Heterozygous RB1 mutation enhanced ATP production in human iPSC-derived retinal organoids.

BACKGROUND: Recent in vitro studies using RB1+/- fibroblasts and MSCs have shown molecular and functional disruptions without the need for biallelic loss of RB1. However, this was not reflected in the recent in vitro studies employing RB1+/- retinal organoids. To gain further insights into the molecular disruptions in the RB1+/- retinal organoids, we performed a high throughput RNA sequencing analysis. METHODS AND RESULTS: iPSCs were generated from RB1+/+ and RB1+/- OAMSCs derived from retinoblastoma patients. RB1+/+ and RB1+/- iPSCs were subjected to a step-wise retinal differentiation protocol. Retinal differentiation was evaluated by Real-time PCR and flow cytometry analysis of the retinal markers. To gain further insights into the molecular differences in RB1+/- retinal organoids, a high throughput RNA sequencing followed by differential gene expression analysis and gene set enrichment analysis (GSEA) was performed. The analysis revealed a shift from the regular metabolic process of glycolysis to oxidative phosphorylation in the RB1+/- retinal organoids. To investigate further, we performed assays to determine the levels of pyruvate, lactate and ATP in the retinal organoids. The results revealed significant increase in ATP and pyruvate levels in RB1+/- retinal organoids of day 120 compared to that of the RB1+/+. The results thus revealed enhanced ATP production in the RB1+/- retinal organoids. CONCLUSION: The study provides novel insights into the metabolic phenotype of heterozygous RB1 mutant suggesting dysregulation of energy metabolism and glycolytic pathways to be first step even before the changes in cellular proliferation or other phenotypic consequences ensue.

The pRb/RBL2-E2F1/4-GCN5 axis regulates cancer stem cell formation and G0 phase entry/exit by paracrine mechanisms.

The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.

RB1 loss induces quiescent state through downregulation of RAS signaling in mammary epithelial cells.

While loss of function (LOF) of retinoblastoma 1 (RB1) tumor suppressor is known to drive initiation of small-cell lung cancer and retinoblastoma, RB1 mutation is rarely observed in breast cancers at their initiation. In this study, we investigated the impact on untransformed mammary epithelial cells given by RB1 LOF. Depletion of RB1 in anon-tumorigenic MCF10A cells induced reversible growth arrest (quiescence) featured by downregulation of multiple cyclins and MYC, upregulation of p27KIP1, and lack of expression of markers which indicate cellular senescence or epithelial-mesenchymal transition (EMT). We observed a similar phenomenon in human mammary epithelial cells (HMEC) as well. Additionally, we found that RB1 depletion attenuated the activity of RAS and the downstream MAPK pathway in an RBL2/p130-dependent manner. The expression of farnesyltransferase ß, which is essential for RAS maturation, was found to be downregulated following RB1 depletion also in an RBL2/p130-dependent manner. These findings unveiled an unexpected mechanism whereby normal mammary epithelial cells resist to tumor initiation upon RB1 LOF.

Retinoblastoma-associated protein is important for TRIM24-mediated activation of the mTOR signaling pathway through DUSP2 action in prostate cancer.

RB transcriptional corepressor 1 (RB) deletion is the most important genomic factor associated with the prognosis of castration-resistant prostate cancer (CRPC) patients receiving androgen receptor (AR) signaling inhibitor therapy. Loss of RB could support prostate cancer cell growth in a hormone-independent manner, but the underlying mechanism by which RB regulates tumor progression extends far beyond the cell cycle pathway. A previous study indicated that RB inactivates AKT signaling but has no effect on mTOR signaling in cancer cells. Here, we found that the S249/T252 site in RB is key to regulating the transcriptional activity of the tumor-promoting factor TRIM24 in CRPC, as identified through FXXXV mapping. The RB/TRIM24 complex functions through DUSP2, which serves as an intermediate bridge, to activate the mTOR pathway and promote prostate cancer progression. Accordingly, we designed RB-linker-proteolysis-targeting chimera (PROTAC) molecules, which decreased TRIM24 protein levels and inactivated the mTOR signaling pathway, thereby inhibiting prostate cancer. Therefore, this study not only elucidates the novel function of RB but also provides a theoretical basis for the development of new drugs for treating prostate cancer.

Immunohistochemical p16 overexpression and Rb loss correlate with high-risk human papillomavirus infection in endocervical adenocarcinomas.

AIMS: p16 is a sensitive surrogate marker for transcriptionally active high-risk human papillomavirus (HR-HPV) infection in endocervical adenocarcinoma (ECA); however, its specificity is not perfect. METHODS AND RESULTS: We examined p16 and Rb expressions by immunohistochemistry (IHC) and the transcriptionally active HR-HPV infection by mRNA in-situ hybridisation (ISH) with histological review in 108 ECA cases. Thirteen adenocarcinomas of endometrial or equivocal origin (six endometrioid and seven serous carcinomas) were compared as the control group. HR-HPV was detected in 83 of 108 ECA cases (77%), including five HPV-associated adenocarcinomas in situ and 78 invasive HPV-associated adenocarcinomas. All 83 HPV-positive cases showed consistent morphology, p16 positivity and partial loss pattern of Rb. Among the 25 cases of HPV-independent adenocarcinoma, four (16%) were positive for p16, and of these four cases, three of 14 (21%) were gastric type adenocarcinomas and one of 10 (10%) was a clear cell type adenocarcinoma. All 25 HPV-independent adenocarcinomas showed preserved expression of Rb irrespective of the p16 status. Similarly, all 13 cases of the control group were negative for HR-HPV with preserved expression of Rb, even though six of 13 (46%) cases were positive for p16. Compared with p16 alone, the combination of p16 overexpression and Rb partial loss pattern showed equally excellent sensitivity (each 100%) and improved specificity (100 versus 73.6%) and positive predictive values (100 versus 89.2%) in the ECA and control groups. Furthermore, HR-HPV infection correlated with better prognosis among invasive ECAs. CONCLUSIONS: The results suggest that the combined use of p16 and Rb IHC could be a reliable method to predict HR-HPV infection in primary ECAs and mimics. This finding may contribute to prognostic prediction and therapeutic strategy.

Functional characterization of D-type cyclins involved in cell division in rice.

BACKGROUND: D-type cyclins (CYCD) regulate the cell cycle G1/S transition and are thus closely involved in cell cycle progression. However, little is known about their functions in rice. RESULTS: We identified 14 CYCD genes in the rice genome and confirmed the presence of characteristic cyclin domains in each. The expression of the OsCYCD genes in different tissues was investigated. Most OsCYCD genes were expressed at least in one of the analyzed tissues, with varying degrees of expression. Ten OsCYCD proteins could interact with both retinoblastoma-related protein (RBR) and A-type cyclin-dependent kinases (CDKA) forming holistic complexes, while OsCYCD3;1, OsCYCD6;1, and OsCYCD7;1 bound only one component, and OsCYCD4;2 bound to neither protein. Interestingly, all OsCYCD genes except OsCYCD7;1, were able to induce tobacco pavement cells to re-enter mitosis with different efficiencies. Transgenic rice plants overexpressing OsCYCD2;2, OsCYCD6;1, and OsCYCD7;1 (which induced cell division in tobacco with high-, low-, and zero-efficiency, respectively) were created. Higher levels of cell division were observed in both the stomatal lineage and epidermal cells of the OsCYCD2;2- and OsCYCD6;1-overexpressing plants, with lower levels seen in OsCYCD7;1-overexpressing plants. CONCLUSIONS: The distinct expression patterns and varying effects on the cell cycle suggest different functions for the various OsCYCD proteins. Our findings will enhance understanding of the CYCD family in rice and provide a preliminary foundation for the future functional verification of these genes.

Analysis of E2F1 single-nucleotide polymorphisms reveals deleterious non-synonymous substitutions that disrupt E2F1-RB protein interaction in cancer.

Cancer is a medical condition that is caused by the abnormal growth and division of cells, leading to the formation of tumors. The E2F1 and RB pathways are critical in regulating cell cycle, and their dysregulation can contribute to the development of cancer. In this study, we analyzed experimentally reported SNPs in E2F1 and assessed their effects on the binding affinity with RB. Out of 46, nine mutations were predicted as deleterious, and further analysis revealed four highly destabilizing mutations (L206W, R232C, I254T, A267T) that significantly altered the protein structure. Molecular docking of wild-type and mutant E2F1 with RB revealed a docking score of -242 kcal/mol for wild-type, while the mutant complexes had scores ranging from -217 to -220 kcal/mol. Molecular simulation analysis revealed variations in the dynamics features of both mutant and wild-type complexes due to the acquired mutations. Furthermore, the total binding free energy for the wild-type E2F1-RB complex was -64.89 kcal/mol, while those of the L206W, R232C, I254T, and A267T E2F1-RB mutants were -45.90 kcal/mol, -53.52 kcal/mol, -55.67 kcal/mol, and -61.22 kcal/mol, respectively. Our study is the first to extensively analyze E2F1 gene mutations and identifies candidate mutations for further validation and potential targeting for cancer therapeutics.

Patterns in the tapestry of chromatin-bound RB.

The retinoblastoma protein (RB)-mediated regulation of E2F is a component of a highly conserved cell cycle machine. However, RB's tumor suppressor activity, like RB's requirement in animal development, is tissue-specific, context-specific, and sometimes appears uncoupled from cell proliferation. Detailed new information about RB's genomic distribution provides a new perspective on the complexity of RB function, suggesting that some of its functional specificity results from context-specific RB association with chromatin. Here we summarize recent evidence showing that RB targets different types of chromatin regulatory elements at different cell cycle stages. RB controls traditional RB/E2F targets prior to S-phase, but, when cells proliferate, RB redistributes to cell type-specific chromatin loci. We discuss the broad implications of the new data for RB research.

CDK4/6 Inhibition With Lerociclib is a Potential Therapeutic Strategy for the Treatment of Pediatric Sarcomas.

BACKGROUND: Sarcomas are a heterogenous collection of bone and soft tissue tumors. The heterogeneity of these tumors makes it difficult to standardize treatment. CDK 4/6 inhibitors are a family of targeted agents which limit cell cycle progression and have been shown to be upregulated in sarcomas. In the current preclinical study, we evaluated the effects of lerociclib, a CDK4/6 inhibitor, on pediatric sarcomas in vitro and in 3D bioprinted tumors. METHODS: The effects of lerociclib on viability, proliferation, cell cycle, motility, and stemness were assessed in established sarcoma cell lines, U-2 OS and MG-63, as well as sarcoma patient-derived xenografts (PDXs). 3D printed biotumors of each of the U-2 OS, MG-63, and COA79 cells were utilized to study the effects of lerociclib on tumor growth ex vivo. RESULTS: CDK 4/6, as well as the intermediaries retinoblastoma protein (Rb) and phosphorylated Rb were identified as targets in the four sarcoma cell lines. Lerociclib treatment induced cell cycle arrest, decreased proliferation, motility, and stemness of sarcoma cells. Treatment with lerociclib decreased sarcoma cell viability in both traditional 2D culture as well as 3D bioprinted microtumors. CONCLUSIONS: Inhibition of CDK 4/6 activity with lerociclib was efficacious in traditional 2D sarcoma cell culture as well as in 3D bioprints. Lerociclib holds promise and warrants further investigation as a novel therapeutic strategy for management of these heterogenous groups of tumors.

Targeting the E2F1/Rb/HDAC1 axis with the small molecule HR488B effectively inhibits colorectal cancer growth.

Colorectal cancer (CRC), the third most common cancer worldwide, remains highly lethal as the disease only becomes symptomatic at an advanced stage. Growing evidence suggests that histone deacetylases (HDACs), a group of epigenetic enzymes overexpressed in precancerous lesions of CRC, may represent promising molecular targets for CRC treatment. Histone deacetylase inhibitors (HDACis) have gradually become powerful anti-cancer agents targeting epigenetic modulation and have been widely used in the clinical treatment of hematologic malignancies, while only few studies on the benefit of HDACis in the treatment of CRC. In the present study, we designed a series of small-molecule Thiazole-based HDACis, among which HR488B bound to HDAC1 with a high affinity and exerted effective anti-CRC activity both in vitro and in vivo. Moreover, we revealed that HR488B specifically suppressed the growth of CRC cells by inducing cell cycle G0/G1 arrest and apoptosis via causing mitochondrial dysfunction, reactive oxygen species (ROS) generation, and DNA damage accumulation. Importantly, we noticed that HR488B significantly decreased the expression of the E2F transcription factor 1 (E2F1), which was crucial for the inhibitory effect of HR488B on CRC. Mechanistically, HR488B obviously decreased the phosphorylation level of the retinoblastoma protein (Rb), and subsequently prevented the release of E2F1 from the E2F1/Rb/HDAC1 complex, which ultimately suppressed the growth of CRC cells. Overall, our study suggests that HR488B, a novel and efficient HDAC1 inhibitor, may be a potential candidate for CRC therapy in the future. Furthermore, targeting the E2F1/Rb/HDAC1 axis with HR488B provides a promising therapeutic avenue for CRC.

Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity.

Cyclin-dependent kinases 4 and 6 (CDK4/6) are critical for initiating cell proliferation by inactivating the retinoblastoma (Rb) protein. However, mammalian cells can bypass CDK4/6 for Rb inactivation. Here we show a non-canonical pathway for Rb inactivation and its interplay with external signals. We find that the non-phosphorylated Rb protein in quiescent cells is intrinsically unstable, offering an alternative mechanism for initiating E2F activity. Nevertheless, this pathway incompletely induces Rb-protein loss, resulting in minimal E2F activity. To trigger cell proliferation, upregulation of mitogenic signaling is required for stabilizing c-Myc, thereby augmenting E2F activity. Concurrently, stress signaling promotes Cip/Kip levels, competitively regulating cell proliferation with mitogenic signaling. In cancer, driver mutations elevate c-Myc levels, facilitating adaptation to CDK4/6 inhibitors. Differentiated cells, despite Rb-protein loss, maintain quiescence through the modulation of c-Myc and Cip/Kip levels. Our findings provide mechanistic insights into an alternative model of cell-cycle entry and the maintenance of quiescence.

Combined inactivation of RB and Hippo converts differentiating Drosophila photoreceptors into eye progenitor cells through derepression of homothorax.

The retinoblastoma (RB) and Hippo pathways interact to regulate cell proliferation and differentiation. However, the mechanism of interaction is not fully understood. Drosophila photoreceptors with inactivated RB and Hippo pathways specify normally but fail to maintain their neuronal identity and dedifferentiate. We performed single-cell RNA sequencing to elucidate the cause of dedifferentiation and to determine the fate of these cells. We find that dedifferentiated cells adopt a progenitor-like fate due to inappropriate activation of the retinal differentiation suppressor homothorax (hth) by Yki/Sd. This results in the activation of a distinct Yki/Hth transcriptional program, driving photoreceptor dedifferentiation. We show that Rbf physically interacts with Yki and, together with the GAGA factor, inhibits the hth expression. Thus, RB and Hippo pathways cooperate to maintain photoreceptor differentiation by preventing inappropriate expression of hth in differentiating photoreceptors. Our work highlights the importance of both RB and Hippo pathway activities for maintaining the state of terminal differentiation.

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.

CSE1L is a negative regulator of the RB-DREAM pathway in p53 wild-type NSCLC and can be targeted using an HDAC1/2 inhibitor.

P53 represses transcription by activating p21 expression and promoting formation of RB1-E2F1 and RBL1/RBL2-DREAM transcription repressor complexes. The DREAM complex is composed of DP1, RB-family proteins RBL1 or RBL2 (p107/p130), E2F4/5, and MuvB. We recently reported RBL2-DREAM contributes to improved therapy responses in p53 wild-type NSCLC cells and improved outcomes in NSCLC patients whose tumors express wild-type p53. In the current study we identified CSE1L as a novel inhibitor of the RBL2-DREAM pathway and target to activate RBL2-DREAM in NSCLC cells. CSE1L is an oncoprotein that maintains repression of genes that can be reactivated by HDAC inhibitors. Mocetinostat is a HDAC inhibitor in clinical trials with selectivity against HDACs 1 and 2. Knockdown of CSE1L in NSCLC cells or treatment with mocetinostat increased p21, activated RB1 and RBL2, repressed DREAM target genes, and induced toxicity in a manner that required wild-type p53. Lastly, we found high levels of CSE1L and specific DREAM-target genes are candidate markers to identify p53 wild-type NSCLCs most responsive to mocetinostat. Thus, we identified CSE1L as a critical negative regulator of the RB-DREAM pathway in p53 wild-type NSCLC that can be indirectly targeted with HDAC1/2 inhibitors (mocetinostat) in current clinical trials. High expression of CSE1L and DREAM target genes could serve as a biomarker to identify p53 wild-type NSCLCs most responsive to this HDAC1/2 inhibitor.

Metastatic Competency and Tumor Spheroid Formation Are Independent Cell States Governed by RB in Lung Adenocarcinoma.

Inactivation of the retinoblastoma (RB) tumor suppressor in lung adenocarcinoma is associated with the rapid acquisition of metastatic ability and the loss of lung cell lineage commitment. We previously showed that restoration of RB in advanced lung adenocarcinomas in the mouse was correlated with a decreased frequency of lineage decommitted tumors and overt metastases. To identify a causal relationship for RB and its role in reprogramming lineage commitment and reducing metastatic competency in lung adenocarcinoma, we developed multiple tumor spheroid forming lines where RB restoration could be achieved after characterization of the degree of each spheroid's lineage commitment and metastatic ability. Surprisingly, we discovered that RB inactivation dramatically promoted tumor spheroid forming potential in tumors that arise in the KrasLSL-G12D/+; p53flox/flox lung adenocarcinoma model. However, RB reactivation had no effect on the maintenance of tumor spheroid lines once established. In addition, we show that RB-deficient tumor spheroid lines are not uniformly metastatically competent but are equally likely to be nonmetastatic. Interestingly, unlike tumor spheroid maintenance, RB restoration could functionally revert metastatic tumor spheroids to a nonmetastatic cell state. Thus, strategies to reinstate RB pathway activity in lung cancer may reverse metastatic ability and have therapeutic potential. Finally, the acquisition of tumor spheroid forming potential reflects underlying cell state plasticity, which is often predictive of, or even conflated with metastatic ability. Our data support that each is a discrete cell state restricted by RB and question the suitability of tumor spheroid models for their predictive potential of advanced metastatic tumor cell states. SIGNIFICANCE: Members of the RB pathway are frequently mutated in lung adenocarcinoma. We show that RB regulates cell state plasticity, tumor spheroid formation, and metastatic competency. Our data indicate that these are independent states where spheroid formation is distinct from metastatic competency. Thus, we caution against conflating spheroid formation and other signs of cell state plasticity with advanced metastatic cell states. Nevertheless, our work supports clinical strategies to reactivate RB pathways.

RBL2-E2F-GCN5 guide cell fate decisions during tissue specification by regulating cell-cycle-dependent fluctuations of non-cell-autonomous signaling.

The retinoblastoma family proteins (RBs) and E2F transcription factors are cell-autonomous regulators of cell-cycle progression, but they also impact fate choice in addition to tumor suppression. The range of mechanisms involved remains to be uncovered. Here, we show that RBs, particularly RBL2/p130, repress WNT ligands such as WNT4 and WNT8A, thereby directing ectoderm specification between neural crest to neuroepithelium. RBL2 achieves this function through cell-cycle-dependent cooperation with E2Fs and GCN5 on the regulatory regions of WNT loci, which direct neuroepithelial versus neural crest specification by temporal fluctuations of WNT/ß-catenin and DLL/NOTCH signaling activity. Thus, the RB-E2F bona fide cell-autonomous axis controls cell fate decisions, and RBL2 regulates field effects via WNT ligands. This reveals a non-cell-autonomous function of RBL2-E2F in stem cell and tissue progenitor differentiation that has broader implications for cell-cycle-dependent cell fate specification in organogenesis, adult stem cells, tissue homeostasis, and tumorigenesis.

FRMD8 targets both CDK4 activation and RB degradation to suppress colon cancer growth.

Cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (RB) are both important cell-cycle regulators that function in different scenarios. Here, we report that FERM domain-containing 8 (FRMD8) inhibits CDK4 activation and stabilizes RB, thereby causing cell-cycle arrest and inhibiting colorectal cancer (CRC) cell growth. FRMD8 interacts separately with CDK7 and CDK4, and it disrupts the interaction of CDK7 with CDK4, subsequently inhibiting CDK4 activation. FRMD8 competes with MDM2 to bind RB and attenuates MDM2-mediated RB degradation. Frmd8 deficiency in mice accelerates azoxymethane/dextran-sodium-sulfate-induced colorectal adenoma formation. The FRMD8 promoter is hypermethylated, and low expression of FRMD8 predicts poor prognosis in CRC patients. Further, we identify an LKCHE-containing FRMD8 peptide that blocks MDM2 binding to RB and stabilizes RB. Combined application of the CDK4 inhibitor and FRMD8 peptide leads to marked suppression of CRC cell growth. Therefore, using an LKCHE-containing peptide to interfere with the MDM2-RB interaction may have therapeutic value in CDK4/6 inhibitor-resistant patients.

Dexamethasone induces cancer mitigation and irreversible senescence in lung cancer cells via damaging cortical actin and sustained hyperphosphorylation of pRb.

Activation of the glucocorticoid receptors by its cognate ligand, dexamethasone (DEX) is commonly used as an adjuvant treatment in solid tumors. However, its direct effect on cancerous phenotype is not fully understood. We explored the effect and molecular mechanisms of DEX action in lung cancer. In in vitro experiments, DEX treatment causes decrease in migration, invasion and colony formation ability of A549 cells even at lower doses. DEX also decreased adhesion of A549 cells by reducing the formation of cortical actin. Treatment with RU486, a GR antagonist, indicated that these effects are partially mediated through GR. Further; DEX induces G0/G1 arrest of A549 cells. Mechanistically, DEX induces expression of both CDK inhibitors (p21Cip1, p27Kip1) and cyclin-dependent kinases (CDK4, CDK6). Due to this compensatory activation of CDKs and CDKIs, DEX induces the hyper phosphorylation state of Rb protein (pRb) leading to irreversible senescence as confirmed by ß-gal staining. Next, in clinical dataset of NSCLC (Non-small cell lung cancer), GR was lowly expressed in cancer patients as compared to the normal group, where higher expression of GR led to higher overall survival of NSCLC indicating for a protective role of GR. Interestingly, when combined with chemotherapeutic agents, DEX can modulate the drug-sensitivity of cells. Taken together, these data indicate that DEX through GR activation may suppress tumor growth by decreasing proliferation and inducing irreversible senescence and combination of standard chemotherapy and DEX can be a potential treatment for NSCLC.

Loss of CDK4/6 activity in S/G2 phase leads to cell cycle reversal.

In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle1,2, when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein3-5. Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell's progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling.

Preferential killing of melanoma cells by a p16-related peptide.

We report the identification of a synthetic, cell-penetrating peptide able to kill human melanoma cells efficiently and selectively, while being less toxic to normal human melanocytes and nontoxic to human fibroblasts. The peptide is based on the target-binding site of the melanoma suppressor and senescence effector p16 (also known as INK4A or CDKN2A), coupled to a cell-penetrating moiety. The killing is by apoptosis and appears to act by a route other than the canonical downstream target of p16 and CDK4, the retinoblastoma (RB) protein family, as it is also effective in HeLa cells and a melanocyte line expressing HPV E7 oncogenes, which both lack any active RB. There was varying toxicity to other types of cancer cell lines, such as glioblastoma. Melanoma cell killing by a p16-derived peptide was reported once before but only at a higher concentration, while selectivity and generality were not previously tested.