RNAi-mediated downregulation of CDKL1 inhibits growth and colony-formation ability, promotes apoptosis of human melanoma cells
Introduction
Malignant melanoma is a highly aggressive skin tumor with increasing incidence and poor prognosis [1]. Many attempts have been made to treat the disease. BRAF V600E inhibitors (e.g. vemurafenib, dabrafenib), the first target therapy, have improved both progression-free survival and overall survival, compared with chemotherapy in patients with BRAF V600E-mutated metastatic melanoma [2], [3], [4], [5], [6], [7]. However, clinical evidence of tumor resistance developed 5–7 months later in a subset of patients [8], [9], due to MAPK reactivation, driven by secondary mutations in NRAS and MEK1 genes [10].
Malignant tumors are characterized by inappropriate cell proliferation, which arises when checkpoint mechanisms that limit proliferation of cells with damaged DNA are degraded, permitting expansion of clones with genetic instability [11]. Disturbed regulation of the cell cycle is a hallmark of cancer [12]. Melanomagenesis is associated with not only defects in nucleotide excision repair of solar radiation-induced DNA damage, but also cell cycle checkpoints that arrest growth after DNA damage [13]. Defective checkpoints may represent potentially selective anti-melanoma therapeutic targets.
The cyclin-dependent protein kinase (CDK) protein family has been demonstrated to be an important regulator of cell division at the G1/S and G2/M checkpoints [14]. The CDK family regulates a wide range of cellular functions such as cell cycle progression, differentiation, and apoptosis [15]. CDKs form active complexes with a specific cyclin, thus controlling the expression of downstream genes involved in the cell cycle [14]. Cyclin-dependent kinase 1 (CDK1), also known as cell division control protein 2 (CDC2), is a key molecule among them. Cyclin-dependent kinase-like 1 (CDKL1) is a member of CDK1-related serine–threonine protein kinase family [16]. It was cloned on the basis of its similarity to the CDK1 kinase domain [17], and named after the amino acid sequence corresponding to the PSTAIRE motif of CDK1/CDC2 [18], [19]. CDKL kinase family is not known to interact with cyclins, but considered as a separate branch of CDK family that is similar to the MAP kinases group of signal transducing enzymes [23]. CDKL1 contains the conserved MAP kinase dual phosphorylation motif Thr-Xaa-Tyr (Thr-Asp-Tyr), and may therefore contribute to signal transduction [24]. Recent studies reported that CDKL1 was overexpressed in human breast cancer and gastric cancer, and loss of CDKL1 function in these cancer cell lines resulted in inhibition of cell proliferation and increase of apoptosis [20], [21]. However, up to now, the expression of CDKL1 in malignant melanoma and its function in melanoma cell regulation are not known.
In this study, we investigated the expression of CDKL1 in human melanoma cell lines A375 and MV3. Furthermore, we constructed CDKL1-siRNA expressing lentivirus, and evaluated the effects of RNAi-mediated CDKL1 downregulation on A375 and MV3 cell proliferation, colony-formation ability, cell cycle and apoptosis.
Section snippets
Cell culture
Human melanoma cell lines, A375 and MV3 were obtained from Shanghai institutes for biological science (SIBS) cell bank. A375 cells were maintained in DMEM and MV3 cells were maintained in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) plus 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) in a 5% CO2 incubator at 37 °C.
Lentiviral CDKL-siRNA vector construction and packaging
Small interfering RNA (siRNA) target sequence (CTACTGTGATACCAAGAAA) for CDKL1 gene (NM_004196) was designed and a non-silencing siRNA sequence (TTCTCCGAACGTGTCACGT) was
Knockdown efficiency of CDKL1 by siRNA lentivirus system in human melanoma cells
To investigate the role of CDKL1in melanoma, siRNA targeting CDKL1 or non-silencing sequences were cloned into pGCSIL-GFP plasmid vector, respectively. Then, CDKL1 siRNA lentivirus or non-silencing siRNA lentivirus (negative control) expressing GFP were generated and infected into two human melanoma cell lines, A375 and MV3 cells. As shown in Fig. 1a, three days after infection, more than 80% of GFP expressing cells were observed under the fluorescence microscope, suggesting an infection
Discussion
Our study showed that CDKL1 is expressed in melanoma cell lines A375 and MV3. We then employed lentivirus-mediated knockdown to specifically inhibit CDKL1 gene expression and demonstrated that loss of CDKL1 function significantly inhibited cell proliferation and colony formation, and promoted apoptosis in A375 and MV3 cells. These results suggested that CDKL1 might be associated with tumor progression in malignant melanoma. It may become a probable target of anti-melanoma therapy. Besides, our
Acknowledgements
This work was funded by National Natural Science Foundation of China (No. 81102070).
References (27)
- et al.
Safety and efficacy of vemurafenib in BRAF (V600E) and BRAF(V600K) mutation-positive melanoma (BRIM-3): extended follow-up of a phase 3, randomised, open-label study
Lancet Oncol
(2014) - et al.
Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial
Lancet
(2012) Targeting cell cycle and apoptosis for the treatment of human malignancies
Curr Opin Cell Biol
(2004)- et al.
Dysregulation of cellular signaling in gastric cancer
Cancer Lett
(2010) - et al.
Defective cell cycle checkpoint functions in melanoma are associated with altered patterns of gene expression
J Invest Dermatol
(2008) - et al.
Identification and characterization of a novel serine–threonine kinase gene from the Xp22 region
Genomics
(1998) - et al.
MLL3 is a Haploinsufficient 7q tumor suppressor in acute myeloid leukemia
Cancer Cell
(2014) - et al.
Melanoma incidence and mortality among US whites, 1969–1999
JAMA
(2002) - et al.
BRAF(V600E) protein expression and outcome from BRAF inhibitor treatment in BRAF(V600E) metastatic melanoma
Br J Cancer
(2013) - et al.
Survival in BRAF V600—advanced melanoma treated with vemurafenib
N Engl J Med
(2012)
Improved survival with vemurafenib in melanoma with BRAF V600E mutation
N Engl J Med
Phase II trial (BREAK-2) of the BRAF inhibitor dabrafenib (GSK2118436) in patients with metastatic melanoma
J Clin Oncol
Treatment of BRAF-mutant melanoma: the role of vemurafenib and other therapies
Clin Pharmacol Ther
Cited by (18)
CDKL1 promotes the chemoresistance of human oral squamous cell carcinoma cells to hydroxycamptothecin
2019, Molecular and Cellular ProbesCitation Excerpt :However, few studies have focused on the specific factors influencing the sensitivity of HCPT in OSCC. Since CDKL1 has high sequence similarity with the cell cycle controllers CDKs, accumulating data have pointed to a critical role for CDKL1 in the development and metastasis of several distinct cancers [9–11]. In this study, RT-qPCR and western blot analysis revealed that HCPT treatment induced CDKL1 expression in OSCC CAL27 cells, and the effect was exaggerated with higher doses.
CDKL1 potentiates the antitumor efficacy of radioimmunotherapy by binding to transcription factor YBX1 and blocking PD-L1 expression in lung cancer
2024, Journal of Experimental and Clinical Cancer ResearchTherapeutic Effects of Self-Assembled Tetrahedral Framework Nucleic Acids on Liver Regeneration in Acute Liver Failure
2022, ACS Applied Materials and Interfaces