Stable Knockdown Cell Research Articles

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA.

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.

The synthetic oleanane triterpenoid CDDO-Me binds and inhibits pyruvate kinase M2.

The M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2) is one of the key components in the Warburg effect, and an important regulator of cancer cell metabolism. Elevated PKM2 expression is a hallmark of numerous tumor types, making it a promising target for cancer therapy. Migration of H1299 lung tumor cells treated with synthetic oleanane triterpenoid derivatives CDDO-Me and CDDO-Im was monitored using scratch and transwell assays. Direct binding and inhibition of PKM2 activity by CDDO-Me was demonstrated by pull-down and activity assays. PKM2 localization in the absence and presence of CDDO-Me or CDDO-Im was determined by subcellular fractionation and immunofluorescence microscopy. Involvement of PKM2 in tumor cell migration was assessed using a stable PKM2 knockdown cell line. We demonstrate that migration of H1299 lung tumor cells is inhibited by CDDO-Me and CDDO-Im in scratch and transwell assays. CDDO-Me binds directly and specifically to recombinant PKM2, leading to a reduction of its catalytic activity. PKM2 knockdown cells exhibit significantly lower migration compared to control cells when subjected to glucose and oxygen deprivation, but not under regular conditions. The results suggest that PKM2 expression in a tumor-like environment contributes to cell migration, and that PKM2 activity can be down regulated by synthetic triterpenoid derivatives.

CTNNB1 Knockdown Inhibits Cell Proliferation and Aldosterone Secretion Through Inhibiting Wnt/β-Catenin Signaling in H295R Cells.

Aldosterone-producing adenomas (APA) is one of the causative factors of primary aldosteronism. Previous studies have suggested that there are somatic CTNNB1 mutations in APA, but the specific mechanism of CTNNB1 mutation in APA tumorigenesis and aldosterone secretion remains unclear. In the present study, human adrenocortical carcinoma cell line H295 R was used to establish stable CTNNB1 knockdown cell lines. Cell proliferation and aldosterone secretion of H295 R cells in response to angiotensin Ⅱ (Agn Ⅱ) were analyzed. We found that CTNNB1 knockdown reduced β-catenin expression and inhibited proliferation of H295 R cells. CTNNB1 knockdown inhibited Wnt/β-catenin signaling pathway and downregulated expression of downstream genes including axin 2, lymphoid enhancer binding factor 1 (LEF1), and cyclin D1. In addition, CTNNB1 knockdown decreased responses of H295 R cells to Agn Ⅱ and decreased aldosterone secretion. Our findings suggest that CTNNB1 knockdown can inhibit H295 R cell proliferation and decrease aldosterone secretion in the responses of H295 R cells to Ang II through inhibiting Wnt/β-catenin signaling pathway, indicating that targeting Wnt/β-catenin signaling pathway may be an important approach to decrease aldosterone secretion in the treatment of aldoster-producing adenomas.

The TGFβ-Induced Long Non-coding RNA TBULC Promotes the Invasion and Migration of Non-small Cell Lung Cancer Cells and Indicates Poor Prognosis.

Objective: To investigate the biological function and clinicopathological significance of the TGFβ-induced long non-coding RNA (lncRNA) TBULC in non-small cell lung cancer (NSCLC) and to analyze its potential value in clinical diagnosis and treatment.Methods: RT-qPCR was used to detect the expression level of TBULC in NSCLC cells and tissues, and the correlation between the TBULC expression level and clinicopathological characteristics was analyzed. A cytoplasmic/nuclear fractionation assay was performed to define the cellular localization of the TBULC. A rapid amplification of cDNA ends (RACE) assay was performed to acquire the full-length sequence of the TBULC. Stable TBULC overexpression and TBULC knockdown cell clones were constructed by lentiviral infection, and Transwell assays were used to explore the effect of the TBULC on cell invasion and migration.Results: Stimulation with TGFβ in NSCLC cell lines significantly upregulated the expression level of the nuclear-localized lncRNA TBULC. The RACE assay indicated that the full-length TBULC sequence was 1,020 nucleotides, and the sequence was located on chromosome 15. Cell function experiments showed that the TBULC played a crucial role in promoting NSCLC metastasis. Knockdown of TBULC significantly suppressed the invasion and migration of NSCLC cells, and overexpression of TBULC had the opposite effects. The expression level of TBULC in 106 NSCLC tumor tissues was significantly higher than that in adjacent normal tissues, and TBULC was proven to be an independent prognostic factor in NSCLC patients [p = 0.030, OR = 0.513 (0.281–0.936)].Conclusion: The TGFβ-induced lncRNA TBULC was upregulated in NSCLC and promoted the invasion and migration of NSCLC cells. TBULC was an independent prognostic factor and might be a potential biomarker for predicting the prognosis of NSCLC patients.

UCP2 promotes proliferation and chemoresistance through regulating the NF-κB/β-catenin axis and mitochondrial ROS in gallbladder cancer

Uncoupling protein 2 (UCP2) is a mitochondrial anion carrier which plays a key role in energy homeostasis. UCP2 is deregulated in several human cancers and has been suggested to regulate cancer metabolism. However, the role of UCP2 in gallbladder cancer has not been defined.Using clinical samples, we found highly expressed UCP2 in gallbladder cancer tissues, and higher expression levels of UCP2 correlated with worse clinical characteristics. To study whether UCP2 promotes gallbladder cancer growth, UCP2 stable knockdown cells were generated, and cell proliferation was suppressed in these knockdown cells. Further studies demonstrated that glycolysis was inhibited and IKKβ, as well as the downstream signaling molecules NF-κB/FAK/β-catenin, were downregulated in UCP2 knockdown cells. More importantly, gallbladder cancer cells became sensitive to gemcitabine treatments when UCP2 was inhibited. UCP2 knockdown suppressed the activation of the NF-κB/β-catenin axis and promoted the increases in mitochondrial ROS in gallbladder cancer cells exposed to gemcitabine treatments. The UCP2 inhibitor genipin suppressed xenograft tumor growth and sensitized grafted tumors to gemcitabine treatments. These results suggest targeting UCP2 as a novel therapeutic strategy for the treatment of gallbladder cancer.

Long non-coding RNA RP11-480I12.5 promotes cervical carcinoma progression by regulating the Wnt/β-catenin signaling pathway.

The long non-coding RNA (lncRNA), RP11-480I12.5 is one of the most dysregulated lncRNAs, which is believed to contribute to the progression of cervical carcinoma (CC); however, the exact function of RP11-480I12.5 in human CC remains unknown. The present study aimed to investigate the function and underlying molecular mechanism of RP11-480I12.5 in CC. First, reverse transcription-quantitative PCR was implemented in order to detect differences in the expression of RP11-480I12.5 between normal and CC tissues. The present study used in vitro analysis to establish RP11-480I12.5 stable knockdown and overexpressing cell lines, in order to investigate the function and potential molecular mechanism of RP11-480I12.5 in the progression of CC. RP11-480I12.5 was upregulated in CC tissue compared with normal tissue. Furthermore, RP11-480I12.5 was associated with clinical stage, tumor size and lymph node metastasis. RP11-480I12.5 promoted the proliferation, migration and invasion of CC cell lines. Subsequently, the present study investigated the association between RP11-480I12.5 and the epithelial-to-mesenchymal transition (EMT) and Wnt/β-catenin pathways. RP11-480I12.5 promoted EMT through the Wnt/β-catenin pathway. Overall, the results of the present study demonstrate that RP11-480I12.5 promotes cercical cancer cell migration, invasion and EMT through the Wnt/β-catenin pathway.

The Role of Proteasome Activator PA28α in Multiple Myeloma

Multiple myeloma (MM) is a commonly occurring hematologic malignancy in the United States with poor prognosis. Among all treatments, proteasome inhibitor (PI) based regimens have been a major breakthrough for patients' outcomes. Available PIs all target 20S proteasome core complex, and the duration of response is limited by toxicity and resistance development. Until now, the underlying mechanism of drug resistance remains unclear. The proteasome is the major proteolytic machinery in protein homeostasis which is pivotal for myeloma cell survival. A functional proteasome consists of 20S proteasome core particle with regulatory particle on one or both ends. There are 3 types of proteasome regulators that could activate a 20S proteasome, PA700 (19S), 11S REG (PA28) and PA200. The 11S REG (PA28) protein family consists of three members, α, β, and γ. PA28 α/β are IFN-γ inducible and with higher expression in antigen presenting cells. Currently, the function of 11S subunit remains largely unknown. Our analysis of plasma cells from MM patients and healthy donors has demonstrated that expression of 11S proteasome is higher in myeloma cells than normal plasma cells and progressively upregulated with disease progression. To further identify the function of 11S proteasome especially PA28α in MM, we generate PA28α knockdown stable MM cell lines. We have found that knockdown of PA28α inhibits MM cell growth and proliferation, also induces myeloma cell resistance to PIs. The mechanism of PI resistance is different from knocking down of 19S or 20S proteasome subunits. Silencing of PA28α inhibits proteasome activity and decreases proteasome work load concurrently, resulting in a favorable proteasome load vs capacity ratio. Altogether, in this report, we describe the function of PA28α in MM cells, also provide novel insights into regulating PIs sensitivity through modulation of the 11S proteasome subunit PA28α. Disclosures Hofmeister: Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Kaufman:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Incyte: Consultancy; Celgene: Consultancy; Winship Cancer Institute of Emory University: Employment; AbbVie: Consultancy; Takeda: Consultancy; TG Therapeutics: Consultancy. Nooka:Amgen: Honoraria, Other: advisory board participation; GSK: Honoraria, Other: advisory board participation; Celgene: Honoraria, Other: advisory board participation; Takeda: Honoraria, Other: advisory board participation; Spectrum pharmaceuticals: Honoraria, Other: advisory board participation; BMS: Honoraria, Other: advisory board participation; Janssen: Honoraria, Other: advisory board participation; Adaptive technologies: Honoraria, Other: advisory board participation. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Lonial:Takeda: Consultancy, Research Funding; Amgen: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; GSK: Consultancy; Karyopharm: Consultancy; Genentech: Consultancy; Celgene Corporation: Consultancy, Research Funding.

PRMT5 Promotes Human Lung Cancer Cell Apoptosis via Akt/Gsk3β Signaling Induced by Resveratrol

Protein arginine methyltransferase 5 (PRMT5) is implicated in various types of human cancer and tumor development, especially in lung cancer. Nevertheless, it is still unclear whether suppression of PRMT5 could promote lung cancer cell apoptosis and chemosensitivity induced by resveratrol, and the underlying molecular mechanism remains completely unknown. Here, we showed that PRMT5 was overexpressed in human lung cancer tissues and different types of lung cancer cell lines. Moreover, we constructed PRMT5 stable knockdown cell lines (A549 and ASCT-a-1) and investigated the roles of PRMT5 and the related signaling pathway in lung cancer cell apoptosis induced by resveratrol. Our results indicated that inhibition or down-regulation of PRMT5 by GSK591, a PRMT5-specific inhibitor, or shRNAs markedly enhanced cell apoptosis and chemosensitivity stimulated by resveratrol. Further investigation showed that inhibition or down-regulation of PRMT5 further reduced Akt/GSK3β phosphorylation and the downstream targets cyclin D1 and E1 expression upon resveratrol treatment. Our findings suggest that PRMT5 is a pivotal mediator for human lung cancer cell death induced by resveratrol, which also reveals that PRMT5 may serve as a new therapeutic target for the treatment of human lung cancer.

Roles of p38α and p38β mitogen‑activated protein kinase isoforms in human malignant melanoma A375 cells.

Skin cancer is one of the most common cancers worldwide. Melanoma accounts for ~5% of skin cancers but causes the large majority of skin cancer-related deaths. Recent discoveries have shown that the mitogen-activated protein kinase (MAPK) signaling pathway is critical for melanoma development and progression. Many oncogenic pathways that cause melanoma tumorigenesis have been identified, most of which are due to RAF/MEK/ERK (MAPK) pathway activation. However, the precise role of p38 remains unclear. Using specific short hairpin (sh) RNA to silence p38α and p38β, the present findings demonstrated that p38α was a crucial factor in regulating cell migration in the A375 melanoma cell line. Silencing p38α downregulated the expression of epithelial-mesenchymal transition markers, such as matrix metallopeptidase (MMP) 2, MMP9, twist family bHLH transcription factor 1, snail family transcriptional repressor 1 and vimentin, while mesenchymal-epithelial transition markers, such as E-cadherin, were upregulated. Of note, the results also demonstrated that p38α silencing impaired vascular endothelial growth factor expression, which regulates tumor angiogenesis. Furthermore, p38α knockdown inhibited cell proliferation in melanoma cells. In addition, silencing p38α induced senescence-like features, but not cell cycle arrest. Expression of the senescence markers p16, p21, p53 and β-galactosidase was upregulated, and an increase in the number of senescence-associated β-galactosidase-positive cells was observed in a p38α knockdown stable clone. However, no significant difference was found between control and p38β stable knockdown cells. Taken together, the present results suggested that p38α knockdown impaired migration and proliferation, and increased senescence, in A375 melanoma cells. However, p38β may not be involved in melanoma tumorigenesis. Therefore, targeting p38α may be a valuable approach towards inhibiting tumor growth and metastasis in patients with melanoma.

Knockdown of mitofilin inhibits autophagy and facilitates starvation-induced apoptosis in HeLa cells.

Objective(s):Mitofilin contributes to the maintenance of mitochondrial structure and functions. This study was undertaken to determine the mechanisms underlying its regulation of apoptosis. Materials and Methods:Mitofilin was knockdowned by specific short hairpin RNA (shRNA) and the stable HeLa cell clone was selected. The autophagy activity were assessed with LC3-II conversion and puncta formation by western blot and fluorescence imaging in starved and normal cultured HeLa cells. Autophagy flux was measured in the presence of NH4Cl. Wortmannin was used to inhibit autophagy. Cell viability and apoptosis were detected with cell counting kit-8 (CCK-8) and fluorescence-activated cell sorting (FACS) assay, respectively. Results:Mitofilin expression was down-regulated in starved HeLa cells. In established mitofilin stable knockdown cell lines, LC3-II conversion and puncta formation were detected, which are both hallmarks of autophagy, under both basal and starvation conditions. Mitofilin down-regulation decreased LC3-II conversion and puncta formation, which indicates that loss of mitofilin function inhibits both basal and starvation-induced autophagy activity. CCK-8 and FACS analysis confirmed mitofilin involvement in the regulation of cell survival since mitofilin down-regulation facilitated starvation-induced apoptosis in HeLa cells. Conclusion:Taken together, mitofilin is a potent regulator of autophagy and it may modulate cell survival through regulation of autophagy.

Non-SMC condensin I complex subunit H enhances proliferation, migration, and invasion of hepatocellular carcinoma.

Non‐SMC condensing I complex subunit H (NCAPH) is a member of the Barr protein family and part of the condensin I complex. The upregulation of NCAPH is associated with poor prognosis in patients with colon cancer. However, the relationship between NCAPH and hepatocellular carcinoma (HCC) remains unclear. This study aimed to explore NCAPH expression in HCC tissues and to investigate NCAPH functions in HCC cells. In this study, we found that high expression of NCAPH in HCC indicated worse prognosis via bioinformatics analysis. Consistently, quantitative real‐time polymerase chain reaction assays in 20 pairs of HCC specimens and the immunohistochemical analysis of 100 HCC tissues showed the upregulation of NCAPH. We established stable NCAPH‐overexpressing and NCAPH knockdown cell lines. Cell Counting Kit‐8 assays and colony formation assay were performed to analyze cell proliferation. Migration and invasion were analyzed by Transwell assays. Subcutaneous xenograft models were used to explore the role of NCAPH in tumor formation in vivo. Our results showed that NCAPH promoted tumor proliferation, migration, and invasion in vitro and in vivo. In conclusion, our findings indicate that NCAPH could serve as a novel prognostic biomarker and a potential therapeutic target for patients with HCC.

Ensamblaje y liberación del virus dengue: controversia sobre la participación de la proteína Alix

Algunos virus envueltos usurpan la maquinaria celular ESCRT (complejo de clasificación endosomal requerido para el transporte) para llevar a cabo funciones como la transcripción, la traducción, el ensamblaje y la liberación de partículas virales desde las células huésped. Aunque esta estrategia ha sido estudiada principalmente en retrovirus, son varios los virus envueltos que la usan. El objetivo del trabajo fue explorar la participación de una proteína accesoria de ESCRT, la proteína Alix, en la transcripción, traducción, ensamblaje y liberación del virus dengue (DENV), así como su interacción con la proteína viral NS3. Células A549 infectadas con DENV2 fueron tratadas con pequeños ARN de interferencia (siRNA) para disminuir la expresión (“knock-down”) de la proteína Alix. Simultáneamente, se obtuvo una línea A549 que expresaba una proteína NS3 recombinante y sobre este sistema se hicieron ensayos de inmunoprecipitación y “pull-down” para detectar interacción entre NS3 y Alix. Los resultados mostraron que el “knock-down” de Alix no tuvo efecto notable en la transcripción o la traducción viral, pero sí en el ensamblaje y la liberación de DENV2, mientras que los ensayos de “pull-down” revelaron la interacción entre NS3 y Alix. La participación de Alix en la producción de DENV2 y su interacción con NS3 constituyen un potencial blanco para el diseño de estrategias dirigidas a controlar la propagación de DENV.

DJ-1 overexpression confers the multidrug resistance phenotype to SGC7901 cells by upregulating P-gp and Bcl-2

Gastric cancer (GC) is one of the most malignant tumors with high incidence and mortality worldwide, and the multidrug resistance (MDR) often results in chemotherapy failure in GC. DJ-1 has been well indicated to be associated with drug resistance in multiple cancers. However, the role of DJ-1 in the MDR of gastric cancer cells and its possible mechanism remain to be elucidated. Therefore, the current study was investigated whether DJ-1 expression is differential in parental gastric cancer cell SGC7901 and vincristine (VCR)-induced gastric cancer MDR cell SGC7901/VCR, and whether DJ-1 plays a significant role in development of MDR in gastric cancer. The results showed that DJ-1 expression in SGC7901/VCR cells was significantly higher than its sensitive parental SGC7901 cells. Furthermore, DJ-1 overexpressed gastric cancer cell line SGC7901/LV-DJ-1 led to the increase of cell survival rate, the IC50 of chemotherapeutic drugs and number of cell clones as well as decrease of cell cycle G0/G1 phase ratio compared with its parental cells under the treatment of VCR, adriamycin (ADR), 5-Fluorouracil (5-FU) and cisplatin (DDP). However, the DJ-1 knockdown stable cell line SGC7901/VCR/shDJ-1 reversed the above mentioned series of MDR. Moreover, it was found that upregulation of DJ-1 protein expression promoted the pumping rate of GC cells to ADR and reduced the apoptotic index of GC cells treated with chemotherapeutic drugs by upregulating P-gp and Bcl-2. Similarly, knocking down DJ-1, P-gp or Bcl-2 displayed a converse effect. In conclusion, the current study demonstrated that DJ-1 overexpression confers the MDR phenotype to SGC7901 cells and this process is related to DJ-1 promoting active efflux of drugs and enhancing the anti-apoptotic ability of MDR GC cells by upregulating P-gp and Bcl-2.

DZIP1 Promotes Proliferation, Migration, and Invasion of Oral Squamous Carcinoma Through the GLI1/3 Pathway

BACKGROUND: DZIP1 is an oncogene involved in the progression and stemness of carcinoma through the Wnt/β-catenin pathway, and the potential mechanism of DZIP1 in oral squamous cancer remains unknown. The aim of this study was to uncover the effect and mechanism of DZIP1 in the progression of oral squamous carcinoma. METHODS: TCGA database scanning was applied to verify dysregulated genes in oral squamous carcinoma. quantitative real-time polymerase chain reaction, immunohistochemistry, and Western blotting assays were used to detect the expression of DZIP1 in tissues and cell lines. We established stable DZIP1-overexpressing and DZIP1 knockdown cell lines. We investigated the biological function and the underlying mechanism of DZIP1 through a series of experiments. RESULTS: DZIP1 was one of the genes discovered by the scanning strategy to be upregulated in cancer tissue and negatively correlated with the overall survival (OS) of patients. DZIP1 promotes proliferation, migration, and invasion in an oral squamous carcinoma cell line through EMT in a GLI1/3-dependent manner. CONCLUSIONS: DZIP1 promotes the proliferation, migration, and invasion of oral squamous carcinoma through the GLI1/3 pathway.

Skp2-mediated ubiquitination and mitochondrial localization of Akt drive tumor growth and chemoresistance to cisplatin.

The E3 ligase S-phase kinase-associated protein 2(Skp2) is overexpressed in human cancers and correlated with poor prognosis, but its contributions to tumorigenesis and chemoresistance in nasopharyngeal carcinoma (NPC) are not evident. Herein we show that Skp2 is highly expressed in NPC tumor tissues and cell lines. Knockdown of Skp2 suppresses tumor cell growth, colony formation, glycolysis, and in vivo tumor growth. Skp2 promotes Akt K63-mediated ubiquitination and activation, which is required for EGF-induced Akt mitochondrial localization. Importantly, K63-linked ubiquitination enhances the interaction between Akt and HK2 and eventually increases HK2 phosphorylation on Thr473 and mitochondrial localization. Overexpression of Skp2 impaired the intrinsic apoptotic pathway and confers cisplatin resistance. Moreover, ectopic expression of Myr-Akt1 or phosphomimetic HK2-T473D rescued cisplatin-induced tumor suppression in Skp2 knockdown stable cells. Also, depletion of Akt ubiquitination enhances the antitumor efficacy of cisplatin in vitro and in vivo. Finally, we demonstrated that Skp2 is positively correlated with p-Akt and HK2 in NPC tumor tissues. This study highlights the clinical value of Skp2 targeting in NPC treatment.

Abstract 5219: Kaiso as a novel therapeutic target in castration-resistant prostate cancer (CRPC)

Abstract Androgen receptor (AR) is a critical driver in the progression of prostate cancer (PCa). Androgen deprivation therapy(ADT) has been a standard treatment of PCa. However, the PCa develops to castration resistant prostate cancer (CRPC) in almost 53% of patients after 18-32 month’s therapy. Metastatic CRPC has poor prognosis and mean survival time is fewer than 2 years. Kaiso belongs to a BTB/POZ zinc finger protein family and is known as a transcriptional repressor. Kaiso expression and localization have been reported to correlate with the prognosis and metastatic potential in several human malignancies. Previous works from our lab have demonstrated that transcription factor Kaiso was upregulated in the progression of PCa. Therefore, our objective is to explore the interrelationship between Kaiso and AR, further to clarify Kaiso as a potential therapeutic target in PCa progression and CRPC. To investigate molecular mechanisms underlying how aberrant expression of Kaiso contributes to CRPC, the androgen sensitive human prostate cancer LNCaP cells were treated with 10µM anti-androgen Enzalutamide (MDV3100). Kaiso expression levels were increased as detected by RT-PCR and immunofluorescence. To systematically investigate Kaiso targets in PCas, we performed Kaiso ChIP-Seq assay using prostate cancer cell lines LNCaP, C4-2B and PC3. The Kaiso ChIP-Seq peaks indicate androgen receptor motif enrichment in AR expressing LNCaP and C4-2B cells but not in AR-negative PC3 cells, indicating Kaiso could cooperate with AR as co-regulator and potentially control transcription of a subset of its target genes. The interaction of them was confirmed by co-immunoprecipitation of AR using Kaiso antibody in LNCaP cells. To identify Kaiso target genes that interacted with AR pathway, Kaiso knockdown and overexpression stable cell lines were established in LNCaP cells and C42B cells. RT2 Profiler PCR array of AR pathway were performed using LNCaP scramble (LNCaP-Scr) cells and shKaiso cells. The results showed multiple genes were upregulated and downregulated, such as FOLH1, PTEN, RAC3 and IGF1. More importantly, LNCaP Kaiso overexpression cells were more resistant to MDV3100 treatment compared to resistance observed in LNCaP-Scr cells. There were significant negative correlations between Kaiso expression levels and castration-sensitivity. Our results suggest that Kaiso is a novel AR-interacting protein. Kaiso regulates the genes in AR pathway. Targeting Kaiso presents a potential therapeutic strategy for disrupting AR signaling in CRPC and may prevent CRPC development. These studies were supported by grants U54MD007585, (NIH/RCMI) [CY], U54CA118623-01 (NIH/NCI) [CY] Citation Format: Hui-Xian Lin, Honghe Wang, Jason White, Balasubramanyam Karanam, Anghesom Ghebremedhin, Benjamin Adu Addai, William E. Grizzle, Clayton Yates. Kaiso as a novel therapeutic target in castration-resistant prostate cancer (CRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5219.

Abstract 4651: Polo-like kinase 1 phosphorylates and stabilizes KLF4 to promote tumorigenesis in nasopharyngeal carcinoma

Abstract INTRODUCTION Advanced nasopharyngeal carcinoma (NPC) is an aggressive disease with no targeted therapies and poor outcomes. New innovative targets are urgently needed. Krüppel-like factor 4 (klf4, gklf) is a zinc-finger transcription factor involved in a wide variety of cellular processes, including apoptosis, cell cycle progression and stem cell renewal. Emerging data indicate that klf4 dysregulation either facilitates or impedes tumor progression. The function of klf4 in NPC remains elusive. METHODS we examined the expression of klf4 in human NPC (n=152) with a tissue immunohistochemistry (IHC) assay, and performed klf4 knockdown stable cell line with two different small hairpin RNAs (shRNAs) to evaluate the impact of klf4 on NPC. Global gene expression experiments were performed to explore the molecular mechanisms underlying klf4-dependent tumorigenesis. Small-molecule kinase inhibitor screening was performed to identify potential upstream kinases of klf4. Co-immunoprecipitation assay used to test the interaction between plk1 and klf4. Mass spectrometry analysis was performed to determine the specific site at which klf4 is phosphorylated by plk1. Statistical analyses were conducted using GraphPad Prism, SPSS software and JavaGSEA Desktop Application. A p value of <0.05 was considered to indicate statistical significance. RESULTS Our investigation showed that high expression of klf4 was correlated with poor prognosis in NPC. Moreover, genome-wide profiling revealed that klf4 directly activated oncogenic programmes, including gene sets associated with KRAS, VEGF, and MYC signalling. We further found that inhibition of polo-like kinase 1 could downregulate the expression of klf4 and that plk1 directly phosphorylated klf4 at Ser234. Notably, phosphorylation of klf4 by plk1 caused the recruitment and binding of the E3 ligase traf6, which resulted in klf4 K32 K63-linked ubiquitination and klf4 stabilization. Moreover, klf4 could enhance traf6 expression at the transcriptional level, thus initiating a klf4-traf6 feed-forward loop. Treatment with the plk1 inhibitor volasertib (BI6727) significantly inhibited tumor growth in nude mice. CONCLUSION In this study, we revealed that klf4 is upregulated in NPC and is correlated with poor prognoses. Our study demonstrated for the first time that plk1 can directly interact with klf4 and phosphorylate Ser234, alter the affinity of klf4 for the E3 ligase traf6, and promote the K63-linked ubiquitination of klf4 K32. In addition, klf4 can enhance traf6 expression at the transcriptional level and further promote klf4 expression. The resulting increase in klf4 ubiquitination leads to stabilization and upregulation of klf4, which leads to tumorigenesis in NPC. These results expand our understanding of the role of KLF4 in NPC and validate PLK1 inhibitors as potential therapeutic agents for NPC. Citation Format: Jia Mai, Zhuo-Yan Zhong, Xiu-Xing Chen, Yan-Qun Xiang, Xuan Li, Hai-Liang Zhang, Rong Deng, Xiao-Feng Zhu. Polo-like kinase 1 phosphorylates and stabilizes KLF4 to promote tumorigenesis in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4651.

Histone demethylase Jmjd3 modulates osteoblast apoptosis induced by tumor necrosis factor-alpha through directly targeting RASSF5

ABSTRACT Purpose: Regulation of gene expression is fine-tuned by a dynamic equilibrium between repressive modifications and transcriptional activation of histone tails. Jumonji domain-containing 3 (Jmjd3), also known as KDM6B, is a specific histone demethylase for trimethylation on histone H3 lysine 27 (H3K27me3) that specifically removes the methylation of H3K27me3 and promotes gene expression. Our previous study showed that Jmjd3 inhibits serum deprivation-induced osteoblast apoptosis. In this study, we clarified the role of Jmjd3 in tumor necrosis factor-alpha (TNF-α)-induced osteoblast apoptosis. Materials and Methods: Jmjd3 activity was inhibited by GSK-J4. Transfection of osteoblastic murine MC3T3-E1 cells with short hairpin RNA (shRNA) was used to establish stable Jmjd3 knockdown cells. Osteoblast apoptosis was detected using Annexin V-APC/PI staining, cysteinyl aspartate specific protease-3 (caspase-3) activity assays, and Western blot. Real-time polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) assays were performed to clarify the mechanism responsible for Jmjd3-regulated osteoblast apoptosis induced by TNF-α. Results: Based on Annexin V-APC/PI staining, caspase-3 activation, and poly ADP-ribose polymerase (PARP) cleavage, pretreatment with GSK-J4 and knockdown of Jmjd3 by shRNA transfection each inhibited osteoblast apoptosis. Furthermore, knockdown of Jmjd3 decreased the expression of Ras association domain family 5 (RASSF5), which is a pro-apoptotic gene of the Ras associated domain family. H3K27me3 levels in the promoter region of RASSF5 were up-regulated in the Jmjd3 knockdown cells. Conclusions: Jmjd3 regulated TNF-α-induced osteoblast apoptosis by targeting RASSF5.

Characterization of the epithelial membrane protein 3 interaction network reveals a potential functional link to mitogenic signal transduction regulation.

Epithelial Membrane Protein 3 (EMP3), a 4-transmembrane glycoprotein, first gained attention as a putative tumor suppressor. Accumulating evidence, however, points to a more tumor promotive function of EMP3. The biological function of EMP3 remains largely unclear. To elucidate more of EMP3's interaction network, we performed a Yeast-Two-Hybrid (Y2H) screening, followed by validation of candidate interactors by Biomolecular Fluorescence Complementation (BiFC) and Proximity Ligation Assay (PLA). Furthermore, we generated stable EMP3 knockdown cell lines and measured cell proliferation, migration and sensitivity to apoptosis induction as well as the expression and activation levels of important signal pathway components. The Y2H screening yielded 10 novel interactions of EMP3, eight of which could also be detected by BiFC and PLA interaction assays. All newly discovered interaction partners are involved in signaling or trafficking regulation. Most notably, FLOT1 and HTATIP2 have well described roles in the regulation of EGFR signaling. In addition, knockdown of EMP3 resulted in reduced levels of p-AKT, p-ERK and p-EGFR, attenuated cell proliferation and migration and sensitized cells to apoptosis induction by TRAIL and Staurosporine. Based on these observations we hypothesize that EMP3 might be involved in the regulation of receptor-tyrosine-kinase mediated mitogenic signaling.

HCP5 is a SMAD3-responsive long non-coding RNA that promotes lung adenocarcinoma metastasis via miR-203/SNAI axis.

Introduction: Transforming growth factor-beta (TGFβ) signaling plays a vital role in lung adenocarcinoma (LUAD) progression. However, the involvement of TGFβ-regulated long non-coding RNAs (lncRNAs) in metastasis of LUAD remains poorly understood.Methods: We performed bioinformatic analyses to identify putative lncRNAs regulated by TGF-β/SMAD3 and validated the results by quantitative PCR in LUAD cells. We performed luciferase reporter and chromatin immunoprecipitation assays to demonstrate the transcriptional regulation of the lncRNA histocompatibility leukocyte antigen complex P5 (HCP5) we decided to focus on. Stable HCP5 knockdown and HCP5-overexpressing A549 cell variants were generated respectively, to study HCP5 function and understand its mechanism of action. We also confirmed our findings in mouse xenografts and metastasis models. We analyzed the correlation between the level of lncRNA expression with EGFR, KRAS mutations, smoke state and prognostic of LUAD patients.Results: We found that the lncRNA HCP5 is induced by TGFβ and transcriptionally regulated by SMAD3, which promotes LUAD tumor growth and metastasis. Moreover, HCP5 is overexpressed in tumor tissues of patients with LUAD, specifically in patients with EGFR and KRAS mutations and current smoker. HCP5 high expression level is positively correlated with poor prognosis of patients with LUAD. Finally, we demonstrated that upregulation of HCP5 increases the expression of Snail and Slug by sponging the microRNA-203 (miR-203) and promoting epithelial-mesenchymal transition (EMT) in LUAD cells.Conclusions: Our work demonstrates that the lncRNA HCP5 is transcriptionally regulated by SMAD3 and acts as a new regulator in the TGFβ/SMAD signaling pathway. Therefore, HCP5 can serve as a potential therapeutic target in LUAD.