pRb Phosphorylation Research Articles

PRb phosphorylation regulates the proliferation of supporting cells in gentamicin-damaged neonatal avian utricle: Erratum.

pRb phosphorylation regulates the proliferation of supporting cells in gentamicin-damaged neonatal avian utricle: Erratum.

Anticancer Tetrahydrocarbazoles: A Wide Journey from 2000 Till Date

Abstract: Tetrahydrocarbazoles (THCz) are widespread in numerous indole alkaloids and have been reported since time for exhibiting profound pharmacological potential. Many pharmaceuticals drugs have tetrahydrocarbazole nucleus in their structure e.g. vinca alkaloids (Vincristine, Vinblastine, Vinorelbine), Frovatriptan, (R)-Ramatroban, Ondansetron, etc. that are used in various multifactorial diseases. In this review article, the anticancer potential of tetrahydrocarbazole based derivatives has been covered, enumerating their vast journey from the year 2000 to 2021. Since the last twenty-one years, tetrahydrocarbazoles have been a matter of focus among researchers worldwide, whereby several novel tetrahydrocarbazole derivatives have been synthesized and reported for their anticancer potential against various cancer cell lines. Tetrahydrocarabzole and its derivatives have exhibited profound anticancer potential mediated via various cancer pathways like apoptosis, cell cycle arrest, microtubule inhibition, Nrf2 Modulators, DNA intercalators, pERK and pRb phosphorylation, VEGF (Vascular Endothelial Growth Factor) and TNF-α inhibition, TPSO (translocator protein), Histone Deacetylase (HDAC) Inhibitors also discussed. The present review entails the synthesis, SAR studies, and anticancer mechanism of tetrahydrocarbazoles derivatives reported in review literature till date, and would provide a strong database to the medicinal chemist world over in discovering newer potential anticancer agent against various types of cancer diseases.

A54 SHP2 FUNCTION IN NORMAL AND APC-MUTANT INTESTINAL EPITHELIAL CELLS

Abstract Background Somatic SHP2 gain-of-function mutations were found in some colorectal tumours. However, its function in colorectal epithelium remains to be determined. SHP2 is a tyrosine phosphatase necessary for RAS/MAPK pathway activation by most, if not all, tyrosine kinase receptors, as well as by GPCRs and cytokine receptors. SHP2 can also regulate the PI3K, Jak/Stat, RhoA and Hippo pathways in different cell settings. As a result, SHP2 can control cellular processes including growth, migration and differentiation. Previously, we observed that SHP2 levels are enhanced in human colorectal tumours, notably in late adenomas. Likewise, SHP2 silencing inhibited ERK activation and tumour properties of established human CRC cell lines. Hence, these findings support a model in which SHP2 activation promotes tumorigenesis in the intestinal epithelium. But how? Aims To analyze the exact cellular and molecular mechanisms underlying SHP2 pro-oncogenic function in intestinal epithelial cells. Methods The impact of SHP2 pharmaco-inhibition (RMC-4550 and/or SHP099) on intracellular signaling, cell cycle, proliferation and survival was evaluated in three complementary models: 1) HIEC-6 cells, normal human fetal intestinal crypt-like cells; 2) organoids derived from normal mouse small intestine or ApcMin/+ tumors; 3) Caco-2/15 cells, colorectal cancer cells with APC mutation. Results 1- Pharmaco-inhibition of SHP2 blocks proliferation of HIEC-6 cells (loss of pRb phosphorylation, decreased EdU staining) and induces cell enlargement and darkening of the outside of perinuclear area. This phenotype was reminiscent of cellular senescence, and we indeed observed increased senescence-associated β-galactosidase activity (SA-β-Gal) and INK4A gene expression. 2- As expected, SHP2 inhibition in normal mouse organoids resulted in a reduction in the number of protrusions and EdU positive cells. RNA sequencing was performed, and gene set enriched revealed significant down regulation of several Ras signaling-dependant genes including cell cycle regulated genes (E2F targets, G2/M checkpoint) and Myc targets 48h after SHP2 inhibition when compared to DMSO treated organoids. Genes associated with PI3K signaling were also diminished in SHP099 treated organoids but no significant modulation of genes associated with Wnt/b-catenin signaling was observed. 3- Notably, SHP2 pharmaco-inhibition in Caco-2/15 cells as well as APC-mutant organoids rapidly induced cell death associated with increased expression of stem cell markers. Conclusions These results suggest that SHP2 is required for proliferation of normal IEC and protects against senescence. Notably, its inhibition restrains the proliferative and tumorigenic potential conferred by APC inactivation. Overall, our data suggest that SHP2 could represent a potential target to counter colorectal tumour initiation. Funding Agencies CIHRFRQS

Targeting Non-Kinase Function of CDK6 to Overcome CDK4/6 Inhibitor Resistance

CDK4/6 inhibitors have revolutionized the care of patients with Estrogen Receptor-positive (ER+) breast cancer (BC). However, most patients develop resistance to these agents, commonly through overexpression of CDK6. The objective of this study is to define the mechanism by which CDK6 overexpression confers resistance to CDK4/6 inhibitors and to develop a new therapeutic strategy to overcome such resistance. ER+ MCF-7 and T-47D cells overexpressing wild-type CDK6 or kinase dead (K43M) mutant of CDK6 (and the corresponding isogenic cell lines expressing an empty vector) were used assess response to palbociclib in vitro and in vivo. pRB phosphorylation was assessed by Western blot analysis. A selective CDK4/6 degrader, BSJ-1-184, was used to evaluate the therapeutic benefit of eliminating both kinase and non-kinase functions of CDK6 (and CDK4) to overcome palbociclib resistance using standard cell growth and tumor growth assays. Surprisingly, analysis of multiple publicly available databases, including the Human Proteome Atlas, revealed that primary ER+ breast tumors show no detectable expression of CDK6. Similarly, Western blot analysis of a panel of human ER+ BC cell lines showed no detectable expression of CDK6. Despite no detectable expression of CDK6 in primary ER+ breast tumors, resistance to CDK4/6 inhibitors in many cases is driven by overexpression of CDK6 but not CDK4. Based on these findings, we hypothesized that CDK6 may carry additional functions (relative to CDK4) that confer evolutionary benefit for survival of BC cells in the face of treatment with CDK4/6 inhibitors. To test this hypothesis, we generated a panel of stable ER+ BC cell lines overexpressing CDK6. As expected, these cells exhibited complete resistance to palbociclib, a CDK4/6 inhibitor. However, palbociclib completely inhibited phosphorylation of pRB (retinoblastoma protein), a primary substrate for phosphorylation for both CDK4 and 6, suggesting that non-kinase function of CDK6 may play a role in conferring palbociclib resistance. Using a kinase dead mutant of CDK6 (K43M), we demonstrated that non-kinase function of CDK6 was sufficient to confer partial resistance to palbociclib both in vitro and in vivo. Furthermore, BSJ-1-184, a selective CDK4/6 degrader (which eliminates both kinase and non-kinase functions of these proteins) completely overcame resistance to CDK6 overexpression both in vitro and in vivo, further highlighting the dual kinase and non-kinase functions of CDK6 in conferring palbociclib resistance. The kinase function of CDK4/6 has been the primary target of current CDK4/6 inhibitors used in clinic. Our studies show that non-kinase function of CDK6 plays a critical role in palbociclib resistance. We also provide a novel therapeutic strategy to overcome CDK4/6 inhibitor resistance, thereby setting the stage for development of next generation drugs that eliminate both the kinase and non-kinase functions of this class of proteins.

CDKN2A/p16 evaluation in cytology specimens.

CDKN2A/p16 evaluation in cytology specimens.

Decreased sensitivity of cyclin-dependent kinase 4/6 inhibitors, palbociclib and abemaciclib to canine lymphoma cells with high p16 protein expression and low retinoblastoma protein phosphorylation.

Canine lymphoma/leukemia cell lines with p16 protein expressions: high (17-71 and GL-1) and low (CLBL-1, CLC, Nody-1, and UL-1) were treated in vitro with cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, palbociclib or abemaciclib. Cell proliferation decreased as a result, with higher IC50 levels observed in the high p16 (17-71 and GL-1) and one low p16 (UL-1) cell lines compared with the low p16 cells (CLBL-1, CLC, and Nody-1). As expected, palbociclib and abemaciclib treatment reduced pRb phosphorylation in a dose-dependent manner, especially in cells with low p16. These results suggest that CDK4/6 inhibitors have potential as new chemotherapeutic agents for canine lymphoma and high p16 protein expression may be used as a biomarker for resistance to CDK4/6 inhibitor therapy.

Genome-wide analysis of DNaseI hypersensitivity unveils open chromatin associated with histone H3 modifications after areca nut with lime exposure.

Research over the years revealed that precocious anaphase, securin overexpression, and genome instability in both target and nontarget cells are significantly associated with the increased risk of areca nut (AN) and lime-induced oral, esophageal, and gastric cancers. Further, hyperphosphorylation of Rb and histone H3 epigenetic modifications both globally and in the promoter region of the securin gene were demonstrated after AN + lime exposure. This study aims whether the extract of raw AN + lime relaxes chromatin structure which further facilitates the histone H3 epigenetic modifications during the initial phase of carcinogenesis. Three groups of mice (10 in each group) were used. The treated group consumed 1 mg/day/mice of AN extract with lime ad libitum in the drinking water for 60 days. The dose was increased by 1 mg every 60 days. Isolated nuclei were digested with DNaseI and 2 kb and below DNA was eluted from the agarose gel, purified and PCR amplified by using securin and GAPDH primers. Securin and E2F1 expression, pRb phosphorylation, and histone epigenetic modifications were analyzed by immunohistochemistry. The number of DNA fragments within 2 kb in size after DNaseI treatment was higher significantly in AN + lime exposed tissue samples than in the untreated one. The PCR result showed that the number of fragments bearing securin gene promoter and GAPDH gene was significantly higher in AN + lime exposed DNaseI-treated samples. Immunohistochemistry data revealed increased Rb hyperphosphorylation, upregulation of E2F1, and securin in the AN + lime-treated samples. Increased trimethylation of histone H3 lysine 4 and acetylation of H3 lysine 9 and 18 were observed globally in the treated samples. Therefore, the results of this study have led to the hypothesis that AN + lime exposure relaxes the chromatin, changes the epigenetic landscape, and deregulates the Rb-E2F1 circuit which might be involved in the upregulation of securin and some other proto-oncogenes that might play an important role in the initial phases of AN + lime mediated carcinogenesis.

The transcription factor E2F1 controls the GLP-1 receptor pathway in pancreatic β cells.

The glucagon-like peptide 1 (Glp-1) has emerged as a hormone with broad pharmacological potential in type 2 diabetes (T2D) treatment, notably by improving β cell functions. The cell-cycle regulator and transcription factor E2f1 is involved in glucose homeostasis by modulating β cell mass and function. Here, we report that β cell-specific genetic ablation of E2f1 (E2f1β-/-) impairs glucose homeostasis associated with decreased expression of the Glp-1 receptor (Glp1r) in E2f1β-/- pancreatic islets. Pharmacological inhibition of E2F1 transcriptional activity in nondiabetic human islets decreases GLP1R levels and blunts the incretin effect of GLP1R agonist exendin-4 (ex-4) on insulin secretion. Overexpressing E2f1 in pancreatic β cells increases Glp1r expression associated with enhanced insulin secretion mediated by ex-4. Interestingly, ex-4 induces retinoblastoma protein (pRb) phosphorylation and E2f1 transcriptional activity. Our findings reveal critical roles for E2f1 in β cell function and suggest molecular crosstalk between the E2F1/pRb and GLP1R signaling pathways.

Simultaneous Analysis of the p16 Gene and Protein in Canine Lymphoma Cells and Their Correlation with pRb Phosphorylation.

Simple SummaryLymphoma is one of the most frequently diagnosed malignancies in dogs. The most common epigenetic alteration is gene methylation. Methylation of the p16 gene leads to decreased expression of its protein. The p16 protein inhibits the activity of cyclin-dependent kinase, as a negative control of the cell cycle to prevent phosphorylation of the retinoblastoma (pRb) protein. The methylation of the p16 gene has been reported in canine lymphomas, however, p16 protein expression has not been examined in previous studies. In this study, the gene and protein expression of p16, and phosphorylation of pRb, were examined simultaneously in canine lymphoma/leukemia cell lines treated with or without a demethylation drug in vitro. We identified the hypermethylation of the p16 gene, the decreased expression of p16 protein and the hyperphosphorylation of pRb in four out of eight cell lines. Furthermore, we revealed that the expression of the p16 protein was more stable than that of the p16 gene and more closely related to the phosphorylation of pRb. In conclusion, the p16 protein expression is suggested as a promising biomarker for canine lymphoma cells, and the p16–pRb pathway could be a target for the better treatment of canine lymphomas.Cyclin-dependent kinase inhibitor p16 (CDKN2A) primarily functions as a negative regulator of the retinoblastoma protein (pRb) pathway to prevent pRb phosphorylation, thus playing a critical role in cell cycle arrest. In canine lymphoma cells, methylation due to inactivation of the p16 gene has been reported. However, its protein expression has not been examined in previous studies. In our in vitro study, the gene and protein expression of p16 and phosphorylated pRb were examined simultaneously in eight canine lymphoma and leukemia cell lines (17-71, CLBL-1, GL-1, CLC, CLGL-90, Ema, Nody-1, and UL-1). Methylation of the p16 gene was also explored using the demethylation drug 5-Aza-2′-deoxycytidine (5-Aza). After 5-Aza treatment, p16 gene and protein expression increased and pRb phosphorylation decreased, suggesting that both hypermethylation of the p16 gene and pRb hyperphosphorylation occurred in four out of eight cell lines (CLBL-1, CLC, Nody-1, and UL-1). Moreover, the estimation of p16’s protein expression was better than that of p16’s mRNA expression because the expression of the protein was more stable than those of the gene, and highly related to the phosphorylation of pRb. These results revealed that p16’s protein expression could be a promising biomarker for canine lymphoma cells.

An immature, dedifferentiated, and lineage-deconstrained cone precursor origin of N-Myc–initiated retinoblastoma

Most retinoblastomas develop from maturing cone precursors in response to biallelic RB1 loss and are dependent on cone maturation-related signaling. Additionally, ∼2% lack RB1 mutations but have MYCN amplification (MYCNA), N-Myc protein overexpression, and more rapid and invasive growth, yet the MYCNA retinoblastoma cell of origin and basis for its responses to deregulated N-Myc are unknown. Here, using explanted cultured retinae, we show that ectopic N-Myc induces cell cycle entry in cells expressing markers of several retinal types yet induces continuous proliferation and tumorigenesis only in cone precursors. Unlike the response to RB1 loss, both immature cone arrestin-negative (ARR3-) and maturing ARR3+ cone precursors proliferate, and maturing cone precursors rapidly dedifferentiate, losing ARR3 as well as L/M-opsin expression. N-Myc-overexpressing retinal cells also lose cell lineage constraints, occasionally coexpressing the cone-specific RXRγ with the rod-specific NRL or amacrine-specific AP2α and widely coexpressing RXRγ with the progenitor and Müller cell-specific SOX9 and retinal ganglion cell-specific BRN3 and GAP43. Mechanistically, N-Myc induced Cyclin D2 and CDK4 overexpression, pRB phosphorylation, and SOX9-dependent proliferation without a retinoma-like stage that characterizes pRB-deficient retinoblastoma, despite continuous p16INK4A expression. Orthotopic xenografts of N-Myc-overexpressing retinal cells formed tumors with retinal cell marker expression similar to those in MYCN-transduced retinae and MYCNA retinoblastomas in patients. These findings demonstrate the MYCNA retinoblastoma origin from immature and lineage-deconstrained cone precursors, reveal their opportunistic use of an undifferentiated retinal progenitor cell feature, and illustrate that different cancer-initiating mutations cooperate with distinct developmental stage-specific cell signaling circuitries to drive retinoblastoma tumorigenesis.

Hypophosphorylated pRb knock‐in mice exhibit hallmarks of aging and vitamin C‐preventable diabetes

Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock‐in mice, in which either four or all seven phosphorylation sites in the C‐terminal region of pRb, respectively, have been abolished by Ser/Thr‐to‐Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin‐sensitive and associated with failure of quiescent pancreatic β‐cells to re‐enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence‐associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre‐treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re‐entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK‐inhibitor therapeutics, diabetes, and longevity.

The antipsychotic drug pimozide is effective against human T-cell leukemia virus type 1-infected T cells

Patients with adult T-cell leukemia (ATL), caused by the human T-cell leukemia virus type 1 (HTLV-1), exhibit poor prognosis owing to drug resistance. Pimozide is a dopamine D2 receptor antagonist and antipsychotic shown to exhibit anticancer activity. Herein, we investigated whether pimozide exerts anti-ATL effects and explored the mechanisms underlying these effects. Pimozide inhibited cell growth and survival in HTLV-1-infected T cells but not in the uninfected T cells. The dopamine D2 receptor subfamily mRNA expression levels in HTLV-1-infected T cells were high. Pimozide induced G1 cell cycle arrest concomitant with the upregulation of p21/p27/p53, and suppression of cyclin D2/E, cyclin-dependent kinase 2/4/6 and c-Myc expression, and pRb phosphorylation. Pimozide also induced apoptosis by activating caspases, upregulating pro-apoptotic proteins and downregulating anti-apoptotic proteins. Additionally, it promoted reactive oxygen species (ROS) generation and increased the expression of the endoplasmic reticulum stress marker activating transcription factor 4 and the DNA damage-inducible protein GADD45α and the phosphorylation of the DNA damage marker H2AX. Furthermore, pimozide-induced cytotoxicity was partially inhibited by a ROS scavenger, and pan-caspase and necroptosis inhibitors, indicating the involvement of caspase-dependent and -independent lethal pathways. The activities of the nuclear factor-κB, Akt, STAT3/5 and AP-1 signaling pathways were inhibited via the dephosphorylation of IκBα, IκB kinase α/β, Akt and STAT3/5, in addition to reduced JunB and JunD expression in HTLV-1-infected T cells. Pimozide also exhibited potent anti-ATL activity in the xenograft mouse model. These findings demonstrated the efficacy of pimozide as a potential therapeutic agent for ATL.

Abstract 1017: Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells

Abstract Arsenic trioxide (ATO) has been used for the treatment of acute promyelocytic leukemia (APL). Although ATO modulates cell cycle progression and apoptosis in APL cells, its exact mechanism of action remains elusive. In this research, we investigated its effects on E2F1, cyclin E, p53, pRb and PI3K signaling molecules by western blotting, immunocytochemistry and/or confocal imaging. We found that ATO inhibited the proliferation of APL cells through down-regulation of E2F1 and cyclin E expression, and stimulation of pRb. It also reduced the interaction of pRb and E2F1with binding to the E2F1 promoter, by stimulating pRb association. ATO also effected the phosphorylation of pRb at S608 and T373 residues and association of E2F1, pRb, and p53, simultaneously. However, in p53-knockdown NB4 cells, ATO did not significantly reduce E2F1 and cyclin E expression. Our findings demonstrate that ATO inhibits APL cell growth through reduced expression of E2F1, cyclin E, and stimulation of pRb. It also effected both interaction and association of E2F1, pRb and p53 by phosphorylation of pRb at T373 and S608 residues and reduced phosphorylation of PI3K signaling molecules. This novel mode of action of ATO in APL cells may be useful for designing new APL drugs. Citation Format: Paul B. Tchounwou, Sanjay Kumar. Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1017.

Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells.

Arsenic trioxide (ATO) has been used for the treatment of acute promyelocytic leukemia (APL). Although ATO modulates cell cycle progression and apoptosis in APL cells, its exact mechanism of action remains elusive. In this research, we investigated its effects on E2F1, cyclin E, p53, pRb, and PI3K signaling molecules by western blotting, immunocytochemistry and/or confocal imaging. We found that ATO inhibited the proliferation of APL cells through down‐regulation of E2F1 and cyclin E expression, and stimulation of pRb. It also reduced the interaction of pRb and E2F1with binding to the E2F1 promoter, by stimulating pRb association. ATO also effected the phosphorylation of pRb at S608 and T373 residues and association of E2F1, pRb, and p53, simultaneously. However, in p53‐knockdown NB4 cells, ATO did not significantly reduce E2F1 and cyclin E expression. Our findings demonstrate that ATO inhibits APL cell growth through reduced expression of E2F1, cyclin E, and stimulation of pRb. It also effected both interaction and association of E2F1, pRb, and p53 by phosphorylation of pRb at T373 and S608 residues and reduced phosphorylation of PI3K signaling molecules. This novel mode of action of ATO in APL cells may be useful for designing new APL drugs.

Effect of cyclin-dependent kinase 4 and 6 inhibitors (CDKIs) on body composition (BC) in patients (pts) with hormone receptor-positive, HER2-negative (HR+/HER2-) metastatic breast cancer (MBC).

e13033 Background: CDKIs with endocrine therapy (ET) is first-line treatment in HR+/HER2- MBC. Mouse models have shown that CDKIs prevent pRB phosphorylation in the mediobasal hypothalamus, a pathway hyper-activated in diet-induced obesity; and CDKIs lead to fat mass decrease without significant effect on lean mass. We aimed to assess the impact of CDKIs on weight (wt) and BC in pts with HR+/HER2- MBC. Methods: We identified pts with HR+/HER2- MBC who received CDKIs and ET from 2015-2018. To isolate the effect of CDKIs on BC, we identified another cohort of pts who only received ET. Body mass index (BMI), wt, and computed tomography (CT) records were reviewed. BC was analyzed at L3 on CT scans using Tomovision’s SliceOmatic v5.0 and included skeletal muscle area (SMA), skeletal muscle density (SMD), subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), and muscle adiposity (MA). Total adipose tissue (TAT) was defined as SAT+VAT+MA. BC changes at 3 and 6 months of therapy were evaluated using paired t-tests. Results: There were 107 pts who received CDKI plus ET - 43% were Black, and 41% were Hispanic. CDKIs used were palbociclib (85%), abemaciclib (9%), and ribociclib (6%). ETs used were letrozole (47%), fulvestrant (39%), anastrozole (12%), and exemestane (2%). Median number of prior chemo and ET lines was 0 (range 0-5). 63 pts received ET alone. There was no difference in age (63 vs. 65 years, p = 0.26), BMI (28.80 vs. 28.12kg/m2, p = 0.48), and visceral disease (69% vs. 65%, p = 0.64) between CDKI plus ET and ET alone group. At month 3 of CDKI plus ET, there was a significant decrease in wt (-0.30kg, Interquartile range [IQR] -2.55-0.95, p = 0.02), BMI (-0.12kg/m2, IQR -1.06-0.46, p = 0.02), SAT (-8.05cm2, IQR -32.58-14.74, p = 0.01), and TAT (-8.51cm2, IQR -50.42-17.84, p < 0.01), with similar results at month 6. These findings were not seen in pts on ET only at 3 months (wt: 0.00kg, IQR -2.65-2.38, p = 0.98; BMI: 0.00kg/m2, IQR -1.07-0.91, p = 0.93; SAT: -2.97cm2, IQR -26.10-25.15, p = 0.60; TAT: -0.58cm2, IQR -44.39-27.43, p = 0.18), or at 6 months. There were no significant changes in VAT, SMA, SMD, or MA in both groups at 3 or 6 months. In the CDKI plus ET group, baseline wt (74.64 vs. 72.72kg, p = 0.60), BMI (29.21 vs. 27.88kg/m2, p = 0.31), and SAT (280.29 vs. 252.75cm2, p = 0.31) were not significantly different for those who did or did not develop grade 3/4 toxicities. We obtained similar results when stratifying toxicities into hematological- and GI-related events. Conclusions: CDKIs are associated with decrease in BMI and SAT with no significant effect on VAT, SMA, or SMD. Given the known effect of obesity on breast cancer prognosis, CDKIs may have an additional effect on breast cancer prognosis by modulating body fat. Further studies are required to determine if decrease in SAT is associated with breast cancer outcomes or toxicities in pts on CDKIs.

Characterization of Signalling Pathways That Link Apoptosis and Autophagy to Cell Death Induced by Estrone Analogues Which Reversibly Depolymerize Microtubules

The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds’ effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.

MiR-449a Sponges Cyclin D1 Expression and Suppresses Phosphorylation of Retinoblastoma Protein against Osteosarcoma.

MiR-449a has tumor-regulatory properties in different cancers, but its role in osteosarcoma had not been clearly understood. This research aimed to study the underlying mechanism of how miR-449a regulates osteosarcoma cells. The expression of miR-449a, cyclin D1, and pRb in osteosarcoma tissues and cultivated cells was assessed by qRT-PCR, western blot, and immunofluorescent assay. Dual-luciferase reporter assay was conducted to verify the target of miR-449a. Western blot, CCK-8, colony formation, and flow cytometry assays were applied to examine the regulatory effects of miR-449a on osteosarcoma cells and the molecular mechanism. MiR-449a and pRb expression was impeded whereas cyclin D1 expression was enhanced in osteosarcoma tissues and cells. Cyclin D1 was confirmed as a target of miR-449a. MiR-449a impeded the viability and proliferation of osteosarcoma cells and controlled the cell cycle through targeting cyclin D1 to reduce pRB phosphorylation. These findings provided evidence that miR-449a played an anticancer role in osteosarcoma cells by directly targeting cyclin D1 to prevent pRb from phosphorylation. Our study suggested that miR-449a might have the potential to become a new therapeutic biomarker involved in the diagnosis, prevention, and treatment of osteosarcoma.

Pharmacological inactivation of CDK2 inhibits MYC/BCL-XL-driven leukemia in vivo through induction of cellular senescence

ABSTRACT Deregulated expression of the MYC oncogene is a frequent event during tumorigenesis and generally correlates with aggressive disease and poor prognosis. While MYC is a potent inducer of apoptosis, it often suppresses cellular senescence, which together with apoptosis is an important barrier against tumor development. For this latter function, MYC is dependent on cyclin-dependent kinase 2 (CDK2). Here, we utilized a MYC/BCL-XL-driven mouse model of acute myeloblastic leukemia (AML) to investigate whether pharmacological inhibition of CDK2 can inhibit MYC-driven tumorigenesis through induction of senescence. Purified mouse hematopoietic stem cells transduced with MYC and BCL-XL were transplanted into lethally irradiated mice, leading to the development of massive leukemia and subsequent death 15–17 days after transplantation. Upon disease onset, mice were treated with the selective CDK2 inhibitor CVT2584 or vehicle either by daily intraperitoneal injections or continuous delivery via mini-pumps. CVT2584 treatment delayed disease onset and moderately but significantly improved survival of mice. Flow cytometry revealed a significant decrease in tumor load in the spleen, liver and bone marrow of CVT2584-treated compared to vehicle-treated mice. This was correlated with induced senescence evidenced by reduced cell proliferation, increased senescence-associated β-galactosidase activity and heterochromatin foci, expression of p19ARF and p21CIP1, and reduced phosphorylation (activation) of pRb, while very few apoptotic cells were observed. In addition, phosphorylation of MYC at Ser-62 was decreased. In summary, inhibition of CDK2 delayed MYC/BCL-XL-driven AML linked to senescence induction. Our results suggest that CDK2 is a promising target for pro-senescence cancer therapy, in particular for MYC-driven tumors, including leukemia.

Abstract 295: Protein phosphatase, Mg2+/Mn2+ dependent 1G promotes proliferation via modulating phosphorylation of retinoblastoma protein and associates with poor outcome in hepatocellular carcinoma

Abstract The serine/threonine protein phosphatase family has been demonstrated involved in a variety of cellular processes. Here, we identified that protein phosphatase, Mg2+/Mn2+ -dependent 1G (PPM1G) is an oncogene-associated protein in hepatocellular carcinoma (HCC), and is associated with the progression of HCC. In this study, we carried out a series of in vitro (MTT, colony formation, immunoblot analysis, IHC, and Q-PCR) as well as in vivo (Hepatocellular carcinoma cell xenograft mouse model) experiments to determine the biological roles of PPM1G in HCC. Our results showed that PPM1G expression was significantly increased in HCC tissues, compared with the adjacent non-tumorous tissues. Further analysis revealed that high expression of PPM1G is associated with advanced clinical characters including advanced clinical stage and tumor recurrence. Kaplan-Meier analysis suggested that a high level of PPM1G is correlated to unfavorable overall survival (P < 0.0001) as well as disease-free survival (P = 0.004) in patients with HCC. Furthermore, overexpression of PPM1G promoted cell proliferation while this effect can be attenuated by the knockdown of PPM1G in in vitro and in vivo studies. Notably, our findings showed that the tumor suppressor gene expression (e.g. pRb phosphorylation and p53 protein) was inhibited upon the silencing of PPM1G. Taken together, our study demonstrated that PPM1G plays an crucial role in HCC cell proliferation by modulating pRb phosphorylation as well as p53 expression. Furthermore, this study provides strong clinical implications of PPM1G in prognostic prediction in patients with HCC. Citation Format: Wen Hu, Xin Yuan Guan, Lei Li, Yinghui Zhu, Yan Li, Ting Ting Zeng, Yun Fei Wen. Protein phosphatase, Mg2+/Mn2+ dependent 1G promotes proliferation via modulating phosphorylation of retinoblastoma protein and associates with poor outcome in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 295.

Novel Dihydropyrimidinone-Derived Selenoesters as Potential Cytotoxic Agents to Human Hepatocellular Carcinoma: Molecular Docking and DNA Fragmentation.

Evidence point out promising anticancer activities of Dihydropyrimidinones (DHPM) and organoselenium compounds. This study aimed to evaluate the cytotoxic and antiproliferative potential of DHPM-derived selenoesters (Se-DHPM), as well as their molecular mechanisms of action. Se-DHPM cytotoxicity was evaluated against cancer lines (HeLa, HepG2, and MCF-7) and normal cells (McCoy). HepG2 clonogenic assay allowed verifying antiproliferative effects. The propidium iodide/ orange acridine fluorescence readings showed the type of cell death induced after treatments (72h). Molecular simulations with B-DNA and 49H showed docked positions (AutoDock Vina) and trajectories/energies (GROMACS). In vitro molecular interactions used CT-DNA and 49H applying UV-Vis absorbance and fluorescence. Comet assay evaluated DNA fragmentation of HepG2 cells. Flow cytometry analysis verified HepG2 cell cycle effects. Levels of proteins (β-actin, p53, BAX, HIF-1α, γH2AX, PARP-1, cyclin A, CDK-2, and pRB) were quantified by immunoblotting. Among Se-DHPM, 49H was selectively cytotoxic to HepG2 cells, reduced cell proliferation, and increased BAX (80%), and p53 (66%) causing apoptosis. Molecular assays revealed 49H inserted in the CT-DNA molecule causing the hypochromic effect. Docking simulations showed H-bonds and hydrophobic interactions, which kept the ligand partially inserted into the DNA minor groove. 49H increased the DNA damage (1.5 fold) and γH2AX level (153%). Besides, treatments reduced PARP-1 (60%) and reduced pRB phosphorylation (21%) as well as decreased cyclin A (46%) arresting cell cycle at the G1 phase. Together all data obtained confirmed the hypothesis of disruptive interactions between Se-DHPM and DNA, thereby highlighting its potential as a new anticancer drug.