Skip to main content

Main menu

  • Home
  • Current Issue
  • Archive
  • Info for
    • Authors
    • Subscribers
    • Advertisers
    • Editorial Board
  • Other Publications
    • In Vivo
    • Cancer Genomics & Proteomics
    • Cancer Diagnosis & Prognosis
  • More
    • IIAR
    • Conferences
    • 2008 Nobel Laureates
  • About Us
    • General Policy
    • Contact
  • Other Publications
    • Anticancer Research
    • In Vivo
    • Cancer Genomics & Proteomics

User menu

  • Register
  • Subscribe
  • My alerts
  • Log in
  • My Cart

Search

  • Advanced search
Anticancer Research
  • Other Publications
    • Anticancer Research
    • In Vivo
    • Cancer Genomics & Proteomics
  • Register
  • Subscribe
  • My alerts
  • Log in
  • My Cart
Anticancer Research

Advanced Search

  • Home
  • Current Issue
  • Archive
  • Info for
    • Authors
    • Subscribers
    • Advertisers
    • Editorial Board
  • Other Publications
    • In Vivo
    • Cancer Genomics & Proteomics
    • Cancer Diagnosis & Prognosis
  • More
    • IIAR
    • Conferences
    • 2008 Nobel Laureates
  • About Us
    • General Policy
    • Contact
  • Visit us on Facebook
  • Follow us on Linkedin
Research ArticlePROCEEDINGS OF THE 20th ANNUAL MEETING OF THE SOCIETY OF BIOTHERAPEUTIC APPROACHES, 10 December, 2016 (Fukuoka, Japan)

An Alpha-kinase 2 Gene Variant Disrupts Filamentous Actin Localization in the Surface Cells of Colorectal Cancer Spheroids

KENSUKE NISHI, HAO LUO, KAZUHIKO NAKABAYASHI, KEIKO DOI, SHUHEI ISHIKURA, YURI IWAIHARA, YASUHIRO YOSHIDA, KUMPEI TANISAWA, TOMIO ARAI, SEIJIRO MORI, MOTOJI SAWABE, MASAAKI MURAMATSU, MASASHI TANAKA, TOSHIFUMI SAKATA, SENJI SHIRASAWA and TOSHIYUKI TSUNODA
Anticancer Research July 2017, 37 (7) 3855-3862;
KENSUKE NISHI
1Department, of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
2Department, of Otorhinolaryngology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
HAO LUO
1Department, of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
KAZUHIKO NAKABAYASHI
3Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Tokyo, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
KEIKO DOI
1Department, of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
4Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
SHUHEI ISHIKURA
1Department, of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
4Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
YURI IWAIHARA
1Department, of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
YASUHIRO YOSHIDA
1Department, of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
KUMPEI TANISAWA
5Departments of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
TOMIO ARAI
5Departments of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
SEIJIRO MORI
6Center for Promotion of Clinical Investigation, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
MOTOJI SAWABE
7Department of Moleculo-genetic Sciences, Division of Biomedical Laboratory Sciences Molecular Pathophysiology, Graduate School of Health Care Sciences, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
MASAAKI MURAMATSU
8Department of Molecular Epidemiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
MASASHI TANAKA
9Department of Clinical Laboratory, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
TOSHIFUMI SAKATA
2Department, of Otorhinolaryngology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
SENJI SHIRASAWA
1Department, of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
4Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
TOSHIYUKI TSUNODA
1Department, of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
4Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: tsunoda@fukuoka-u.ac.jp
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

Background/Aim: Alpha-kinase 2 (ALPK2), suggested to be a novel tumour-suppressor gene down-regulated by oncogenic KRAS, plays a pivotal role in luminal apoptosis in normal colonic crypts. The aim of this study was to determine the association between ALPK2 germline variants and colorectal cancer. Materials and Methods: Missense single nucleotide variants in the exons of the ALPK2 gene in 2,343 consecutive autopsy cases (1,446 cases with cancer and 897 cases without cancer) were screened using HumanExome BeadChip arrays. To address the functional effect of a missense ALPK2 variant, a 3D floating cell culture was performed using HCT116-derived human colorectal cancer cells stably expressing wild-type (wt) ALPK2 (HCT116-wtALPK2) or amino acid-substituted (sub) ALPK2 (HCT116-subALPK2). Results: We identified that one of the ALPK2 germline variants, rs55674018 (p.Q1853E), was significantly associated with the presence of cancer (adjusted odds ratio(OR)=4.39; 95% confidence interval(CI)=1.31-14.78, p=0.001). The p.Q1853E variant was present in the East Asian population and located in the immunoglobulin-like domain. Notably, the basolateral polarity of actin in the surface of HCT116-wtALPK2 spheroids was more attenuated compared to that of HCT116-subALPK2 spheroids. Furthermore, luminal apoptosis and cell aggregation were promoted by wtALPK2, but not by subALPK2 in 3D culture. Conclusion: The p.Q1853E variant of ALPK2, which had been accumulating in the Japanese population, induced a metastatic phenotype by disrupting ALPK2 function.

  • ALPK2
  • missense variation
  • luminal apoptosis
  • basolateral polarity
  • intercellular interaction
  • three-dimensional floating culture
  • colorectal cancer

Human tumorigenesis is associated with multiple genetic alterations. Mutations of Kirsten rat sarcoma viral oncogene homolog (KRAS) are frequently observed during the early stages of colorectal cancer (CRC) development, and even in adenomas (1-3), suggesting that oncogenic KRAS plays several crucial roles in the adenoma–carcinoma sequence (4). We previously investigated the behavior of HKe3 cells, which are HCT116 human CRC cells with a disruption in oncogenic KRAS (5), in three-dimensional (3D) culture and reported that they formed an organized structure resembling a colonic crypt (6). In this model, we found that the alpha-kinase 2 (ALPK2) gene was one of the genes down-regulated by oncogenic KRAS (7).

ALPKs constitute a recently discovered family of protein kinases that have no detectable sequence homology to conventional protein kinases (8). To date, six human ALPKs have been identified, namely eukaryotic elongation factor-2 kinase (9), ALPK1 (lymphocyte ALPK, LAK), ALPK2 (heart ALPK, HAK), and ALPK3 (muscle ALPK, MAK), transient receptor potential cation channel subfamily M member 6 (TRPM6) and TRPM7 (9, 10).

The ALPK2 gene is mapped to 18q21.31, the distal end of a minimal region of loss of heterozygosity frequently observed in colonic adenomas and colon cancer (11-13). We showed that ALPK2 induces luminal apoptosis and cell polarity and up-regulates DNA-repair genes (including TP53) in a 3D-specific manner (6, 7), suggesting ALPK2 to be a novel tumor-suppressor gene. Indeed, Lawrence et al. recently reported ALPK2 to be one of 33 genes whose somatic point mutations were associated with 21 cancer cell lines (14) (http://www.tumorportal.org). Furthermore, a recent study showed that ALPK2 is down-regulated by miRNA-214, which is overexpressed in human non-small cell lung cancer with metastatic potential (15). These studies strongly suggest that ALPK2 has pivotal roles as a tumor-suppressor gene. However, the functional details of ALPK2 action still remain unknown.

In this study, we hypothesized that certain germline variations in the ALPK2 gene are associated with cancer and examined nonsynonymous variants of ALPK2 in 2,343 autopsied individuals, for whom the presence or absence of cancer was pathologically confirmed (16).

Materials and Methods

Study population. The study group comprised 2,343 consecutive autopsy cases, which were collected from the Tokyo Metropolitan Geriatric Hospital between 1995 and 2012 (1,293 men and 1,050 women; the mean age at the time of death was 80.6±8.8 years) (16). The subjects were enrolled in the online database of Japanese single nucleotide polymorphisms for Geriatric Research (JG-SNP) (17). The presence or absence of any disease was determined by a thorough autopsy on approximately 29% of patients who died in the hospital.

Genotyping. Genomic DNA was extracted from the renal cortex using a standard procedure. All samples were genotyped using Infinium HumanExome BeadChip Kit Version 1.1 (Illumina, San Diego, CA, USA) as previously described (16).

Association analysis. In the exome array, 14 probes were available for nonsynonymous variants of the ALPK2 gene: rs7240666 (p.I2157V), rs723499 (p.H1767Y), rs17065127 (p.K1730E), rs33910491 (p.Q1579R), rs3809977 (p.H1174P), rs3809975 (p.S977T), rs4940404 (p.N916K), rs3826593 (p.T891I), rs3809973 (p.K829N), rs3809970 (p.G810S), rs12103986 (p.H719Q), rs9944810 (p.R136S), rs6566987 (p.K2T) and rs55674018 (p.Q1853E). Multiple logistic regression analyses under a dominant model were performed to determine the association between the genotypes and phenotypes as described previously (16). By applying the Bonferroni method for multiple testing correction, the significance threshold was set to 0.00357.

Ethics statement. This study was approved by the Tokyo Medical and Dental University Ethics Committee (approval no. 2009-19-4) and the Tokyo Metropolitan Geriatric Hospital Ethics Committee (approval no. 230405). Written informed consent was obtained from a family member of each participant involved in this study before autopsy.

Cell culture. HCT116 human CRC cells were obtained from the American Type Culture Collection (Frederick, MD, USA). HKe3 cells, HCT116 and HCT116-derived cells were maintained as previously described (5, 6, 18). All cell lines used were confirmed to be mycoplasma-free using the MycoAlert system (Lonza, Verviers, Belgium). Cell morphology was checked regularly to ensure that the cell lines were not cross-contaminated.

Retroviral production and generation of stable cell lines. Dasher Green Fluorescent Protein (DGFP) cDNA from DNA2.0's Cas9 vectors (pD1401-AD) was subcloned into the retroviral pMSCVpuro vector (Clontech,, Palo Alto, CA, USA) at the multi-cloning site (pMSCV-DGFP). Each of the cDNAs for human wtALPK2 (accession number NM052947.3) and subALPK2 (Q1853E) was inserted into the pMSCVpuro and pMSCV-DGFP vectors. Retroviruses were produced by the transfection of the retrovirus vectors as previously described (19). HCT116 cells in 6-well plates were infected with the viruses by being spun for 2 h at 32°C and 2000 × g. Approximately 48 h after infection, the cells were treated with 2 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA) for 1 week to establish HCT116-derived cells stably expressing wtALPK2 or subALPK2. The cells were further maintained in medium containing 2 μg/ml puromycin.

Antibodies and reagents. The antibodies used were monoclonal antibody to ALPK2 (4E5) from Abnova (Taipei, Taiwan), anti-heat-shock protein 90 (HSP90; 68/Hsp90) from BD Biosciences (San Jose, CA, USA), anti-E-cadherin from BD Biosciences, anti-cleaved caspase-3 (5A1) from Cell Signaling Technology (Beverly, MA, USA) and anti--actin (A2066) from Sigma-Aldrich. 4’,6-Diamidino-2-phenylindole (DAPI) was obtained from Sigma-Aldrich.

Subcellular fractionation. Subcellular fractions from mouse thymus were obtained as described previously (20).

3D Floating (3DF) culture. 1×103 cells were seeded in a 96-well plate with an ultra-low attachment surface and round bottom (product number 7007; Corning Inc., Corning, NY, USA). Cells were cultured for 2 to 6 days with or without EtOH in CO2 incubator as previously described (19). Photomicrographs of cells were taken using a BIOREVO BZ9000 microscope (Keyence, Osaka, Japan), while the area of spheroids was measured using a BZ Analyzer (Keyence) as previously described (6, 7, 20, 21).

Immunoblotting. Cells grown in two-dimensional culture (2D) were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail; Roche, Basel, Switzerland) and subjected to immunoblotting as previously described (20, 22). For 3DF culture, cells were seeded in 96-well plates and cultured. At day six, by inverting the 96-well plate, the resultant spheroids were collected into plastic dishes. The collected spheroids from the plastic dishes were transferred to 50 ml tubes and centrifuged for 3 min at 200 × g. The spheroid pellets were then lysed in RIPA buffer for immunoblotting. Quantitative analysis of immunoblotting was performed using ImageJ software (National Institute of Health, Bethesda, MD, USA).

Immunofluorescence labelling and confocal microscopy. Immunofluorescence experiments were performed as described previously (6, 7). To examine 3D structures, a TCS-SP5 Laser Scanning Confocal Microscope (Leica, Wetzlar, Germany) was used.

Statistical analyses in cell culture experiments. Data are presented as the mean±standard deviation. Statistical analyses were performed using unpaired two-tailed Student's t-test. All p-values less than 0.05 were considered to be statistically significant.

Results

The p.Q1853E variant is associated with the presence of cancer at the time of death. In this study, we analyzed nonsynonymous germline variants of the ALPK2 gene in a study population previously described (16): the 2,343 subjects included 1,446 cases with and 897 without cancer, respectively. Regardless of the cause of death, 61% of the patients had at least one cancer at the time of death. The top three cancer types were lung (18.9%, n=274), gastric (18.1%, n=262) and colonic (12.0%, n=173) cancer.

Among the 14 nonsynonymous variants in the ALPK2 gene, there was a significant association between the presence of cancer at the time of death and the p.Q1853E variant (p=0.001; Table I). For individual cancer types, p.Q1853E was associated with several types of cancer, including those of the prostate, lung, biliary tract and rectum (data not shown).

p.Q1853E is a rare and potentially rare functional variant present in the East Asian population. We then examined the association between the ALPK2 variants, 14 of which were analyzed here, and four different ethnic populations using the Exome Aggregation Consortium (ExAC) Browser (http://www.http://exac.broadinstitute.org/). As shown in Table II, the ALPK2 locus contains many missense variants with a high variant-allele frequency. Notably, the p.Q1853E variant was only found in the Asian populations.

Many of the rare missense variants are potentially functional and may be crucial for yielding insights into the genetic basis of human disease (23). Amino acid residue 1853 of APLK2 is located in the immunoglobulin-like domain, which is known to be involved in intercellular interactions (24). Therefore, there is the possibility that the accumulation of p.Q1853E variant in the East Asian population is functional. We examined the potential effect of the p.Q1853E variant on APLK2 protein function using the SIFT browser (http://sift.jcvi.org.), which is a sequence homology-based tool that sorts-out intolerant amino acid substitutions from tolerant ones and predicts the extent of their phenotypic effects (25). The amino acid substitution of p.Q1853E variant was predicted as damaging as shown in Table II, suggesting that the p.Q1853E variant was functionally important.

The p.Q1853E variant affects luminal apoptosis of HCT116 spheroids. In order to address the effects of the p.Q1853E variant on cell proliferation in 3D culture, we established HCT116 cells stably expressing wtALPK2 or subALPK2. Expression levels of HCT116-wtALPK2 and -subALPK2 cells were similar to those of endogenous ALPK2 in HKe3 cells (Figure 1A). The areas of HCT116-wtALPK2 and HCT116-subALPK2 spheroids were compared. Although no significant differences in the growth rates were observed between HCT116-wtALPK2 and HCT116-subALPK2 spheroids (Figure 1B), the signal for cleaved caspase-3 was higher in HCT116-wtALPK2 than in HCT116-subALPK2 spheroids, suggesting that the p.Q1853E variant affects luminal apoptosis in the 3D microenvironment, as previously reported (7).

View this table:
  • View inline
  • View popup
  • Download powerpoint
Table I.

Distribution of p.Q1853E variants in autopsy cases with and without cancer.

The p.Q1853E variant alters the localization of filamentous-actin (F-actin) in the surface cells of spheroids. The intracellular localization of ALPK2 is not well-characterized. Staining of HKe3 cells with phalloidin and antibody to ALPK2 revealed that the endogenous ALPK2 proteins co-localized with F-actin, which is recognized by phalloidin, and localized in the cytoplasm and membrane in 2D culture (Figure 2A). To further characterize the subcellular localization pattern of ALPK2, cells were lysed with RIPA buffer, and the lysate was separated into the supernatant containing the soluble fraction, including actin and the pellet containing the insoluble fraction, including E-cadherin. The immunoblotting detected the ALPK2 protein predominantly in the supernatant fraction (Figure 2B). Therefore, consistent with the results of the confocal microscopic analysis (Figure 2A), our results suggest that the majority of the ALPK2 protein is localized in the cytoplasm, while a little is localized in the membrane (Figure 2B). To examine whether the p.Q1853E variant changes the subcellular localization of ALPK2, we prepared HCT116-wtALPK2-DGFP and HCT116-subALPK2-DGFP cells grown in 3DF culture and stained the surface of the spheroids using DAPI and phalloidin. ALPK2 colocalized with F-actin in HCT116-wtALPK2-DGFP and HCT116-subALPK2-DGFP cells. Notably, although ALPK2 was localized near the basolateral membrane (upper panels) in HCT116-wtALPK2 cells, that in HCT116-subALPK2 cells was thoroughly localized in the cytoplasmic region (lower panels), suggesting that ALPK2 is involved in the basolateral polarity of the surface cells of spheroids (Figure 2C).

View this table:
  • View inline
  • View popup
  • Download powerpoint
Table II.

Allelic frequencies of alpha-kinase-2 (ALPK2) variants in different ethnic populations.

The p.Q1853E variant induces the disassembly of HCT116 spheroids. The p.Q1853E variant is located in the immunoglobulin-like domain, which is known to be involved in intercellular interactions (24). In order to further understand the function of the p.Q1853E variant in vitro, we developed a 3DF culture system for examining cellular aggregation. Ethanol is known to cause redistribution and intracellular mislocalization of tight junction proteins, including zonula occludens-1, and occluding without affecting the levels of protein expression (26). To examine whether ALPK2 was associated with spheroid assembly, HCT116 cells overexpressing wt ALPK2 or variant ALPK2 were used. The addition of ethanol partially prevented the formation of HCT116-subALPK2 spheroids in a dose-dependent manner, but did not affect that of HCT116-wtALPK2 spheroids (Figure 3). These results suggest that the p.Q1853E variant attenuates intercellular interaction.

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

The p.Q1853E variant of alpha-kinase 2 (ALPK2) reduces caspase-3 cleavage in HCT116 spheroids. A: Western blot analyses of ALPK2 expression in HKe3, parental (Par) HCT116 and HCT116 cells stably expressing wild-type (wt)ALPK2 (HCT116-wtALPK2) or amino acid-substituted (sub) ALPK2 (HCT116-subALPK2) grown in 2D culture. B: The area of spheroids formed by HCT116-derived cells at day 8 in 3D floating (3DF) culture. One thousand each of HCT116-wtALPK2 and -subALPK2 cells were seeded for 3DF culture. The areas of spheroids were measured at day 8. n.s.: Not significantly different. C: Expression of cleaved caspase-3 in HKe3, HCT116, HCT116-wtALPK2 and HCT116-subALPK2 cells at day 6 in a 3DF culture. β-Actin was used as a loading control. The upper panel shows western blotting results and the lower panel shows quantitative analyses of the relative expression levels. Bars represent the relative signal intensities of cells normalized to the signal of HCT116 cells. Significantly different at *p<0.05 and **p<0.005.

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

The p.Q1853E variant of alpha-kinase-2 (ALPK2) alters the localization of ALPK2 and filamentous-actin (F-actin) in the surface cells of spheroids. A: Signals for antibody to ALPK2 (green), phalloidin-labelled F-actin (red) and 4’,6-diamidino-2-phenylindole (DAPI)-labelled nuclei (blue) in HKe3 cells grown in 2D culture. Scale bar=10 μm. B: Western blot analyses for the expression of ALPK2 (left panel), E-cadherin and β-actin (right panel) in different fractions of HKe3 cells. C: The signals for Dasher Green Fluorescent Protein (DGFP) (green), phalloidin-labelled F-actin (red) and DAPI-labelled nuclei (blue) in HCT116 cells stably expressing wild-type ALPK2-DGFP (upper panels) or amino acid-substituted (sub) ALPK2-DGFP (lower panels) cells at day six in 3D floating culture. Scale bar=20 μm.

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3.

The p.Q1853E variant of alpha-kinase 2 (ALPK2) attenuates cellular aggregation. Representative images of HCT116 spheroids stably expressing wild-type (wt) ALPK2 or amino acid-substituted (sub) ALPK2 grown in the presence or absence of ethanol (EtOH, 0.2% or 1%) at day two in 3D floating culture. Scale bar=100 μm.

Discussion

To our knowledge, this is the first report to focus on the association between the germline genetic variants of ALPK2 and the presence of cancer in Japanese autopsy cases. The genotype data available in the ExAC Browser clearly demonstrate that the p.Q1853E variant is specific to the Asian population: its frequencies in East and South Asian populations are 0.48% and 0.02% but 0% in the other ethnic populations (Table II). Surprisingly, the frequency of the p.Q1853E variant in individuals with cancer in our study population was 1.5% (Table I). The East Asian population is a population showing rapid growth. Rare variants accumulating in such a population tend to be functional due to weak purifying selection (23). The other ALPK2 missense variants, whose total allelic frequency is less than 0.1% (Table II), are also accumulating in the East Asian population and localized in the functional domains of ALPK2, suggesting that these variants cooperatively disrupt ALPK2 function.

We previously showed that siRNA inhibition of ALPK2 expression prevents luminal apoptosis (7). In this study, we demonstrated that overexpression of wt ALPK2 protein also induces luminal apoptosis of HCT116 cells in a 3DF culture, while the overexpression of variant ALPK2 did not (Figure 1). These results indicate that that the 1853rd glutamine residue of ALPK2 protein is essential for luminal apoptosis.

Wild-type and variant ALPK2 both co-localized with F-actin in 2D and 3D cultures, indicating the physical association between ALPK2 and the cytoskeleton. Notably, although wt ALPK2 was localized near the basolateral membrane, variant ALPK2 was thoroughly localized in the cytoplasmic region. These results show that ALPK2 is involved in the basolateral polarity of the surface cells of spheroids (Figure 2C). A recent study of invertebrate ALPK2 family proteins, such as myosin heavy chain kinase (MHCK) A, MHCK-B and MHCK-C, suggested that these ALPK family members target proteins involved in filament disassembly, including myosin II (10). Ivanov et al. reported that myosin II regulates the spherical shape of epithelial cysts by controlling actin polymerization at the cyst surface (27). Furthermore, in smooth muscle cells, the inhibition of myosin light chain kinase induces apoptosis in vitro and in vivo (28). A recent study also showed that the inhibition of non-muscle myosin IIA (NMIIA) reduces the nuclear localization of wt TP53 (29), suggesting the increased induction of apoptosis. Interestingly, we previously reported that siRNA inhibition of ALPK2 expression leads to the down-regulation of DNA repair-related genes that are regulated by TP53 (7), suggesting the functional association between ALPK2 and TP53 via NMIIA. Collectively, our results and other reports suggest that the p.Q1853E variant affects ALPK2 kinase activity and induces F-actin depolarization and luminal apoptosis.

We also demonstrated that the p.Q1853E variant prevents cell aggregation under conditions where tight junction protein function is attenuated (Figure 3), suggesting that p.Q1853E is involved in intercellular interaction through the immunoglobulin-like domain.

Taken together, the intact intercellular interaction, which includes apical and basolateral membranes, induces luminal apoptosis (6). These results strongly suggest that p.Q1853E plays an essential role in disrupting normal cyst formation. Further elucidation of ALPK2 function and its substrates, as well as the validation of the association between ALPK2 variants and cancer progression, will help in establishing diagnosis, therapeutics and prognosis for patients with cancer having rare ALPK2 variants or the deletion of ALPK2 gene.

Acknowledgements

The Authors thank Takami Danno, Shiori Yamano and Yumiko Hirose for their technical assistance.

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Funding

    This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan; by GMEXT/JSPS KAKENHI Grants (Numbers: A-25242062, A-22240072, B-21390459, C-26670481, C-21590411 and CER-24650414 to MT); by Grants-in-Aid for Research on Intractable Diseases Mitochondrial Disorders (grant numbers 23-016, 23-116 and 24-005 to MT) from the Ministry of Health, Labor, and Welfare of Japan; by the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development, AMED (15ek0109088h0001 and 15ek0109088s0401 to MT); by the Takeda Science Foundation (to MT); by the Smoking Research Foundation (to TA, and MS); and by the Joint Usage/Research Program of the Medical Research Institute, Tokyo Medical and Dental University (to MM).

  • Received May 2, 2017.
  • Revision received May 25, 2017.
  • Accepted May 29, 2017.
  • Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved

References

  1. ↵
    1. Hanahan D,
    2. Weinberg RA
    : The hallmarks of cancer. Cell 100: 57-70, 2000.
    OpenUrlCrossRefPubMed
    1. Rosin-Arbesfeld R,
    2. Ihrke G,
    3. Bienz M
    : Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells. Embo J 20: 5929-5939, 2001.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Cho KR,
    2. Vogelstein B
    : Genetic alterations in the adenoma – carcinoma sequence. Cancer 70: 1727-1731, 1992.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Vogelstein B,
    2. Kinzler KW
    : Cancer genes and the pathways they control. Nat Med 10: 789-799, 2004.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Shirasawa S,
    2. Furuse M,
    3. Yokoyama N,
    4. Sasazuki T
    : Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science 260: 85-88, 1993.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Tsunoda T,
    2. Takashima Y,
    3. Fujimoto T,
    4. Koyanagi M,
    5. Yoshida Y,
    6. Doi K,
    7. Tanaka Y,
    8. Kuroki M,
    9. Sasazuki T,
    10. Shirasawa S
    : Three-dimensionally specific inhibition of DNA repair-related genes by activated KRAS in colon crypt model. Neoplasia 12: 397-404, 2010.
    OpenUrlPubMed
  6. ↵
    1. Yoshida Y,
    2. Tsunoda T,
    3. Doi K,
    4. Fujimoto T,
    5. Tanaka Y,
    6. Ota T,
    7. Ogawa M,
    8. Matsuzaki H,
    9. Kuroki M,
    10. Iwasaki A,
    11. Shirasawa S
    : ALPK2 is crucial for luminal apoptosis and DNA repair-related gene expression in a three-dimensional colonic-crypt model. Anticancer Res 32: 2301-2308, 2012.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Drennan D,
    2. Ryazanov AG
    : Alpha-kinases: analysis of the family and comparison with conventional protein kinases. Prog Biophys Mol Biol 85: 1-32, 2004.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Ryazanov AG,
    2. Ward MD,
    3. Mendola CE,
    4. Pavur KS,
    5. Dorovkov MV,
    6. Wiedmann M,
    7. Erdjument-Bromage H,
    8. Tempst P,
    9. Parmer TG,
    10. Prostko CR,
    11. Germino FJ,
    12. Hait WN
    : Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proc Natl Acad Sci USA 94: 4884-4889, 1997.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Middelbeek J,
    2. Clark K,
    3. Venselaar H,
    4. Huynen MA,
    5. van Leeuwen FN
    : The alpha-kinase family: an exceptional branch on the protein kinase tree. Cell Mol Life Sci 67: 875-890, 2010.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Johnson-Pais TL,
    2. Nellissery MJ,
    3. Ammerman DG,
    4. Pathmanathan D,
    5. Bhatia P,
    6. Buller CL,
    7. Leach RJ,
    8. Hansen MF
    : Determination of a minimal region of loss of heterozygosity on chromosome 18q21.33 in osteosarcoma. Int J Cancer 105: 285-288, 2003.
    OpenUrlCrossRefPubMed
    1. Vogelstein B,
    2. Fearon ER,
    3. Hamilton SR,
    4. Kern SE,
    5. Preisinger AC,
    6. Leppert M,
    7. Nakamura Y,
    8. White R,
    9. Smits AM,
    10. Bos JL
    : Genetic alterations during colorectal-tumor development. N Engl J Med 319: 525-532, 1988.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Cunningham JL,
    2. Diaz de Stahl T,
    3. Sjoblom T,
    4. Westin G,
    5. Dumanski JP,
    6. Janson ET
    : Common pathogenetic mechanism involving human chromosome 18 in familial and sporadic ileal carcinoid tumors. Genes Chromosomes Cancer 50: 82-94, 2011.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Lawrence MS,
    2. Stojanov P,
    3. Mermel CH,
    4. Robinson JT,
    5. Garraway LA,
    6. Golub TR,
    7. Meyerson M,
    8. Gabriel SB,
    9. Lander ES,
    10. Getz G
    : Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 505: 495-501, 2014.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Salim H,
    2. Arvanitis A,
    3. de Petris L,
    4. Kanter L,
    5. Haag P,
    6. Zovko A,
    7. Ozata DM,
    8. Lui WO,
    9. Lundholm L,
    10. Zhivotovsky B,
    11. Lewensohn R,
    12. Viktorsson K
    : miRNA-214 is related to invasiveness of human non-small cell lung cancer and directly regulates alpha protein kinase 2 expression. Genes Chromosomes Cancer 52: 895-911, 2013.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Yamada M,
    2. Sato N,
    3. Ikeda S,
    4. Arai T,
    5. Sawabe M,
    6. Mori S,
    7. Yamada Y,
    8. Muramatsu M,
    9. Tanaka M
    : Association of the chromodomain helicase DNA-binding protein 4 (CHD4) missense variation p.D140E with cancer: potential interaction with smoking. Genes Chromosomes Cancer 54: 122-128, 2015.
    OpenUrl
  15. ↵
    1. Sawabe M,
    2. Arai T,
    3. Kasahara I,
    4. Esaki Y,
    5. Nakahara K,
    6. Hosoi T,
    7. Orimo H,
    8. Takubo K,
    9. Murayama S,
    10. Tanaka N
    : Developments of geriatric autopsy database and Internet-based database of Japanese single nucleotide polymorphisms for geriatric research (JG-SNP). Mech Ageing Dev 125: 547-552, 2004.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Baba I,
    2. Shirasawa S,
    3. Iwamoto R,
    4. Okumura K,
    5. Tsunoda T,
    6. Nishioka M,
    7. Fukuyama K,
    8. Yamamoto K,
    9. Mekada E,
    10. Sasazuki T
    : Involvement of deregulated epiregulin expression in tumorigenesis in vivo through activated Ki-Ras signaling pathway in human colon cancer cells. Cancer Res 60: 6886-6889, 2000.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Tsunoda T,
    2. Ishikura S,
    3. Doi K,
    4. Iwaihara Y,
    5. Hidesima H,
    6. Luo H,
    7. Hirose Y,
    8. Shirasawa S
    : Establishment of a Three-dimensional Floating Cell Culture System for Screening Drugs Targeting KRAS-mediated Signaling Molecules. Anticancer Res 35: 4453-4459, 2015.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Tsunoda T,
    2. Ota T,
    3. Fujimoto T,
    4. Doi K,
    5. Tanaka Y,
    6. Yoshida Y,
    7. Ogawa M,
    8. Matsuzaki H,
    9. Hamabashiri M,
    10. Tyson DR,
    11. Kuroki M,
    12. Miyamoto S,
    13. Shirasawa S
    : Inhibition of phosphodiesterase-4 (PDE4) activity triggers luminal apoptosis and AKT dephosphorylation in a 3-D colonic-crypt model. Mol Cancer 11: 46, 2012.
    OpenUrlPubMed
  19. ↵
    1. Yoshida Y,
    2. Tsunoda T,
    3. Doi K,
    4. Tanaka Y,
    5. Fujimoto T,
    6. Machida T,
    7. Ota T,
    8. Koyanagi M,
    9. Takashima Y,
    10. Sasazuki T,
    11. Kuroki M,
    12. Iwasaki A,
    13. Shirasawa S
    : KRAS-mediated up-regulation of RRM2 expression is essential for the proliferation of colorectal cancer cell lines. Anticancer Res 31: 2535-2539, 2011.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Tsunoda T,
    2. Inokuchi J,
    3. Baba I,
    4. Okumura K,
    5. Naito S,
    6. Sasazuki T,
    7. Shirasawa S
    : A novel mechanism of nuclear factor kappaB activation through the binding between inhibitor of nuclear factor-kappaBalpha and the processed NH(2)-terminal region of Mig-6. Cancer Res 62: 5668-5671, 2002.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Nelson MR,
    2. Wegmann D,
    3. Ehm MG,
    4. Kessner D,
    5. St Jean P,
    6. Verzilli C,
    7. Shen J,
    8. Tang Z,
    9. Bacanu SA,
    10. Fraser D,
    11. Warren L,
    12. Aponte J,
    13. Zawistowski M,
    14. Liu X,
    15. Zhang H,
    16. Zhang Y,
    17. Li J,
    18. Li Y,
    19. Li L,
    20. Woollard P,
    21. Topp S,
    22. Hall MD,
    23. Nangle K,
    24. Wang J,
    25. Abecasis G,
    26. Cardon LR,
    27. Zollner S,
    28. Whittaker JC,
    29. Chissoe SL,
    30. Novembre J,
    31. Mooser V
    : An abundance of rare functional variants in 202 drug target genes sequenced in 14,002 people. Science 337: 100-104, 2012.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Smith DK,
    2. Xue H
    : Sequence profiles of immunoglobulin and immunoglobulin-like domains. J Mol Biol 274: 530-545, 1997.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Ng PC,
    2. Henikoff S
    : Predicting deleterious amino acid substitutions. Genome Res 11: 863-874, 2001.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Elamin E,
    2. Jonkers D,
    3. Juuti-Uusitalo K,
    4. van Ijzendoorn S,
    5. Troost F,
    6. Duimel H,
    7. Broers J,
    8. Verheyen F,
    9. Dekker J,
    10. Masclee A
    : Effects of ethanol and acetaldehyde on tight junction integrity: in vitro study in a three dimensional intestinal epithelial cell culture model. PLoS One 7: e35008, 2012.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Ivanov AI,
    2. Hopkins AM,
    3. Brown GT,
    4. Gerner-Smidt K,
    5. Babbin BA,
    6. Parkos CA,
    7. Nusrat A
    : Myosin II regulates the shape of three-dimensional intestinal epithelial cysts. J Cell Sci 121: 1803-1814, 2008.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Fazal F,
    2. Gu L,
    3. Ihnatovych I,
    4. Han Y,
    5. Hu W,
    6. Antic N,
    7. Carreira F,
    8. Blomquist JF,
    9. Hope TJ,
    10. Ucker DS,
    11. de Lanerolle P
    : Inhibiting myosin light chain kinase induces apoptosis in vitro and in vivo. Mol Cell Biol 25: 6259-6266, 2005.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Coaxum SD,
    2. Tiedeken J,
    3. Garrett-Mayer E,
    4. Myers J,
    5. Rosenzweig SA,
    6. Neskey DM
    : The tumor suppressor capability of p53 is dependent on non-muscle myosin IIA function in head and neck cancer. Oncotarget 8: 22991-23007, 2017.
    OpenUrl
PreviousNext
Back to top

In this issue

Anticancer Research: 37 (7)
Anticancer Research
Vol. 37, Issue 7
July 2017
  • Table of Contents
  • Table of Contents (PDF)
  • Index by author
  • Back Matter (PDF)
  • Ed Board (PDF)
  • Front Matter (PDF)
Print
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for your interest in spreading the word on Anticancer Research.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
An Alpha-kinase 2 Gene Variant Disrupts Filamentous Actin Localization in the Surface Cells of Colorectal Cancer Spheroids
(Your Name) has sent you a message from Anticancer Research
(Your Name) thought you would like to see the Anticancer Research web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
8 + 9 =
Solve this simple math problem and enter the result. E.g. for 1+3, enter 4.
Citation Tools
An Alpha-kinase 2 Gene Variant Disrupts Filamentous Actin Localization in the Surface Cells of Colorectal Cancer Spheroids
KENSUKE NISHI, HAO LUO, KAZUHIKO NAKABAYASHI, KEIKO DOI, SHUHEI ISHIKURA, YURI IWAIHARA, YASUHIRO YOSHIDA, KUMPEI TANISAWA, TOMIO ARAI, SEIJIRO MORI, MOTOJI SAWABE, MASAAKI MURAMATSU, MASASHI TANAKA, TOSHIFUMI SAKATA, SENJI SHIRASAWA, TOSHIYUKI TSUNODA
Anticancer Research Jul 2017, 37 (7) 3855-3862;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Reprints and Permissions
Share
An Alpha-kinase 2 Gene Variant Disrupts Filamentous Actin Localization in the Surface Cells of Colorectal Cancer Spheroids
KENSUKE NISHI, HAO LUO, KAZUHIKO NAKABAYASHI, KEIKO DOI, SHUHEI ISHIKURA, YURI IWAIHARA, YASUHIRO YOSHIDA, KUMPEI TANISAWA, TOMIO ARAI, SEIJIRO MORI, MOTOJI SAWABE, MASAAKI MURAMATSU, MASASHI TANAKA, TOSHIFUMI SAKATA, SENJI SHIRASAWA, TOSHIYUKI TSUNODA
Anticancer Research Jul 2017, 37 (7) 3855-3862;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgements
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

  • No related articles found.
  • PubMed
  • Google Scholar

Cited By...

  • No citing articles found.
  • Google Scholar

More in this TOC Section

  • Design and Synthesis of Novel Anti-metastatic Hypoxic Cytotoxin TX-2137 Targeting AKT Kinase
  • Apremilast Induces Apoptosis of Human Colorectal Cancer Cells with Mutant KRAS
  • HB-EGF Is a Promising Therapeutic Target for Lung Cancer with Secondary Mutation of EGFRT790M
Show more PROCEEDINGS OF THE 20th ANNUAL MEETING OF THE SOCIETY OF BIOTHERAPEUTIC APPROACHES, 10 December, 2016 (Fukuoka, Japan)

Similar Articles

Keywords

  • ALPK2
  • missense variation
  • luminal apoptosis
  • basolateral polarity
  • intercellular interaction
  • three-dimensional floating culture
  • colorectal cancer
Anticancer Research

© 2023 Anticancer Research

Powered by HighWire