mg/kg) + fulvestrant (5 mg/mse). EFM19-PR cells maintained palbociclib resistance in vivo and were included in the xenograft studies. Resistance to palbociclib was confirmed in vitro by a shift in growth inhibitory IC50 from an average of less 50nM to over 1 µM. Significantly reduced Rb and ER protein levels were detected in each of these palbociclib resistant cell lines by RPPA. Cross-resistance to an alternative CDK-4/6 inhibitor (ribociclib) was found in in vitro studies. In addition, switching treatment of the xenografts progressing on palbociclib + fulvestrant to ribociclib (75mg/kg) + fulvestrant did not impact tumor progression. However, when progressing tumors were switched to BYL719 (30mg/kg) + fulvestrant or RAD001 (10mg/kg) + fulvestrant, sustained tumor regression was observed for over 45 days of treatment in every model tested. The triplet combination of ribociclib + fulvestrant + BYL719 or RAD001 provided marginal additional benefit over the doublet combination of BYL719 or RAD001 + fulvestrant. RPPA analysis of xenograft tissue collected from these studies will help to identify additional molecular alterations involved in resistance to CDK-4/6 targeted therapy. These preclinical data indicate that acquired resistance to CDK-4/6 inhibition occurs through a loss of dependence on Rb-signaling. However, targeting an alternative pathway like PI3K/mTOR with molecules such as BYL719 or RAD001 may be a viable strategy for further clinical investigation. Citation Format: Neil A. O'Brien, Dylan Conklin, Tong Luo, Raul Ayala, Shawnt Issakhanian, Ondrej Kalous, Erika Von Euw, Sara A. Hurvitz, Emmanuelle DiTomaso, Faye Su, Ronald Linnartz, Stefan Scherer, Samit Hirawat, Dennis J. Slamon. Anti-tumor activity of the PI3K/mTOR pathway inhibitors alpelisib (BYL719) and everolimus (RAD001) in xenograft models of acquired resistance to CDK-4/6 targeted therapy [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 4150. doi:10.1158/1538-7445.AM2017-4150