Including Cell Cycle Checkpoints Research Articles

SRSF9 promotes cell proliferation and migration of glioblastoma through enhancing CDK1 expression

BackgroundGlioblastoma (GBM) is a highly aggressive and prevalent brain tumor that poses significant challenges in treatment. SRSF9, an RNA-binding protein, is essential for cellular processes and implicated in cancer progression. Yet, its function and mechanism in GBM need clarification.MethodsBioinformatics analysis was performed to explore differential expression of SRSF9 in GBM and its prognostic relevance to glioma patients. SRSF9 and CDK1 expression in GBM cell lines and patients’ tissues were quantified by RT-qPCR, Western blot or immunofluorescence assay. The role of SRSF9 in GBM cell proliferation and migration was assessed by MTT, Transwell and colony formation assays. Additionally, transcriptional regulation of CDK1 by SRSF9 was investigated using ChIP-PCR and dual-luciferase assays.ResultsThe elevated SRSF9 expression correlates to GBM stages and poor survival of glioma patients. Through gain-of-function and loss-of-function strategies, SRSF9 was demonstrated to promote proliferation and migration of GBM cells. Bioinformatics analysis showed that SRSF9 has an impact on cell growth pathways including cell cycle checkpoints and E2F targets. Mechanistically, SRSF9 appears to bind to the promoter of CDK1 gene and increase its transcription level, thus promoting GBM cell proliferation.ConclusionsThese findings uncover the cellular function of SRSF9 in GBM and highlight its therapeutic potential for GBM.

Dual Synergistic Tumor-Specific Polymeric Nanoparticles for Efficient Chemo-Immunotherapy.

Chemo-immunotherapy has made significant progress in cancer treatment. However, the cancer cell self-defense mechanisms, including cell cycle checkpoint and programmed cell death-ligand 1 (PD-L1) upregulation, have greatly hindered the therapeutic efficacy. Herein, norcantharidin (NCTD)-platinum (Pt) codelivery nanoparticles (NC-NP) with tumor-sensitive release profiles are designed to overcome the self-defense mechanisms via synergistic chemo-immunotherapy. NC-NP remains stable under normal physiological conditions but quickly releases 1,2-diaminocyclohexane-platinum(II) (DACHPt, a parent drug of oxaliplatin) and NCTD in response to the tumor acidity. NCTD inhibits protein phosphatase 2A (PP2A) activity to relieve cell cycle arrest and downregulates the tumor PD-L1 expression to disrupt the programmed cell death-1 (PD-1)/PD-L1 interaction, synergistically enhancing Pt-based chemotherapy and immunogenic cell death-induced immunotherapy. As a result, NC-NP exhibits potent synergistic cytotoxicity and promotes T cell recruitment to generate robust antitumor immune responses. The dual synergism exhibits potent antitumor activity against orthotopic 4T1 tumors, providing a promising chemo-immunotherapy paradigm for cancer treatment.

The Significant Role of microRNAs in Gliomas Angiogenesis: A Particular Focus on Molecular Mechanisms and Opportunities for Clinical Application.

MicroRNAs (miRNAs) are non-coding RNAs with only 20-22 nucleic acids that inhibit gene transcription and translation by binding to mRNA. MiRNAs have a diverse set of target genes and can alter most physiological processes, including cell cycle checkpoints, cell survival, and cell death mechanisms, affecting the growth, development, and invasion of various cancers, including gliomas. So optimum management of miRNA expression is essential for preserving a normal biological environment. Due to their small size, stability, and capability of specifically targeting oncogenes, miRNAs have emerged as a promising marker and new biopharmaceutical targeted therapy for glioma patients. This review focuses on the most common miRNAs associated with gliomagenesis and development by controlling glioma-determining markers such as angiogenesis. We also summarized the recent research about miRNA effects on signaling pathways, their mechanistic role and cellular targets in the development of gliomas angiogenesis. Strategies for miRNA-based therapeutic targets, as well as limitations in clinical applications, are also discussed.

Acetylation of MLH1 by CBP increases cellular DNA mismatch repair activity.

The DNA mismatch repair (MMR) proteins recognize and repair DNA base pair mismatches and insertions/deletions of DNA that have occurred during DNA replication. Additionally, they are involved in regulation of the DNA damage response, including cell cycle checkpoints and apoptosis. Therefore, regulation of these proteins is essential for maintaining genomic integrity. It has been recognized that post-translational modifications, such as phosphorylation, ubiquitination, and acetylation, are being used as an important means to regulate the functions and stability of MMR proteins. Here, we report that a histone acetyltransferase CREB binding protein (CBP) interacts with and acetylates MLH1, a component of the MutLα complex (MLH1-PMS2). Moreover, CBP stabilizes MLH1 by preventing it from degradation via the ubiquitin-proteasome degradation pathway. Consistently, acetylation induced by a pan-histone deacetylase inhibitor, Trichostatin A, promotes the assembly between the MutSα (MSH2-MSH6) and MutLα complexes. Furthermore, overexpression of CBP enhances MMR activities in cells. Overall, our results suggest a novel role of CBP in prolonging MLH1 stability and enhancing MutSα-MutLα complex formation, leading to increased cellular MMR activity.

Abstract 5686: Integrative genomic analysis reveals hypermethylation triggered GPER1 loss is associated with genomic instability of endometrial cancer

Abstract Objective: Endometrial cancer is a common gynecologic malignancy, yet carcinogenesis remains elusive. Evidence showed that aberrant genomic methylation profiling contributes to the development of endometrial cancer. G protein-coupled estrogen receptor (GPER1), a membrane-associated estrogen receptor, was reported to play a multifaceted role in tumors. Its function in endometrial cancer is little known. Here, we conducted an integrated analysis of RNA expression, DNA methylation, and somatic mutations data from TCGA to investigate GPER1 upstream regulator and its influence in endometrial cancer biological process. Methods: RNA-seq, DNA methylation, and clinical information of endometrial cancer (n=554) and normal control (n=33) were obtained from TCGA database. RNA-seq profiles of GPER1 low and high groups were analyzed by R 4.1.0 software. KEGG and GSEA based on GPER1 expression were conducted. Correlation analysis of GPER1 and genomic instability were displayed. G1, the GPER1 agonist, was added to observe cell viability and metastatic ability in vitro. DNA methylation data was analyzed. Results: GPER1 expression was decreased in endometrial cancer compared with normal tissues. GPER1 was associated with poor overall survival (HR=0.52, p=0.003) (Figure 1). Low expression group showed a more frequent mutation landscape, higher tumor mutation burden (TMB), and micro-satellite instability (MSI) (Figure 2). Several hypermethylation loci on GPER1 CpG island were revealed. Methylation status is negatively correlative to RNA expression of GPER1 (Figure 3). P-value <0.05 and log2FC >1.5, 473 up-regulated and 114 down-regulated genes were screened. Up-regulated DEGs enriched in the Estrogen signaling pathway, Endometrial cancer, wnt signaling pathway, etc. Down-regulated DEGs enriched in Cell cycle, p53 signaling pathway, etc. (Figure 4). GSEA analysis showed that GPER1 low expression group was enriched in several biological processes including cell cycle checkpoint, Recombinational repair, etc. G1 inhibited endometrial cancer cell growth in a dose-dependent manner, and reduced cell metastatic ability (Figure 5). Conclusions: GPER1 exhibited decreased expression in endometrial cancer and DNA hypermethylation in promoter CpG island locus. The low GPER1 expression group showed prominent genomic instability. GPER1 agonists inhibit endometrial cancer cell growth and metastasis, which may serve as a therapeutic target. Citation Format: Xuan Feng, Ting Huang, Boqiang Shen, Juan Hao, Jiaqi Wang, Yangyang Dong, Jingyi Zhou, Jianliu Wang. Integrative genomic analysis reveals hypermethylation triggered GPER1 loss is associated with genomic instability of endometrial cancer [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 5686.

Anticancer and chemosensitization effects of Wee‐1 kinase inhibitor Adavosertib (MK‐1775; AZD‐1775) in breast cancer cell lines

Current breast cancer classification included cells that express estrogen (ER) and progesterone (PR) receptors, those with ERBB2 (HER‐2/Neu) amplification, and those without expression of ER, PR, or amplification of ERBB2 (referred to as triple‐negative or basal‐like breast cancer). Triple negative breast cancers (TNBC) are generally more difficult to treat, are known to be more aggressive, and with poor prognosis. Several molecular mechanisms have been identified as potential targets for the development of drugs to treat breast cancer including cell cycle checkpoints kinases. The G2/M cell‐cycle protein tyrosine kinase, Wee‐1, plays a crucial role in the checkpoint arrest for DNA repair before mitotic entry. Under normal conditions, cells repair damaged DNA during G1 arrest; however, cancer cells often have a deficient G1/S (p53) checkpoint and depend on a functional G2/M checkpoint for DNA repair. Adavosertib is a well‐characterized Wee‐1 kinase inhibitor with significant evidence supporting its potential use as an anti‐cancer and/or chemosensitizer drug. Working hypothesis proposed for this project was that use of Wee‐1 kinase inhibitors in breast cancer cells can result in profound and significant anticancer and chemosensitization effects. In order to test this hypothesis, we performed a series of dose‐response experiments using a well‐known DNA‐Damaging agent (DNA‐dt: Cisplatin) alone and/or in combination with Adavosertib. Cytotoxic effects of these treatments were evaluated with the MDA‐MB‐231 (TNBC cells) and the non‐tumorigenic MCF10A, as our in‐vitro cell model systems, and the 384‐well plate/96 hours/resazurin (AlamarBlue) cell proliferation assay. Analysis of these results included determination of half‐maximal inhibitory or effective concentration (IC50 or EC50) for each drug treatment and/or combination; and the nature of these interactions (synergy, additivity, antagonism) was determined with the open‐access software CompuSyn, Combenefit, and SynergyFinder. Analysis of the potential for interactions of cisplatin and Adavosertib in MDA‐MB‐231 indicated significant synergism as illustrated by surface and matrix synergy plots, combination index determinations, and isobolograms. However, no significant synergistic interactions were observed when these two drugs are combined in the non‐tumorigenic MCF10A cells. Finally, studies designed to evaluate the possibility that these synergistic effects can also be extended to the levels of expression of several biochemical markers (including pCDC2 and γH2AX) and cell cycle duration are currently underway. These preliminary results are consistent with a potential role of Wee‐1 kinase inhibitors like Adavosertib as anticancer and/or chemosensitizer agents against triple‐negative or basal‐like breast cancer cells.

HSPB11 is a Prognostic Biomarker Associated with Immune Infiltrates in Hepatocellular Carcinoma.

PurposeHeat shock proteins (HSPs) play important roles in oncogenesis and malignant progression. HSPB11 is highly expressed in many malignant tumors, but research on its role in hepatocellular carcinoma (HCC) is insufficient.Patients and MethodsA comprehensive analysis of HSPB11 in HCC was performed based on data of patients with HCC and those from online public databases.ResultsHSPB11 was overexpressed in HCC, with a high discrimination ability between tumor and normal tissues (area under the curve =0.923). HSPB11 overexpression correlated with advanced tumor stage, poorer tumor differentiation, and worse prognosis and was an independent risk factor for HCC prognosis. The nomogram and calibration models composed of HSPB11, T stage, and M stage had good abilities to predict the 1-, 3-, and 5-year survival rates of patients. HSPB11 was determined to be involved in multiple oncogenic processes, including cell cycle checkpoints, the G2M checkpoint, E2F targets, Rho GTPases, and KRAS signaling. HSPB11 expression was related to immune cell infiltration, especially that of Th2 cells and dendritic cells.ConclusionHSPB11 is involved in oncogenesis and immune regulation in HCC and is a potential prognostic biomarker and therapeutic target.

Giants and monsters: Unexpected characters in the story of cancer recurrence.

Polyploid giant cancer cells (PGCC) constitute a dangerous subpopulation of cancer cells and are a driving force in cancer recurrence. These unique cells arise from diploid tumor cells in response to stress encountered in the tumor microenvironment or during cancer therapy. PGCC are greatly dedifferentiated, acquire pluripotency, and are able to replicate through a form of asymmetric division called neosis, which results in new populations that are themselves able to differentiate into new cell types or to re-establish tumors. Progeny tend to be more genetically unstable than the founding population due to the dysregulation required to transition through a PGCC state. Therefore, cancers that escape stressors through this mechanism tend to re-emerge with a more aggressive phenotype that is therapy resistant. This review focuses on the clinical significance of PGCC, the need for standardized nomenclature and molecular markers, as well as possible avenues to develop therapies aimed at PGCC and the process of neosis. The biology underlying the development of PGCC including cell cycle checkpoint dysregulation, stress responses, dedifferentiation, stemness and epithelial-mesenchymal transition is discussed.

Anticancer and Chemosensitizing Effects of WEE‐1 Kinase Inhibitor MK‐1775 in Triple Negative (MB‐231) Breast Cancer Cells

Current breast cancer classification included cells that express estrogen (ER) and progesterone (PR) receptors, those with ERBB2 (HER‐2/Neu) amplification, and those without expression of ER, PR, or amplification of ERBB2 (referred to as triple‐negative or basal‐like breast cancer). Triple negative breast cancers are generally more difficult to treat, are known to be more aggressive and with poor prognosis. Several molecular mechanisms have been identified as potential targets for development of drugs to treat breast cancer including cell cycle checkpoints kinases. The G2/M cell‐cycle protein tyrosine kinase, Wee‐1, plays a crucial role in the checkpoint arrest for DNA repair before mitotic entry. Under normal conditions, cells repair damaged DNA during G1 arrest; however, cancer cells often have a deficient G1/S (p53) checkpoint and depend on a functional G2/M checkpoint for DNA repair. MK‐1775 is a well‐characterized Wee‐1 kinase inhibitor with significant evidence supporting its potential use as anti‐cancer and/or chemosensitizing drug. Working hypothesis proposed for this project was that use of Wee‐1 kinase inhibitors in breast cancer cells can result in profound and significant anticancer and chemosensitizing effects. In order to test this hypothesis, we show previously in a series of dose response experiments using DNA‐Damaging agents or treatments (DNA‐dt: Ex. Cisplatin, 5‐Fluororacil and Doxorubicin) alone and/or in combination with MK‐1775. To continue testing this hypothesis an Immunoblotting bioassay is currently developed for continue monitoring the effects of MK‐1775 in several biochemical markers in triple negative breast cancer cell line. Preliminary results indicated that biochemical marker in triple negative cell line, are time dependent with the different combination of DNA‐dt alone and/or in combination with MK‐1775. Initial preliminary results indicated taken together, our preliminary results are consistent with a potential role of Wee‐1 kinase inhibitors like MK‐17175 as anticancer and/or chemosensitizing agents against triple‐negative or basal‐like breast cancer cells.Support or Funding InformationThe project described was supported by NIGMS/INBRE award P20 GM103475; U.S. Department of Education Title V grant number P031S130068 and Puerto Rico Science Technology and Research Trust. The content is solely the responsibility of the authors and does not necessarily represent the official view of any of the supporting agencies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Defenses against Pro-oxidant Forces - Maintenance of Cellular and Genomic Integrity and Longevity.

There has been enormous recent progress in understanding how human cells respond to oxidative stress, such as that caused by exposure to ionizing radiation. We have witnessed a significant deciphering of the events that underlie how antioxidant responses counter pro-oxidant damage to key biological targets in all cellular compartments, including the genome and mitochondria. These cytoprotective responses include: 1. The basal cellular repertoire of antioxidant capabilities and its supporting cast of facilitator enzymes; and 2. The inducible phase of the antioxidant response, notably that mediated by the Nrf2 transcription factor. There has also been frenetic progress in defining how reactive electrophilic species swamp existing protective mechanisms to augment DNA damage, events that are embodied in the cellular "DNA-damage response", including cell cycle checkpoint activation and DNA repair, which occur on a time scale of hours to days, as well as the implementation of cellular responses such as apoptosis, autophagy, senescence and reprograming that extend the time period of damage sensing and response into weeks, months and years. It has become apparent that, in addition to the initial oxidative insult, cells typically undergo further waves of secondary reactive oxygen/nitrogen species generation, DNA damage and signaling and that these may reemerge long after the initial events have subsided, probably being driven, at least in part, by persisting DNA damage. These reactive oxygen/nitrogen species are an integral part of the pathological consequences of radiation exposure and may persist across multiple cell divisions. Because of the pervasive nature of oxidative stress, a cell will manifest different responses in different subcellular compartments and to different levels of stress injury. Aspects of these compartmentalized responses can involve the same proteins (such as ATM, p53 and p21) but in different functional guises, e.g., in cytoplasmic versus nuclear responses or in early- versus late-phase events. Many of these responses involve gene activation and new protein synthesis as well as a plethora of post-translational modifications of both basal and induced response proteins. It is these responses that we focus on in this review.

Abstract LB-275: Targeting poor-prognosis subtypes of prostate cancer by inhibition of DNA repair gene ribonucleotide reductase small subunit M2

Abstract Studies reveal defective DNA repair contribute to prostate cancer (PC) progression. We hypothesize that overexpression of DNA repair genes could also contribute to poorer outcomes in PC. The nucleotide metabolism enzyme ribonucleotide reductase (RNR) is essential for DNA synthesis and DNA repair by producing dNTPs. The small subunit M2 (RRM2), as the rate-limiting RNR subunit, is frequently up-regulated in cancers. Clinically, targeting of RRM2 with small molecules is being tested in multiple cancers, but there is little knowledge of RRM2 function in PC. The analysis of multiple PC clinical cohorts (total include 1602 cases) revealed that high RRM2 level was associated with poor clinical outcomes, including a higher likelihood of metastasis (p<0.001), biochemical recurrence (p<0.001), and lethality (p<0.0001). In PC cells, knockdown of RRM2 inhibited dNTP production and induced DNA damage, which led to significant cell growth inhibition, major S phase arrest, and apoptosis. Overexpression of RRM2 promoted epithelial-mesenchymal transition (EMT) by increasing the expression of multiple EMT markers. Furthermore, the small molecule RRM2 inhibitor (COH29) induced a similar phenotype as knocking down RRM2 in PC cells. RNA-Seq analysis in siRRM2 or COH29 treated PC cells provided a global assessment of RRM2-regulated transcriptome changes. GSEA analysis revealed that inhibition of RRM2 could activate biological processes including cell cycle checkpoint, DNA damage response, and apoptotic signaling. COH29 treatment could target genes highly enriched in PC. We further applied an RRM2-regulated gene signature (from RNA-Seq datasets) to TCGA and Taylor cohorts. Intriguingly, the RRM2 signature was highly correlated with metastasis and disease free survival (p<0.001). Furthermore, inhibition of RRM2 specifically targets poor prognostic luminal subtypes (PCS1 subtype; Lum B in PAM50 classifier) recently reported. Besides transcriptome changes, protein kinase arrays showed that AKT/mTOR and SFK-STAT signaling were repressed by inhibition of RRM2. These oncogenic signaling pathways are crucial for EMT program. Amplification of RRM2 is rare in PC and transcriptional activation of RRM2 may play a major role in overexpression of RRM2. H3K27ac ChIP-Seq from tissues revealed more activated RRM2 promoter in PC than in normal prostate. 13 potential RRM2-targeting transcription factors (TFs) were identified by integrating clinical cohorts and a TF database. They showed a positive correlation with RRM2 expression in PC cohorts. Among these TFs, FOXM1 was reported to be the master driver of the aggressive luminal subtype of PC. We revealed that FOXM1 expression was associated with clinical outcomes. The ChIP-PCR and luciferase reporter assays provided evidence of physical binding of FOXM1 to the RRM2 promoter in PC cells. Knockdown of FOXM1 significantly repressed RRM2 mRNA and protein levels. Altogether, FOXM1-regulated transcriptional activation contributes to overexpression of RRM2. Intriguingly, COH29 can also repress FOXM1 expression, which leads to transcription repression of RRM2. Altogether, our study elucidated the molecular mechanisms underlying RRM2 oncogenic functions and the transcriptional regulation of RRM2 in PC cells. We suggest that RRM2 can be a novel therapeutic target for PC treatment. Citation Format: Ying Z. Mazzu, Joshua Armenia, Goutam Chakraborty, Yuki Yoshikawa, Travis A. Gerke, Si Ana A. Coggins, Xintao Qiu, Mohammad Atiq, Konrad H. Stopsack, Gwo-Shu Mary Lee, Henry W. Long, Baek Kim, Matthew L. Freedman, Mark M. Pomerantz, Lorelei A. Mucci, Philip W. Kantoff. Targeting poor-prognosis subtypes of prostate cancer by inhibition of DNA repair gene ribonucleotide reductase small subunit M2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-275.

ANTICANCER AND CHEMOSENSITIZING EFFECTS OF WEE‐1 KINASE INHIBITOR MK‐1775 IN TRIPLE NEGATIVE (MB‐231) BREAST CANCER CELLS

Current breast cancer classification included cells that express estrogen (ER) and progesterone (PR) receptors, those with ERBB2 (HER‐2/Neu) amplification, and those without expression of ER, PR, or amplification of ERBB2 (referred to as triple‐negative or basal‐like breast cancer). Triple negative breast cancers are generally more difficult to treat, are known to be more aggressive and with poor prognosis. Several molecular mechanisms have been identified as potential targets for development of drugs to treat breast cancer including cell cycle checkpoints kinases. The G2/M cell‐cycle protein tyrosine kinase, Wee‐1, plays a crucial role in the checkpoint arrest for DNA repair before mitotic entry. Under normal conditions, cells repair damaged DNA during G1 arrest; however, cancer cells often have a deficient G1/S (p53) checkpoint and depend on a functional G2/M checkpoint for DNA repair. MK‐1775 is a well‐characterized Wee‐1 kinase inhibitor with significant evidence supporting its potential use as anti‐cancer and/or chemosensitizing drug. Working hypothesis proposed for this project was that use of Wee‐1 kinase inhibitors in triple negative breast cancer cells can result in profound and significant anticancer and chemosensitizing effects. In order to test this hypothesis we performed a series of dose response experiments using DNA‐Damaging agents or treatments (DNA‐dt: Ex. Cisplatin, 5‐Fluororacil and Doxorubicin) alone and/or in combination with MK‐1775. Cytotoxic effects of these treatments where evaluated with the MDA‐MB‐231 triple negative breast cancer cells as our model system and the 384‐well plate/96 hours/resazurin (AlamarBlue) cell proliferation assay. Analysis of these results included determination of half maximal inhibitory or effective concentration (IC50 or EC50) for each drug treatment and/or combinations followed by assessment of synergy and/or antagonism analysis based on Combenefit software package. Use of this software allows for visualization, analysis and quantification of drug combination effects including surface analyses of drug combinations to identify synergy using classical synergy models. Initial preliminary results indicated IC50/EC50 values for cisplatin (5.88μM), 5‐Fluorouracil (82.55μM), Doxorubicin (0.353μM), and MK‐1775 (0.307μM) similar to the ones reported in the literature. Moreover, initial results are also consistent with a significant degree of potentiation or chemosensitization of MB‐231 cells to the cytotoxic effects of our selected DNA‐dt when combined with low MK‐1775 concentrations. Taken together, our preliminary results are consistent with a potential role of Wee‐1 kinase inhibitors like MK‐17175 as anticancer and/or chemosensitizing agents against triple‐negative or basal‐like breast cancer cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

19p13.3-GADD45B common variants and 19q13.3-PPP1R13L and 19q13.3-CD3EAP in lung cancer risk among Chinese

Lung cancer is the most common cause of cancer-related mortality worldwide. The GADD45 gene family plays important roles in a variety of the responses to cell injury including cell cycle checkpoints, apoptosis, DNA repair and anti-tumor immunity. The 19p13.3-GADD45B encoded protein product is involved in apoptosis and inhibiting tumor growth. To evaluate the association of 19p13.3-GADD45B common variants and lung cancer risk, the present study containing 544 Chinese lung cancer cases and 550 cancer-free controls was conducted. Three htSNPs (haplotype-tagging single nucleotide polymorphism) (rs7354, rs14384, and rs3783501) covering 95% of the common haplotype diversity in 19p13.3-GADD45B and interaction of 19p13.3-GADD45B and 19q13.3-PPP1R13L and 19q13.3-CD3EAP variants and smoking-duration were explored. Genotype and allele frequencies and haplotype distributions of the 19p13.3-GADD45B 3 htSNPs were not associated with lung cancer risk after adjustment for smoking status. 19p13.3-GADD45B rs7354 was associated with lung cancer risk among ≤20 (years) smokers [C/A-A/A versus CC, OR (95% CI) = 3.20 (1.11–9.20), P = 0.025] in a dominant model stratified by smoking duration. MDR (multifactor dimensionality reduction) analyses showed that smoking history as main effect and three-way models (smoking duration, 19p13.3-GADD45B rs3783501, 19q13.3-CD3EAP rs967591) (P = 0.001–0.002) indicated statistically significant association with lung cancer risk. The study identified evidence implicating DNA damage response genes on chromosome 19 in etiology of smoke-exposed lung cancer. In conclusion, our findings demonstrate that 19p13.3-GADD45B rs7354 variant and interaction between 19p13.3-GADD45B rs3783501 and 19q13.3-CD3EAP rs967591 may play a role in association with smoke-exposed lung cancer among Chinese. 19p13.3-GADD45B variants should be further evaluated in large prospective studies with molecular pathological annotations of lung cancer.

Abstract 2355: Palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in squamous cell lung cancer

Abstract Lung cancer remains one of the leading causes of cancer-related mortality. Squamous cell lung cancer (SqCLC) is the second most common subtype of non-small cell lung cancer (NSCLC) and is responsible for ~100,000 deaths in the US and EU. Most SqCLC patients receive chemotherapy as 1st line treatments and have a high un-met medical need for new therapies. Therapeutic approaches that enhance the efficacy of chemotherapy would therefore improve clinical outcomes for this patient population. CDK inhibitors comprise a class of drugs that target the dysregulated cell cycle in malignant cells. Treatment of tumor cells with the CDK4/6 inhibitor palbociclib inhibits tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Based on promising clinical trial results, palbociclib in combination with letrozole was granted accelerated approval by the US FDA for the treatment of postmenopausal women with ER-positive, HER2-negative advanced breast cancer. Like hormone receptor positive breast cancer patients, the vast majority of SqCLC patients harbor wild type RB and thus may also benefit from palbociclib treatment. Previously, we reported robust cytotoxicity and antitumor effects of palbociclib plus taxanes, including nanoparticle albumin-bound paclitaxel (Nab-PTX) or docetaxel (DTX), in several preclinical models of SqCLC. In the present study, we extended our efficacy studies of this combination to additional RB+ SqCLC models with diverse molecular genetic backgrounds. In search of mechanisms of action underlying the observed combinatorial effects, we identified several novel mechanisms, including cell cycle checkpoint abrogation as well as reduction of hypoxia-inducible factor 1 alpha (HIF-1α). Decrease in HIF-1α protein led to strong modulation of downstream genes involved in angiogenesis, resulting in reduced blood vessel size in tumor vasculature. Our results suggest that palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in SqCLC. Citation Format: Joan Cao, Zhou Zhu, Hui Wang, Tim Nichols, Edward Rosfjord, Christine Hopf, Erik Upeslacis, Paul Rejto, Scott Weinrich, Todd Vanarsdale, James Hardwick, Ping Wei. Palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in squamous cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2355. doi:10.1158/1538-7445.AM2017-2355

USP13 regulates the RAP80-BRCA1 complex dependent DNA damage response

BRCA1 regulates multiple cellular pathways that maintain genomic stability including cell cycle checkpoints, DNA repair, protein ubiquitination, chromatin remodelling, transcriptional regulation and apoptosis. Receptor-associated protein 80 (RAP80) helps recruit BRCA1 to double-strand breaks (DSBs) through the scaffold protein CCDC98 (Abraxas) and facilitates DNA damage response (DDR). However, the regulation of RAP80-BRCA1 complex is still unclear. Here we report that a deubiquitinase, USP13, regulates DDR by targeting RAP80. Mechanistically, USP13 is phosphorylated by ATM following DNA damage which, in turn, facilitates its DSB localization. USP13, in turn, deubiquitinates RAP80 and promotes RAP80 recruitment and proper DDR. Depleting or inhibiting USP13 sensitizes ovarian cancer cells to cisplatin and PARP inhibitor (olaparib) while overexpression of USP13 renders ovarian cancer cells resistant to chemotherapy. Overall, we identify USP13 as a regulator of DNA repair and reveal a model in which a phosphorylation-deubiquitination axis dynamically regulates RAP80-BRCA1 complex foci formation and function.

The enigmatic oncogene and tumor suppressor-like properties of RAD54B: Insights into genome instability and cancer.

One of the major challenges to the cell is to ensure genome stability, which can be compromised through endogenous errors or exogenous DNA damaging agents, such as ionizing radiation or common chemotherapeutic agents. To maintain genome stability the cell has a multifaceted line of defense, including cell cycle checkpoints and DNA damage repair pathways. RAD54B is involved in many of these pathways and thus exhibits a role in maintaining and repairing genome stability following DNA damage. RAD54B is involved in cell cycle regulation after DNA damage and participates in homologous recombinational repair, which ensures the precise repair of the most deleterious DNA lesions, double-stranded breaks. This review focuses on structural aspects of RAD54B, molecular functions associated with its cellular roles in preventing genome instability, and how aberrant function contributes to oncogenesis. By understanding how aberrant RAD54B expression and/or function can contribute to oncogenesis, novel therapeutic approaches that specifically exploit these aberrant genetics are now being explored for precision medicine targeting. RAD54B represents an ideal candidate for synthetic genetic therapeutic approaches (synthetic dosage lethality or synthetic lethality), which are designed to target the specific genetics associated with cancer formation. These therapeutic approaches represent a precision-based approach, which is ideal as we are now entering the era of precision medicine.

Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.

The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. We previously showed that Cdk5 (cyclin-dependent kinase 5) is activated by DNA damage and directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM pathway plays a crucial role in DNA damage-induced neuronal injury. This chapter describes protocols used in analyzing ATM phosphorylation by Cdk5 in CGNs (cerebellar granule neurons) and its effects on neuronal survival.

Research progress of checkpoint kinase 1 and DNA damage response pathway in tumors

The main reason of recurrence and metastasis in breast cancer is the resistance for the radiotherapy and chemotherapy, and the mechanism of radio-resistance and chemo-resistance may be related to the DNA damage response(DDR). There is a complicated system of the DDR pathway, including cell cycle checkpoint, DNA repair, transcription and apoptosis to maintain the integrity of cell genes. In the cancer treatment, DDR occurs in various kinds of cytotoxic drugs and radiation to cause genetic damage, which limits the curative effect of chemotherapy and radiotherapy. This promotes the targeted therapy of DDR pathway, especially checkpoint kinase 1 (CHK1). Recently, the new viewpoint supports that CHK1 is a main marker of the DDR pathway activation, which shows that CHK1 not only activates the check point but also affects the DNA repair and apoptosis directly. Thus, the role of CHK1 in DDR will promote CHK1 inhibitor to be one of the new treatment strategies for the cancer patients who resist the radiation and chemotherapy. Key words: Checkpoint kinase 1; Breast neoplasms; DNA damage response pathway

Genome wide expression analysis of radiation-induced DNA damage responses in isogenic HCT116 p53+/+ and HCT116 p53−/− colorectal carcinoma cell lines

Purpose: To study the kinetics of gene expression alterations following radiation exposure of isogenic HCT116 p53 +/+ and HCT116 p53−/− cell lines.Materials and methods: Cells were exposed to 5 Gy of irradiation (Cs-137) and genome-wide temporal expression analysis using Illumina bead chip arrays was performed. Signalling pathways were explored using Metacore (Genego). Biological responses including cell cycle checkpoint activation, centrosome amplification and senescence induction were analyzed.Results: Significant differences in the radiation response were observed between the p53+/+ and the p53−/− cell lines. In p53+/+ cells concurrent G1- and G2-arrests were activated followed by senescence induction. Increased expression of genes associated with senescence, senescence associated secretory phenotype (SASP) and repression of genes essential for G2-M transition were detected. P53−/− cells arrested mainly in G2 followed by centrosome amplification, mitotic slippage and a subsequent increase of polyploid cells. Furthermore, changes in expression correlated well with these signs of mitotic catastrophe.Conclusions: The presence or absence of p53 triggers different signalling cascades with different endpoints. Elucidating these differences is important as it enables improvement of radiation treatment and could be used to develop new combination treatments with specific inhibitors of key regulators of these cell death modalities.

ATR controls the UV‐related upregulation of the CDKN1A mRNA in a Cdk1/HuR‐dependent manner

Ultraviolet (UV) light is a carcinogenic agent that upregulates the expression of several genes involved in various cellular processes, including cell cycle checkpoints and apoptosis. The universal cyclin-dependent kinase inhibitor p21(WAF1/Cip1) plays major roles in these processes, and the level of its corresponding message increases several times in response to UV-induced DNA damage. This upregulation is mainly posttranscriptional owing to HuR-dependent mRNA stabilization. Since the protein kinase Atr plays major roles during the cellular response to UV damage, we sought to investigate its possible implication in the stabilization of the p21(WAF1/Cip1) coding mRNA. We have shown that the UV-dependent accumulation of the CDKN1A mRNA is indeed under the control of the Atr protein kinase. Upon UV damage, Atr allows nuclear-cytoplasmic shuttling of the HuR protein, which binds the CDKN1A mRNA and reduces its turnover. This ATR-dependent effect is mediated through UV-related phosphorylation/inactivation of the Cdk1 protein kinase by Atr, which leads to the dissociation of HuR from Cdk1. Indeed, inhibition or shRNA specific knockdown of CDK1 in ATR-deficient cells enhanced the cytoplasmic level of HuR and restored the CDKN1A mRNA upregulation in response to UV damage. These results show that ATR stabilizes the CDKN1A message in response to UV damage through Cdk1-related cytoplasmic accumulation of HuR.