Upregulation Of p21Cip1 Research Articles

Abstract Mo002: Pharmacological Inhibition of p38 MAPK Attenuates Doxorubicin-induced Cardiotoxicity, Senescence, and Inflammation in C57BL/6 mice

Introduction: Doxorubicin (DOX)-induced cardiotoxicity has been associated with induction of senescence, inflammation, and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Losmapimod (LOSM), an orally active p38 MAPK inhibitor, is an anti-inflammatory agent with cardioprotective effects. Nevertheless, the effect of LOSM against DOX-induced cardiotoxicity has not been reported. The objective of the current work is to determine the effects of LOSM on DOX-induced chronic cardiotoxicity in C57BL/6 mice with particular focus on senescence and inflammatory pathways. Methods: Five-week-old C57BL/6 mice were fed diet containing LOSM (estimated daily intake 12 mg/kg/day) or a control diet for four days. Thereafter, mice were randomized to receive six weekly intraperitoneal injections of either DOX (4 mg/kg/week for 6 weeks) or saline. Three days after the last injection, cardiac function was assessed by trans-thoracic echocardiography. Gene expressions of senescence and inflammatory markers were quantified using real-time PCR and serum inflammatory markers were assessed by Luminex. Results: LOSM ameliorated DOX-induced cardiac dysfunction as evidenced by mitigated DOX-induced reduction in left ventricular fraction and fractional shortening. Notably, LOSM attenuating DOX-induced upregulation of p21 Cip1 , a marker of senescence, in the heart and the liver. Additionally, LOSM demonstrated anti-inflammatory effects, with reduced cardiac interleukin-1α and interleuken-6 gene expression in DOX-treated mice suggesting a senomorphic effect of LOSM. Systemic inflammation, assessed by serum cytokine levels, showed decreased interleukin-6 concentration in both DOX-treated mice and LOSM-treated mice. Conclusion: This is the first study to determine the effect of pharmacological inhibition of p38 MAPK against DOX-induced cardiotoxicity in vivo. Our study underscores the potential therapeutic efficacy of p38 MAPK inhibition in ameliorating DOX-induced cardiotoxicity, offering insights into novel strategies for mitigating chemotherapy-related cardiac complications, and suggesting a possible senomorphic effects of losmapimod.

Crosstalk between protein kinase C α and transforming growth factor β signaling mediated by Runx2 in intestinal epithelial cells

Tight coordination of growth regulatory signaling is required for intestinal epithelial homeostasis. Protein kinase C α (PKCα) and transforming growth factor β (TGFβ) are negative regulators of proliferation with tumor suppressor properties in the intestine. Here, we identify novel crosstalk between PKCα and TGFβ signaling. RNA-Seq analysis of nontransformed intestinal crypt-like cells and colorectal cancer cells identified TGFβ receptor 1 (TGFβR1) as a target of PKCα signaling. RT-PCR and immunoblot analysis confirmed that PKCα positively regulates TGFβR1 mRNA and protein expression in these cells. Effects on TGFβR1 were dependent on Ras-extracellular signal-regulated kinase 1/2 (ERK) signaling. Nascent RNA and promoter-reporter analysis indicated that PKCα induces TGFβR1 transcription, and Runx2 was identified as an essential mediator of the effect. PKCα promoted ERK-mediated activating phosphorylation of Runx2, which preceded transcriptional activation of the TGFβR1 gene and induction of Runx2 expression. Thus, we have identified a novel PKCα→ERK→Runx2→TGFβR1 signaling axis. In further support of a link between PKCα and TGFβ signaling, PKCα knockdown reduced the ability of TGFβ to induce SMAD2 phosphorylation and cell cycle arrest, and inhibition of TGFβR1 decreased PKCα-induced upregulation of p21Cip1 and p27Kip1 in intestinal cells. The physiological relevance of these findings is also supported by The Cancer Genome Atlas data showing correlation between PKCα, Runx2, and TGFβR1 mRNA expression in human colorectal cancer. PKCα also regulated TGFβR1 in endometrial cancer cells, and PKCα, Runx2, and TGFβR1 expression correlates in uterine tumors, indicating that crosstalk between PKCα and TGFβ signaling may be a common mechanism in diverse epithelial tissues.

Chemical Composition, Antitumor Properties, and Mechanism of the Essential Oil from Plagiomnium acutum T. Kop.

Plagiomnium acutum T. Kop. (P. acutum) has been used as a traditional Chinese medicine for thousands of years to treat cancer but lacks evidence. The objective of this work was to reveal the chemical composition of P. acutum essential oil (PEO) and explore its potential antitumor activity and molecular mechanism. PEO was prepared by the simultaneous distillation-extraction method and characterized by gas chromatography/mass spectroscopy. CCK8 assay, flow cytometry, western blot, and immunofluorescence techniques were used to analyze the effects and mechanism of PEO against cancer cells. A total of 74 constituents of PEO were identified, with diterpenes (26.5%), sesquiterpenes (23.89%), and alcohols (21.81%) being the major constituents. Two terpenoids, selina-6-en-4-ol and dolabella-3,7-dien-18-ol, were detected in PEO for the first time. PEO showed significant cell growth inhibitory activity on HepG2 and A549 cells by blocking the G1 phase and inducing apoptosis, which may be attributed to its upregulation of p21Cip1 and p27Kip1 proteins and interference with mitochondrial membrane potential effect. Dolabella-3,7-dien-18-ol accounts for 25.5% of PEO and is one of the main active components of PEO, with IC50 values in HepG2 and A549 cells of (25.820 ± 0.216) µg/mL and (23.597 ± 1.207) μg/mL, respectively. These results confirmed the antitumor medicinal value of P. acutum and showed great application potential in the pharmaceutical industry.

A novel antiproliferative PKCα-Ras-ERK signaling axis in intestinal epithelial cells

We have previously shown that the serine/threonine kinase PKCα triggers MAPK/ERK kinase (MEK)-dependent G1→S cell cycle arrest in intestinal epithelial cells, characterized by downregulation of cyclin D1 and inhibitor of DNA-binding protein 1 (Id1) and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Here, we use pharmacological inhibitors, genetic approaches, siRNA-mediated knockdown, and immunoprecipitation to further characterize antiproliferative ERK signaling in intestinal cells. We show that PKCα signaling intersects the Ras-Raf-MEK-ERK kinase cascade at the level of Ras small GTPases and that antiproliferative effects of PKCα require active Ras, Raf, MEK, and ERK, core ERK pathway components that are also essential for pro-proliferative ERK signaling induced by epidermal growth factor (EGF). However, PKCα-induced antiproliferative signaling differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and involves prolonged rather than transient ERK activation. PKCα forms complexes with A-Raf, B-Raf, and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKCα-induced antiproliferative effects. At least two PKCα–ERK pathways that collaborate to promote growth arrest were identified: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, leads to p21Cip1 upregulation, while additional pathway(s) mediate PKCα-induced cyclin D1 and Id1 downregulation. PKCα also induces ERK-dependent SOS1 phosphorylation, indicating possible negative crosstalk between antiproliferative and growth-promoting ERK signaling. Importantly, the spatiotemporal activation of PKCα and ERK in the intestinal epithelium in vivo supports the physiological relevance of these pathways and highlights the importance of antiproliferative ERK signaling to tissue homeostasis in the intestine.

Effect of 5-aza-2'-deoxycytidine on p27Kip1, p21Cip1/Waf1/Sdi1, p57Kip2, and DNA methyltransferase 1 Genes Expression, Cell Growth Inhibition and Apoptosis Induction in Colon Cancer SW 480 and SW 948 Cell Lines.

Background: Dysregulation of the cell cycle has been reported in various cancers. Inactivation of the cyclin-dependent kinases inhibitors (CDKIs), CIP/KIP family, such as p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 genes because of hypermethylation has been shown in several cancers. Treatment with DNA demethylating agent 5-aza-2ˈ-deoxycytidine (5-Aza-CdR) has been indicated that affect genomic methylation and resulting in silenced genes reactivation in colon cancer. Previously, we evaluated the effect of 5-Aza-CdR on DNA methyltransferase 1 (DNMT1) gene expression in hepatocellular carcinoma (HCC) which encouraged us to design the current study. The present study aimed to evaluate the effect of 5-Aza-CdR on p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, and DNAT1 genes expression, cell growth inhibition and apoptosis induction in colon cancer SW 480 and SW 948 cell lines. Materials and Methods: The effect of 5-aza-CdR on the SW 480 and SW 948 cells growth, apoptosis induction and genes expression were assessed by MTT assay, flow cytometry, and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis respectively. Results: 5-aza-CdR inhibited cell growth as time-and dose-dependent manner significantly (P<0.001). The agent reactivated p15INK4, p16INK4, p18INK4, and p19INK4 genes expression and induced apoptosis at a concentration of 5 μM significantly. Besides, 5-aza-CdR had a more significant effect on the SW 480 cell line in comparison to SW 948 cell line. Conclusion: 5-Aza-CdR plays a key role in the up-regulation of p21Cip1/ Waf1/Sdi1, p27Kip1, and p57Kip2 and down-regulation of DNMT1 genes resulting in cell growth inhibition and apoptosis induction.

Paeonol exerts anti‑tumor activity against colorectal cancer cells by inducing G0/G1 phase arrest and cell apoptosis via inhibiting the Wnt/β‑catenin signaling pathway.

Paeonol is a simple phenolic compound isolated from herbal root bark, which has been reported to possess numerous biological and pharmacological characteristics, including a desirable anti-tumor effect. To date, the effect of paeonol against colorectal cancer (CRC) cells is yet to be fully elucidated. Therefore, the present study aimed to identify the underlying mechanism via which paeonol exerts its anti-tumor activity on HCT116 cells. After incubation with various concentrations of paeonol (7.8125, 15.625, 31.25, 62.5, 125, 250 and 500 µg/ml), the inhibitory effect of paeonol on cell viability was assessed using a Cell Counting Kit-8 assay. Cell apoptosis and cell cycle distribution were measured using flow cytometry. Moreover, caspase activity was measured using a colorimetric caspase assay. Luciferase assay was also used to determine the β-catenin-mediated transcriptional activity of T-cell specific transcription factor/lymphoid-enhancer binding factor (TCF/LEF), and western blotting analysis was performed to measure the related expression of proteins. The results indicated that paeonol exhibited a notable effect against HCT116 cells by inducing G0/G1-phase arrest, as demonstrated by downregulation of the cell cycle regulators cyclin-dependent kinase 4 and cyclin D1 and upregulation of p21Cip1 in a dose-dependent manner. Furthermore, paeonol dose-dependently induced cell apoptosis, accompanied by an increase in the Bax/Bcl-2 ratio, release of cytochrome c and further activation of caspases. Paeonol also dose-dependently blocked the activation of the Wnt/β-catenin signaling pathway by suppressing the expression of β-catenin, resulting in a decrease in β-catenin-mediated activity of TCF/LEF and downregulation of downstream target genes, including cyclin D1, survivin and c-Myc. Therefore, the present results suggested that paeonol exerted its anti-tumor effects on CRC cells, including the inhibition of cell proliferation, induction of cell cycle arrest and initiation of apoptosis, at least partly by suppressing the Wnt/β-catenin pathway, which may offer a promising therapeutic strategy for CRC.

RAGE acts as an oncogenic role and promotes the metastasis of human lung cancer

RAGE (receptor for advanced glycation end-product) is thought to be associated with metastasis and poor prognosis of various types of cancer. However, RAGE is constitutively expressed in the normal lung and down-regulated in cancerous lung, while the opposite evidence shows that RAGE-mediated signaling contributes to the tumorigenesis of lung cancer. Therefore, the role of RAGE in lung cancer progression is still unclear to be further investigated. In this study, RAGE-overexpressed stable clones of human lung cancer A549 cells and two local lung adenocarcinoma cell lines CL1-0 and CL1-5 were utilized to verify the effect of RAGE on lung cancer cells while the in vivo xenograft animal model was further performed to evaluate the role of RAGE in the progression of lung cancer. The growth of A549 cells was inhibited by RAGE overexpression. p53-dependent p21CIP1 expression contributed to RAGE-induced growth inhibition by suppressing CDK2 kinase activity and retinoblastoma protein (RB) phosphorylation in vitro. On the other hand, RAGE overexpression promoted migration, invasion, and mesenchymal features of lung adenocarcinoma cells through ERK signaling. Furthermore, an in vivo xenograft experiment indicated that RAGE promoted the metastasis of lung cancer cells with p21CIP1 up-regulation, ERK activation, and the changes of EMT markers. Regarding to the involvement of tumor-associated macrophage (TAM) in the microenvironment, we monitored the expressions of TAM markers including CD68 and CD163 as well as angiogenesis marker CD31 in xenograft slice. The data showed that RAGE might induce the accumulation of TAM in lung cancer cells and further accelerate the in vivo tumor growth. In summary, our study provides evidence indicating the distinct in vitro and in vivo effects of RAGE and related mechanisms on tumor growth and metastasis, which shed light on the oncogenic role of RAGE in lung cancer.

UNC93B1 promotes tumoral growth by controlling the secretion level of granulocyte macrophage colony-stimulating factor in human oral cancer

Unc-93 homolog B1 (UNC93B1), a transmembrane protein, is correlated with immune diseases, such as influenza, herpes simplex encephalitis, and the pathogenesis of systemic lupus erythematosus; however, the role of UNC93B1 in cancers including human oral squamous cell carcinomas (OSCCs) remains unknown. In the current study, we investigated the UNC93B1expression level in OSCCs using quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, and immunohistochemistry. Our data showed that UNC93B1 mRNA and protein expressions increased markedly (p < 0.05) in OSCCs compared with normal cells and tissues and that high expression of UNC93B1 in OSCCs was related closely to tumoral size. UNC93B1 knockdown (shUNC93B1) OSCC cells showed decreased cellular proliferation by cell-cycle arrest in the G1 phase with up-regulation of p21Cip1 and down-regulation of CDK4, CDK6, cyclin D1, and cyclin E. We also found that granulocyte macrophage colony-stimulating factor (GM-CSF) was down-regulated significantly (p < 0.05) in shUNC93B1 OSCC cells. Moreover, inactivation of GM-CSF using neutralization antibody led to cell-cycle arrest at the G1 phase similar to the phenotype of the shUNC93B1 cells. The current findings indicated that UNC93B1 might play a crucial role in OSCC by controlling the secretion level of GM-CSF involved in tumoral growth and could be a potential therapeutic target for OSCCs.

Centromere Protein N Participates in Cellular Proliferation of Human Oral Cancer by Cell-Cycle Enhancement.

Centromere protein N (CENP-N), an important member of the centromere protein family, is essential for kinetochore assembly and chromosome segregation; however, the relevance of CENP-N in cancers remains unknown. The aim of this study was to investigate CENP-N expression and its functional mechanisms in oral squamous cell carcinoma (OSCC). CENP-N expression was up-regulated significantly in vitro and in vivo in OSCCs. Overexpressed CENP-N was closely (p < 0.05) correlated with tumor growth using quantitative reverse transcriptase-polymerase chain reaction, immunoblot analysis, and immunohistochemistry. CENP-N knockdown (shCENP-N) cells showed depressed cellular proliferation by cell-cycle arrest at the G1 phase with up-regulation of p21Cip1 and p27Kip1 and down-regulation of cyclin D1, CDK2, and CDK4. Interestingly, we newly discovered that calcitriol (1, 25-dihydroxyvitamin D3) controlled the CENP-N expression level, leading to inhibition of tumor growth similar to shCENP-N cells. These results suggested that CENP-N plays a critical role in determining proliferation of OSCCs and that calcitriol might be a novel therapeutic drug for OSCCs by regulating CENP-N.

Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer

Tripartite motif family-like 2 (TRIML2), a member of the TRIM proteins family, is closely related to Alzheimer's disease, however, no studies of TRIML2 have been published in the cancer research literature. In the current study, we investigated the expression level of TRIML2 and its molecular mechanisms in human oral squamous cell carcinoma (OSCC); reverse transcriptase-quantitative polymerase chain reaction, immunoblot analysis, and immunohistochemistry showed that TRIML2 is up-regulated significantly in OSCCs in vitro and in vivo. TRIML2 knockdown OSCC cells showed decreased cellular proliferation by cell-cycle arrest at G1 phase that resulted from down-regulation of CDK4, CDK6, and cyclin D1 and up-regulation of p21Cip1 and p27Kip1. Surprisingly, resveratrol, a polyphenol, led to not only down-regulation of TRIML2 but also cell-cycle arrest at G1 phase similar to TRIML2 knockdown experiments. Taken together, we concluded that TRIML2 might play a significant role in tumoral growth and that resveratrol may be a new drug for treating OSCC by interfering with TRIML2 function.

The histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) as a therapeutic agent in rhabdomyosarcoma

ABSTRACT Rhabdomyosarcoma (RMS) is an aggressive childhood sarcoma with two distinct subtypes, embryonal (ERMS) and alveolar (ARMS) histologies. More effective treatment is needed to improve outcomes, beyond conventional cytotoxic chemotherapy. The pan-histone deacetylase inhibitor, Suberoylanilide Hydroxamic Acid (SAHA), has shown promising efficacy in limited preclinical studies. We used a panel of human ERMS and ARMS cell lines and xenografts to evaluate the effects of SAHA as a therapeutic agent in both RMS subtypes. SAHA decreased cell viability by inhibiting S-phase progression in all cell lines tested, and induced apoptosis in all but one cell line. Molecularly, SAHA-treated cells showed activation of a DNA damage response, induction of the cell cycle inhibitors p21Cip1 and p27Kip1 and downregulation of Cyclin D1. In a subset of RMS cell lines, SAHA promoted features of cellular senescence and myogenic differentiation. Interestingly, SAHA treatment profoundly decreased protein levels of the driver fusion oncoprotein PAX3-FOXO1 in ARMS cells at a post-translational level. In vivo, SAHA-treated xenografts showed increased histone acetylation and induction of a DNA damage response, along with variable upregulation of p21Cip1 and p27Kip1. However, while the ARMS Rh41 xenograft tumor growth was significantly inhibited, there was no significant inhibition of the ERMS tumor xenograft RD. Thus, our work shows that, while SAHA is effective against ERMS and ARMS tumor cells in vitro, it has divergent in vivo effects . Together with the observed effects on the PAX3-FOXO1 fusion protein, these data suggest SAHA as a possible therapeutic agent for clinical testing in patients with fusion protein-positive RMS.

Impaired mammary tumor formation and metastasis by the point mutation of a Smad3 linker phosphorylation site

Triple-negative breast cancer (TNBC) is often aggressive and metastatic. Transforming growth factor-β acts as a tumor-promoter in TNBC. Smad3, a major downstream effector protein in the TGF-β signaling pathway, is regulated by phosphorylation at several sites. The functional significance of the phosphorylation of the linker region in Smad3 is poorly understood for TNBC. Among the four sites in the Smad3 linker region, threonine-179 (T179) appears to be unique as it serves as the binding site for multiple WW-domain-containing proteins upon phosphorylation, suggesting that this phosphorylation is a key for Smad3 to engage other pathways.Using genome editing, we introduced for the first time a knock-in (KI) mutation in the endogenous Smad3 gene in IV2, a lung-tropic subline of the human MDA-MB-231 TNBC cell line. In the resulting cell line, the Smad3 T179 phosphorylation site is replaced by non-phosphorylatable valine (T179V) with the mutation in both alleles.The T179V KI reduced cell growth rate and mammosphere formation. These phenomena were accompanied by a significant upregulation of p21Cip1 and downregulation of c-Myc. The T179V KI also reduced cell migration and invasion in vitro. In the mouse xenograft models, the T179V KI markedly reduced the establishment of primary tumor in the mammary fat pad and the lung metastasis.Our results using gene editing indicate the cancer-promoting role of Smad3 T179 phosphorylation in the human TNBC cells. Our findings highly suggest that controlling this phosphorylation may have therapeutic potential for TNBC.

Critical role of deoxynucleotidyl transferase terminal interacting protein 1 in oral cancer

Deoxynucleotidyl transferase terminal interacting protein 1 (DNTTIP1) forms a complex with histone deacetylase (HDAC); however, the relevance of DNTTIP1 in cancer remains unknown. The aim of this study was to examine DNTTIP1 expression and its functional mechanisms in oral squamous cell carcinomas (OSCCs). DNTTIP1 expression was analyzed by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting analysis, and immunohistochemistry. The expression of DNTTIP1 was upregulated significantly in vitro and in vivo, and in patients with OSCC in whom DNTTIP1 was overexpressed and the expression level was correlated significantly (P < 0.05) with tumoral growth. DNTTIP1 knockdown (siDNTTIP1) cells showed depressed cellular proliferation by cell-cycle arrest at the G1 phase with high acetylation of p53 and upregulation of p21Cip1. Moreover, resveratrol, a HDAC inhibitor, controlled not only acetylated p53 status but also DNTTIP1 expression, leading to a similar phenotype of siDNTTIP1 cells. A marked (P < 0.05) reduction of tumoral growth in mouse xenograft models was observed with lower DNTTIP1 expression under the presence of this chemical reagent. Taken together, our results suggested that DNTTIP1–HDAC interaction promotes tumoral growth through deacetylation of p53 and that DNTTIP1 might be a critical therapeutic target in OSCCs.This study demonstrates that deoxynucleotidyl transferase terminal interacting protein 1 (DNTTIP1), a binding partner of histone deacetylase (HDAC), plays a crucial role in progression of oral squamous cell carcinoma (OSCC) by controlling p53 acetylation. Resveratrol, a HDAC inhibitor, controls both acetylated p53 status and DNTTIP1 expression. Thus, DNTTIP1 might be a novel therapeutic target in OSCC.

Abstract IA08: Kinase-mediated modulation of paclitaxel sensitivity in ovarian cancer

Abstract Epithelial ovarian cancer is the fourth most common cause of cancer death among women in the developed world. Currently only 30% of ovarian cancer patients remain free from disease long term. Paclitaxel is an integral component of primary therapy for ovarian cancer, but less than half of ovarian cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with the disease. In recent years, we have identified several kinases that regulate the sensitivity of cancer cells to paclitaxel by modulating cancer metabolism, enhancing microtubule stability, inhibiting centrosome splitting or blocking AKT/survivin signaling. We performed siRNA kinome-screens to identify molecular targets whose decreased expression overcomes paclitaxel resistance and increases paclitaxel sensitivity in ovarian cancer cells. Upon assessing cell viability, we showed that 20% of the potential kinase targets whose knockdown modulates paclitaxel sensitivity participate in glucose and energy metabolism. Among these, a leading candidate was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), an isoform of the glycolytic enzyme phosphofructokinase (PFK2). Many cancer cells depend on glucose for survival. Glycolysis involves ten metabolic reactions catalyzed by enzymes whose expression is frequently increased during malignant transformation. Many reports document the role of oncogenic signaling in regulating the activity of metabolic enzymes to support the enhanced macromolecule synthesis required for rapid proliferation of cancer cells. Cancer cells express altered levels of the different PFK-2/FBPase-2 isoenzymes and even modulate their relative kinase and bisphosphatase activities according to metabolic needs in a spatial and/or temporal manner. PFKFB2 is a bifunctional glycolytic enzyme that regulates the level of fructose-2,6-bisphosphate (Fru-2,6-BP) and is overexpressed in a fraction of ovarian and breast cancers. We found that knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wtTP553). Additionally, PFKFB2 siRNA or PFKFB2 shRNA significantly inhibited tumor growth and enhanced sensitivity to paclitaxel in xenografts derived from two ovarian cancer cell lines. Knockdown of PFKFB2 increased the rate of glycolysis, but decreased the flow of intermediates through the pentose-phosphate pathway in cancer cell lines with wtTP53, decreasing NADPH. Reactive oxygen species (ROS) accumulated after PFKFB2 knockdown, which stimulated phosphorylation of Janus kinase (JNK), induced G1 cell cycle arrest, and initiated apoptosis that depended upon upregulation of p21Cip1 and Puma. Thus, PFKFB2 is a glycolytic enzyme that drives tumor cell growth and enhances paclitaxel resistance by inhibiting TP53-dependent G1 cell cycle arrest and apoptosis. These findings highlight the interaction of cancer-altered metabolism with cell proliferation and chemosensitivity, which may provide a novel target in patients with ovarian and breast cancers where TP53 function remains intact. We also found that knockdown of kinases that regulate microtubule stability could sensitize ovarian cancer cells to paclitaxel treatment. In previous studies, baseline microtubule stability correlated with response to paclitaxel in ovarian cancer cell lines and both parameters could be enhanced by knockdown of individual kinases. Using the initial 14 kinase targets, we performed assays of microtubule stability, apoptosis, and cell cycle arrest with all possible pairs of kinase siRNA which enhance paclitaxel sensitivity across multiple cell lines and found that dual knockdown of IKBKB/STK39 had the greatest effect on enhancing paclitaxel stability. Our study documents the impact of sequentially silencing IKBKB and STK39 on paclitaxel sensitivity, providing a rationale for siRNA-based therapy. Different siRNAs are in the developmental pipeline for a variety of diseases including cancer, and more than a dozen are in phase I or II clinical trials. One of the most promising candidates to emerge from our kinase screen was salt inducible kinase 2 (SIK2), a serine/threonine kinase that is required for bipolar mitotic spindle formation and normal mitotic progression. With Dr. Ahmed Ahmed, we previously found knockdown of SIK2 induces polyploidy by blocking centrosome splitting and inhibits PI3K, sensitizing cells to paclitaxel. We demonstrated that knockdown of SIK2 inhibits xenograft growth and potentiates paclitaxel activity in vivo. Subsequently, we have partnered with Arrien Pharmaceuticals to test a small-molecule inhibitor of SIK2. We have found that novel SIK2 inhibitors, ARN-3236 and ARN3261, block the activity of SIK2 kinase and inhibit ovarian cancer cell growth, enhancing paclitaxel sensitivity in cultured cells and in xenografts. ARN-3261 will enter first-in-human trials at MD Anderson early next year. Taken together, our results support the development of small-molecule kinase inhibitors that modulate glycolysis, enhance microtubule stability, induce polyploidy, and inhibit AKT/survivin signaling, providing novel routes to enhance primary paclitaxel-based therapy for ovarian cancer and to improve patient outcomes. Citation Format: Zhen Lu. Kinase-mediated modulation of paclitaxel sensitivity in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA08.

The RNA-binding protein Celf1 post-transcriptionally regulates p27Kip1 and Dnase2b to control fiber cell nuclear degradation in lens development.

Opacification of the ocular lens, termed cataract, is a common cause of blindness. To become transparent, lens fiber cells undergo degradation of their organelles, including their nuclei, presenting a fundamental question: does signaling/transcription sufficiently explain differentiation of cells progressing toward compromised transcriptional potential? We report that a conserved RNA-binding protein Celf1 post-transcriptionally controls key genes to regulate lens fiber cell differentiation. Celf1-targeted knockout mice and celf1-knockdown zebrafish and Xenopus morphants have severe eye defects/cataract. Celf1 spatiotemporally down-regulates the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 by interacting with its 5’ UTR and mediating translation inhibition. Celf1 deficiency causes ectopic up-regulation of p21Cip1. Further, Celf1 directly binds to the mRNA of the nuclease Dnase2b to maintain its high levels. Together these events are necessary for Cdk1-mediated lamin A/C phosphorylation to initiate nuclear envelope breakdown and DNA degradation in fiber cells. Moreover, Celf1 controls alternative splicing of the membrane-organization factor beta-spectrin and regulates F-actin-crosslinking factor Actn2 mRNA levels, thereby controlling fiber cell morphology. Thus, we illustrate new Celf1-regulated molecular mechanisms in lens development, suggesting that post-transcriptional regulatory RNA-binding proteins have evolved conserved functions to control vertebrate oculogenesis.

Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells.

The maintenance of ordinal cell cycle phases is a critical biological process in cancer genesis, which is a crucial target for anti-cancer drugs. As an important natural isoquinoline alkaloid from Chinese herbal medicine, Berberine (BBR) has been reported to possess anti-cancer potentiality to induce cell cycle arrest in hepatocellular carcinoma cells (HCC). However, the underlying mechanism remains to be elucidated. In our present study, G0/G1 phase cell cycle arrest was observed in berberine-treated Huh-7 and HepG2 cells. Mechanically, we observed that BBR could deactivate the Akt pathway, which consequently suppressed the S-phase kinase-associated protein 2 (Skp2) expression and enhanced the expression and translocation of Forkhead box O3a (FoxO3a) into nucleus. The translocated FoxO3a on one hand could directly promote the transcription of cyclin-dependent kinase inhibitors (CDKIs) p21Cip1 and p27Kip1, on the other hand, it could repress Skp2 expression, both of which lead to up-regulation of p21Cip1 and p27Kip1, causing G0/G1 phase cell cycle arrest in HCC. In conclusion, BBR promotes the expression of CDKIs p21Cip1 and p27Kip1 via regulating the Akt/FoxO3a/Skp2 axis and further induces HCC G0/G1 phase cell cycle arrest. This research uncovered a new mechanism of an anti-cancer effect of BBR.

Abstract 3564: Silencing pfkfb2 enhances paclitaxel sensitivity by modulating metabolism of p53 wt ovarian and breast cancer cells and xenografts

Abstract Breast cancer is the most common cause and epithelial ovarian cancer the fourth most common cause of cancer death among women in the developed world. While breast cancer can be cured in 70% of cases, only 30% of ovarian cancer patients remain free from disease long term. Paclitaxel is an integral component of primary therapy for both forms of cancer, but less than half of breast and ovarian cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases. In recent years several kinases have been identified that regulate the sensitivity of cancer cells to paclitaxel by inhibiting centrosome splitting or enhancing microtubule stability. Much less attention has been given to kinases that affect paclitaxel sensitivity by modulating cancer cell metabolism. We previously performed siRNA kinome-screens to identify molecular targets whose decreased expression overcomes paclitaxel resistance and increases paclitaxel sensitivity in ovarian cancer cells. We showed that 20% of the potential kinase targets whose knockdown modulates paclitaxel sensitivity participate in glucose and energy metabolism. Among these, a leading candidate was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), an isoform of the glycolytic enzyme phosphofructokinase (PFK2). PFKFB2 is overexpressed in a fraction of ovarian and breast cancers. Knockdown of PFKFB2 inhibited clonogenic growth of ovarian and breast cancer cell lines and enhanced paclitaxel sensitivity in the cell lines with wt TP53. Liposome encapsulated PFKFB2 siRNA significantly inhibited tumor growth and enhanced sensitivity to paclitaxel in xenografts derived from ovarian cancer cell lines. Knockdown of PFKFB2 increased glycolysis, decreasing the flow of glycolytic intermediates to the pentose-phosphate pathway with reduced G6PD activity in wt TP53 cancer cell lines. With decreased NADPH, ROS accumulated after PFKFB2 knockdown, stimulating phosphorylation of JNK, inducing G1 cell cycle arrest, and initiating apoptosis dependent upon upregulation of p21Cip1 and Puma which are downstream targets of TP53. Our studies have shown for the first time that PFKFB2, a glycolytic enzyme, drives tumor cell growth and regulates paclitaxel sensitivity by inducing apoptosis and G1 cell cycle arrest. These findings highlight a remarkable degree of coordination between cancer metabolism with cell proliferation and chemo-sensitivity, which may provide a novel target in patients with ovarian cancers and breast cancers where TP53 function remains intact. Citation Format: Hailing Yang, Shu Zhang, Weiqun Mao, Ahmed A. Ahmed, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Anil K. Sood, Xiao-Feng Le, Zhen Lu, Robert C. Bast. Silencing pfkfb2 enhances paclitaxel sensitivity by modulating metabolism of p53 wt ovarian and breast cancer cells and xenografts [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 3564. doi:10.1158/1538-7445.AM2017-3564

Clarithromycin Interacts with Lenalidomide in the Combination Regimen Bird and Overcomes Drug Resistance in Multiple Myeloma

Introduction:Although the macrolide antibiotic clarithromycin (CAM, or Biaxin) has only minimal single agent activity in MM, the regimens with addition of CAM to IMiDs and steroids, including BLT-D (Biaxin/low-dose thalidomide/dexamethasone [Dex]), BiRD (Biaxin/Revlimid [lenalidomide, Lena]/Dex), t-BiRD (thalidomide/BiRD), Car-BiRD (carfilzomib/BiRD), and ClaPD (CAM/pomalidomide/Dex), appear highly effective in treatment of newly-diagnosed and probably relapsed/refractory multiple myeloma (MM). In this context, two phase 3 trials are currently ongoing to evaluate the efficacy of BiRD vs. RD in newly-diagnosed MM in the United States and Europe, respectively. Of note, recent findings also suggests that addition of CAM to RD might overcome resistance to RD. However, despite increasing clinical evidence for its promising activity, the exact mechanism for such a combination strategy still remain largely unclear. Here, we investigated the mechanisms of action underlying the interaction between CAM and Lena and their capability to overcome drug resistance in MM cells, focusing on the cereblon (CRBN)/IKZF1,3/IRF4/Myc signaling cascade, recently identified as the novel target of IMiDs,Materials and Methods: To test our hypothesis whether and how BiRD overcomes resistance to RD (Lena/Dex), human MM cells lines employed in this study included Dex-sensitive (MM.1S) vs -resistant (MM.1R) cells, drug-naïve RPMI8226 cells vs their Lena-resistant (R10R) or bortezomib (Btz)-resistant counterparts (DR), as well as primary CD138+MM cells isolated from bone marrow samples of newly-diagnosed and relapsed/refractory patients who had received prior IMiDs (including Lena) or Btz. Cells were exposed (72 hr) to CAM (50-100 mg/ml) ± Lena (1-10 mM), after which the CCK-8 assay and flow cytometry with annexin V/7AAD staining were performed to monitor cell viability and apoptosis, respectively. Mechanistic studies included Western blot analyses of the CRBN/IKZF1,3/IRF4/Myc signaling pathway, as well as the apoptotic caspase cascade. Cell cycle was also assessed by flow cytometry.Results: Whereas Lena (1-10 mM) had almost no direct effects on cell viability, CAM (≥ 100 mg/ml) displayed a dose-dependent toxicity in various MM cell lines. Notably, subtoxic concentrations of CAM (e.g., 50 mg/ml) significantly potentiated lethality of Lena in MM.1S (CI value = 0.40-0.86, indicating synergism). Significantly, this effect was even more robust in Dex-resistant MM.1R cells. These events were associated with marked activation of caspase 3, 8, and 9 and PARP cleavage, accompanied by down-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL. While add-on of CAM significantly increased lethality of Lena in RPMI8226 cells, combined treatment was strikingly more effective against Lena-resistant R10R cells. In addition, Btz-resistant RPMI8226 cells were also more sensitive to both CAM alone and in combination with Lena, compared to parental RPMI8226 cells. Consistently, markedly enhanced cell killing by the combination was also observed in primary CD138+ cells, particularly those obtained from patients relapsed after prior IMiDs. Exposure to Lena with or without CAM sharply down-regulated CRBN in MM cells, accompanied by reduced expression of IKZF1, IKZF3, IRF4, and Myc. Interestingly, Lena failed to down-regulate CRBN/IKZF1/IRF4/Myc in Lena-resistant R10R cells, while addition of CAM dramatically resensitized these cells to the action of Lena. Moreover, Lena in the presence or absence of CAM induced cell cycle arrest at G0/G1, in association with marked up-regulation of p21Cip1 and p27Kip1. Last, Lena induced LC3A-II expression (a marker of autophagy), which was clearly increased in the presence of CAM, likely in association with the capability of CAM to impair the late stage process of autophagy e.g., autophagosome clearance by lysosome.Conclusion: Together, these findings indicate that CAM significantly increases the anti-MM activity of Lena in MM cells, especially those resistant to the first-line therapy (e.g., Dex and Btz), and notably overcomes Lena resistance. The mechanisms involves disruption of the CRBN/IKZF1/IRF4/Myc pathway, as well as activation of the apoptotic caspase cascade, induction of cell cycle arrest, and attenuation of autophagy. Collectively, these mechanistic findings support exploring the BiRD regimen in MM, particularly to overcome RD resistance. DisclosuresNo relevant conflicts of interest to declare.

Halofuginone and artemisinin synergistically arrest cancer cells at the G1/G0 phase by upregulating p21Cip1 and p27Kip1.

Combinational drug therapy is one of the most promising strategies in modern anticancer research. Traditional Chinese medicine (TCM) formulas represent a wealth of complex combinations proven successful over centuries of clinical application. One such formula used to treat a variety of diseases, including cancer, contains two herbs, whose main active components are Halofuginone (HF) and Artemisinin (ATS). Here we studied the anticancer synergism of HF and ATS in various cancer cell lines and in a xenograft nude mice model. We found that the HF-ATS combination arrested more cells at the G1/G0 phase than either one alone, with the concomitant increased levels of CDK2 inhibitors, p21Cip1 and p27Kip1. By knocking down p21Cip1 and p27Kip1 separately or simultaneously in HCT116 cells and MCF-7 cells, we found that p21Cip1 was required for HF induced G1/G0 arrest, whereas p21Cip1 and p27Kip1 were both required for ATS or HF-ATS combination-mediated cell cycle arrest. Moreover, HF-ATS combination synergistically inhibited tumor growth in xenograft nude mice, and this was associated with the increased levels of p21Cip1 and p27Kip1. Collectively, these data indicate that the upregulation of p21Cip1 and p27Kip1 contributes to the synergistic anticancer effect of the HF-ATS combination.

Abstract 1346: Immunotherapy against breast cancer based on Stat3 blockade

Abstract The signal transducer and activator of transcription 3 (Stat3) is constitutively active in breast cancer (BC) and contributes to malignant transformation by promoting cell cycle progression, inhibiting apoptosis and mediating tumor immune evasion. Stat3 inhibition induces expression of proinflammatory cytokines and chemokines. On the other hand, it has been shown that oncogene inactivation induces cellular senescence and the secretion of senescence-associated secretome (SAS) in diverse tumor types. We recently described that Stat3 blockade leads to a senescence program in murine BC cells, which is accompanied by the secretion of proinflammatory cytokines. In addition, we demonstrated that immunization of mice with syngeneic Stat3 blocked BC cells induces an antitumoral immune response that involves the participation of CD4+ Th cells and cytotoxic NK cells. In this study our objectives were to study whether immunization with supernatants (SN) produced by Stat3-blocked cells induces an antitumor immune response and to explore the contribution of senescence phenotype in this response. For that purpose we used BC models of ErbB-2-positive, JIMT-1 and KPL-4 cells (human) and C4HD cells (murine), and of triple negative, MDA-MB231 cells (human) and 4T1 cell (murine). Knockdown of Stat3 with siRNA in these cells, induced senescence (assessed by acidic β-galactosidase staining). In human BC cells the senescent phenotype was accompanied by up-regulation of p21cip1 and downregulation of Rb expressions. In mouse BC cells we observed an increased expression of p16ink4a. In addition, simultaneous transfection with siRNAs targeting Stat3 and p16ink4a reverted the senescent phenotype. Then, we used a pre-clinical model in which we embedded the SN of C4HD cells in a slow-delivery depot as an adjuvant of a cellular immunotherapy. We immunized the animals with irradiated C4HD cells together with a depot containing lyophilized SN of C4HD cells transfected in vitro either with Stat3 siRNA (senescent) and Stat3 and p16ink4a siRNA (non-senescent), or a control siRNA. After 3 immunizations, with 15 d of interval between them, animals were challenged with C4HD tumor and tumor growth was monitored for 40 d. We observed that immunization with SN of cells with Stat3 siRNA decreased tumor growth vs. control siRNA group. Interestingly, there was no difference between tumor growth of animals immunized with SN of cells with Stat3 siRNA vs Stat3 and p16ink4a siRNA. We observed in these two groups greater cytotoxic activity of NK cells and an increase in the number of memory CD4+ T cells vs. control. These results suggest that Stat3 blockade drives a senescence program also in human BC cells. The secretome of Stat3-blocked BC cells is an effective adjuvant and could be formulated for immunotherapies independently of the SAS. Defining the protein, peptide, cytokine, chemokine composition of this supernatant opens up new avenues for immunotherapy in cancer patients. Citation Format: Mara De Martino, María Florencia Mercogliano, Mercedes Tkach, Leandro Venturutti, Patricia Virginia Elizalde, Roxana Schillaci. Immunotherapy against breast cancer based on Stat3 blockade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1346. doi:10.1158/1538-7445.AM2015-1346