Depletion of PCAT-1 in head and neck cancer cells inhibits tumor growth and induces apoptosis by modulating c-Myc-AKT1-p38 MAPK signalling pathways
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Head and neck squamous cell carcinoma (HNSCC) represents one of the most common malignancies worldwide with a high mortality rate mainly due to lack of early detection markers, frequent association with metastasis and aggressive phenotype. Recently, long non-coding RNAs (lncRNAs) have been shown to have important regulatory roles in human cancers. The lncRNA prostate cancer-associated transcript 1 (PCAT-1) showed potential oncogenic roles in different cancers, however its role in HNSCC is not known. In this study, we evaluated the role of the PCAT-1 in HNSCC.
The expression of PCAT-1 was measured by quantitative real-time PCR in 23 paired human HNSCC tissues and adjacent non-tumor tissue specimens. Cell proliferation after depleting PCAT-1 was determined. Effect of PCAT-1 depletion in HNSCC cell lines was determined by qRT-PCR and Western blot analyses. Finally, JHU029 HNSCC cells was implanted subcutaneously into athymic nude mice and therapeutic potential of PCAT-1 was investigated.
Up-regulation of PCAT-1 in TCGA dataset of HNSCC was noted. We also observed increased expression of PCAT-1 in archived HNSCC patient samples as compared to adjacent non-tumor tissues. Knockdown of PCAT-1 significantly reduced cell proliferation in HNSCC cell lines. Mechanistic study revealed significant down regulation of c-Myc and AKT1 gene in both RNA and protein levels upon knockdown of PCAT-1. We observed that c-Myc and AKT1 positively correlate with PCAT-1 expression in HNSCC. Further, we observed activation of p38 MAPK and apoptosis signal-regulating kinase 1 upon knockdown of PCAT-1 which induces Caspase 9 and PARP mediated apoptosis. Targeted inhibition of PCAT-1 regresses tumor growth in nude mice.
Together our data demonstrated an important role of the PCAT-1 in HNSCC and might serve as a target for HNSCC therapy.
KeywordsHead and neck squamous cell carcinoma (HNSCC) Long non-coding RNA PCAT-1 c-Myc-AKT1signaling Tumorigenicity
Apoptosis signal- regulating kinase 1
Head and neck squamous cell carcinoma
Long non-coding RNA
MAX dimerization protein 1
Mitogen-activated protein kinase
MYC Associated Factor X
Poly (ADP-ribose) polymerases
Prostate cancer associated transcript 1
Plasmacytoma variant translocation 1
Head and neck squamous cell carcinoma (HNSCC) represents one of the most common malignancies worldwide with a high mortality rate mainly due to lack of early detection markers, frequent association with metastasis and aggressive phenotype . The standard treatment for HNSCC is platinum-based drugs, 5-FU, and cetuximab regimen [2, 3]. Other agents like isotretionin, celecoxib, and erlotinib were in randomized clinical trials; however, no effective and tolerable agent has been identified for prevention of HNSCC . Thus, there is an urgent need to identify biomarkers and therapeutic targets for this disease.
The recent discovery of long non-coding RNAs (lncRNAs) has gained widespread attention as a new layer of regulation in biological processes. It has been shown that lncRNAs, over 200 nucleotides in size, regulate gene functions either interacting with DNA, RNA or directly with proteins [1, 4]. Dysregulation of lncRNAs were found to be associated with various human diseases ranging from neurodegeneration to cancer . To date, numerous cancer-associated lncRNAs are reported to modulate tumour growth, invasion and metastasis, and have been implicated as potential alternative biomarkers and therapeutic targets for cancer . Prostate cancer-associated transcript 1 (PCAT-1) was initially identified in prostate cancer [5, 6]. PCAT-1 is located at the Chr8q24 gene desert approximately 725 kb upstream of the Myc oncogene, the region is frequently amplified in HNSCC [7, 8, 9]. Overexpression of PCAT-1 was reported in several cancers and its function is associated with cell proliferation, invasion, metastasis, survival, cell cycle, chemoresistance, and homologous recombination. . However, the role of PCAT-1 in HNSCC remains unknown.
In this study, we observed overexpression of PCAT-1 in HNSCC patient samples. Knockdown of PCAT-1 inhibited HNSCC cell proliferation. We observed that depletion of PCAT-1 in HNSCC cell lines inhibits c-Myc and AKT1 genes and activates p38 MAPK signalling, resulting in Caspase 9 mediated apoptosis. Targeted inhibition of PCAT-1 reduced in-vivo tumorigenesis in mouse xenograft model. To our knowledge this is first study demonstrating an important role of PCAT-1 in HNSCC.
Patient tissue specimens
Achieved frozen tissues of HNSCC and matched adjacent non-tumor tissues (n = 23) were obtained from Saint Louis University Cancer Center. All the samples were immediately snap frozen in liquid nitrogen for long-term preservation until RNA extraction.
Cell culture and siRNA transfection
HNSCC cell line Cal27 was purchased from the ATCC. JHU029 and JHU022 cell line were obtained from the Johns Hopkins University through MTA. Cal27 cells were maintained in DMEM media, JHU029 and JHU022 cells were in RPMI- 1640 media, and supplemented with 10% FBS and 1% penicillin/ streptomycin in a humidified CO2 incubator at 37 °C. Normal oral keratinocytes (NOK) cells (kindly gifted by Karl Mugner) were maintained in Keratinocyte SFM medium supplemented with epidermal growth factor and bovine pituitary extract (GIBCO, Life technologies). All cells are mycoplasma free, but have not authenticated further.
JHU029 and Cal27 cells (2.5 × 105) were plated using 6-well plates overnight in antibiotic free media. Cells were transfected with either control oligo or siRNAs to PCAT-1 (Assay ID: n511583, ThermoFisher Scientific) mixed with Opti-MEM and LipofectamineRNAiMAX (Invitrogen) according to manufacturer’s protocols for a final concentration of siRNA of 50 nM, and incubated for 48 h. All the analysis was performed in triplicate.
Cell proliferation assay
JHU029 and Cal27 cells were plated using 35 mm dishes and transfected with/ without siRNA. At 0 h, 48 h and 96 h after siRNA transfection cells were harvested with trypsin, stained with trypan blue, and percentage of live cells were counted using a haemocytometer. The cell numbers in control or transfected were compared with the number at 0 h. The experiment was performed in triplicate.
RNA isolation and expression analysis
Total RNA was extracted by TRIzol reagent followed by cDNA synthesis using 1 μg of total RNA by SuperScript III Reverse Transcriptase (Life technology, USA) in accordance with the manufacturer’s protocol. Real-time PCR was performed for quantitation of gene expression using TaqMan Universal PCR master mix and 6-carboxyfluorescein (FAM)-MGB probes for PCAT-1 (assay ID:Hs04275836_s1; ThermoFisher Scientific) as per manufacture’s protocol. 18 s rRNA (assay ID:Hs03928985_g1; ThermoFisher Scientific) was used as an endogenous control. Gene expression analysis of c-Myc (FR: 5′ GCTCGTCTCAGAGAAGCTGG 3′ and RP: 5’GCTCAGATCCTGCAGGTACAA 3′) and AKT1 (FR: 5’TCTATGGCGCTGAGATTGTG 3′ and RP: 5’CTTAATGTGCCCGTCCTTGT 3′) was carried out by SYBR green based detection system as per standard procedure. GAPDH (FR: 5′ CATGTTCGTCATGGGTGTGAACCA 3′ and RP: 5′ AGTGATGGCATGGACTGTGGTCAT 3′) or 18 s (FP: 5′ GTCATAAGCTTGCGTTGATT 3′ and RP: 5′ TAGTCAAGTTCGACCGTCTT 3′) was used as endogenous control. The relative gene expression was analysed by 2-∆∆CT method. Each sample was loaded in triplicate.
Western blot analysis
Cell lysates were prepared using 2× SDS sample buffer, and western blot analysis was performed using specific antibody to c-Myc (1:1000), AKT1 (1:1000), phospho-P38 (Thr180/ Tyr 182) (1:1000), total P38 (1:1000), phospho- ASK1 (Ser83) (1: 1000), total ASK1 (1: 1000), Caspase-9 (1:1000), and PARP (1:1000). All the primary and HRP-conjugated anti mouse or anti-rabbit secondary antibodies (1:2000) were purchased from Cell Signalling Technology. The blot was reprobed with either Actin or GAPDH antibody (1:5000) to compare protein load in each lane. Densitometry analysis was done using Image J software.
Xenograft tumor growth
JHU029 cells were trypsinized, washed, and resuspended in PBS containing 40% BD-Matrigel. Cells (1.4X106) were injected subcutaneously into the flank of BALB/c athymic nude female mice (6 weeks old, n = 6). Mouse number are chosen based on our previous experience. After three weeks of palpable tumor developed (>75mm3), 10 μg of siPCAT-1 or control oligo complexed with siPORTamine (Invitrogen) in 50 μl Opti-MEM were injected intratumorally at an interval of 4 days a total of ten times. Tumors were measured using a Slide Calliper once a week and volume was calculated using the formula V = ½ (L x W2). All the mice were sacrificed at 8-week post injection. After sacrifice tumors were dissected out and snap frozen in liquid nitrogen for RNA extraction and protein isolation. No adverse effect was observed in the animals. Mice were euthanized by asphyxiation by CO2 inhalation using a compressed gas source. This method of euthanasia is consistent with the recommendations of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. All animal experiments were carried out in accordance with NIH guidelines, following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Saint Louis University.
Bioinformatics and statistical analysis
PCAT-1 gene alteration and expression was analysed from TCGA data sets using cBioPortal (www.cbioportal.org) platform and GEPIA (http://gepia.cancer-pku.cn). P value of < 0.05 was considered as cut off value for gene expression analysis. The relationship between PCAT-1 and c-Myc; PCAT-1 and AKT1 in patient samples were evaluated by Pearson’s correlation coefficient (R) (linear regression) using GraphPad Prism 6. Correlation analysis was also performed from the TCGA data set using GEPIA. Results are presented as means ± standard deviations. Data were analysed by Student’s t test. P value of < 0.05 was considered as statistically significant.
Overexpression of PCAT-1 in HNSCC
Knockdown of PCAT-1 inhibits HNSCC cell growth and reduces c-Myc and AKT1 expression
Knockdown of PCAT-1 activates p38 MAP kinase signalling and induces apoptosis
Therapeutic potential of PCAT-1 in HNSCC xenograft model
Our study demonstrated that depletion of PCAT-1 in HNSCC cell lines inhibits cell proliferation and induces apoptosis by i) inhibiting c-Myc and AKT1 expression, ii) activating ASK1 mediated p38 MAPK signalling, and iii) inducing Caspase 9 and PARP cleavage. We also observed that introduction of siRNA to PCAT-1 reduces HNSCC tumor growth in preclinical model.
PCAT-1 was initially identified as an oncogene in prostate cancer [5, 6]. Higher expression of PCAT-1 has been observed in several cancers including colorectal, gastric, esophageal, osteosarcoma, lung, bladder, cervical, hepatocellular carcinoma, cholangiocarcinoma, and multiple myeloma . We observed up-regulation of PCAT-1 in HNSCC patient samples compared to adjacent non-tumor tissues. c-Myc is ubiquitous transcription factor essential for cell proliferation, survival, differentiation, and metabolism in cancers . The AKT pathway is one of the most commonly dysregulated pathways in several cancers . We observed that c-Myc and AKT1 positively correlate with PCAT-1 expression in HNSCC.
LncRNA PVT1, closely located in the c-Myc gene, also regulates c-Myc expression and knockdown of PVT1 resulted in inhibition of cell proliferation and induction of apoptosis in hematologic malignancy via down-regulation of c-Myc . Although Myc inhibition is a powerful approach for cancer therapy, due to its ‘undruggable’ protein structure, targeted therapy has been facing challenges for decades . Therefore, it is plausible that targeting PCAT-1 might have potential for Myc associated cancer therapy. Myc function is dependent on Myc/ MAX complex formation and tumor suppressor protein Mad is recognized as an important cellular antagonist of Myc . Activation of AKT- MAPK signalling was found to induce Mad degradation by phosphorylation resulting functional activation of Myc . Thus, the down-regulation of c-Myc expression may be due to down regulation of AKT1 gene and this mechanism needs to be elucidated in future studies. Modulation of AKT1 gene was noted in the transcriptomic data of PCAT-1 depleted prostate cancer cells . Activation of AKT signalling is a frequent event in human cancers including HNSCC. AKT kinases regulate several important oncogenic events such as cell proliferation, survival, metastasis and angiogenesis [24, 25, 26]. Proliferation and survival signal in cancer is mainly achieved by AKT-MAPK signalling which is also responsible for the drug resistant phenotype [24, 25]. Thus, targeting AKT signalling has potential benefit in cancer therapy. AKT1 inactivates ASK1 by phosphorylating at Ser83 residue. Inhibition of AKT enhances p38 MAPK activation through phosphorylation by ASK1 [10, 11, 12, 13, 14]. Activation of p38 MAPK is also correlated with apoptosis in prostate or colon cancer [15, 27]. AKT1 inhibition by PCAT-1 depletion resulted in p38 MAPK activation via ASK1, resulting in Caspase 9 mediated HNSCC cell apoptosis.
Our results for the first time demonstrated an important role of lncRNA PCAT-1 in HNSCC proliferation and apoptosis in in-vitro and in-vivo systems. Depletion of PCAT-1 reduced c-Myc and AKT1 expression resulting in activation of p38 MAPK signalling and induction of apoptosis (Fig. 5f). Thus, targeting PCAT-1 will have therapeutic advantages against HNSCC.
This work was supported by research grant R01 DE024942 from the National Institutes of Health (R. B. Ray), and Saint Louis University Cancer Center Seed Grant (R. B. Ray).
Availability of data and materials
The data generated for this study is included in this manuscript.
SS and RBR conceived and designed the project, SS, HN, RS and RBR performed the experiments and analysed the data. SS and RBR wrote the manuscript and all authors edited. RBR was responsible for research supervision and funding acquisition. All authors read and approved the manuscript.
Ethics approval and consent to participate
The specimens are collected from discarded tissues samples after surgery at the Department of Otolaryngology, Saint Louis University, Missouri, USA. This research study was approved by the Institutional Review Board of Saint Louis University, and the subjects provided informed consent.
Consent for publication
All authors agreed on the manuscript.
The authors declare that they have no competing interests.
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