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. 2012 Apr;103(4):782-90.
doi: 10.1111/j.1349-7006.2012.02211.x. Epub 2012 Feb 23.

Overexpression of the DNA sensor proteins, absent in melanoma 2 and interferon-inducible 16, contributes to tumorigenesis of oral squamous cell carcinoma with p53 inactivation

Yuudai Kondo  1 , Kentaro Nagai, Shingo Nakahata, Yusuke Saito, Tomonaga Ichikawa, Akira Suekane, Tomohiko Taki, Reika Iwakawa, Masato Enari, Masafumi Taniwaki, Jun Yokota, Sumio Sakoda, Kazuhiro Morishita
Affiliations

Overexpression of the DNA sensor proteins, absent in melanoma 2 and interferon-inducible 16, contributes to tumorigenesis of oral squamous cell carcinoma with p53 inactivation

Yuudai Kondo et al. Cancer Sci. 2012 Apr.

Abstract

The development of oral squamous cell carcinoma (OSCC) is a multistep process that requires the accumulation of genetic alterations. To identify genes responsible for OSCC development, we performed high-density single nucleotide polymorphism array analysis and genome-wide gene expression profiling on OSCC tumors. These analyses indicated that the absent in melanoma 2 (AIM2) gene and the interferon-inducible gene 16 (IFI16) mapped to the hematopoietic interferon-inducible nuclear proteins. The 200-amino-acid repeat gene cluster in the amplified region of chromosome 1q23 is overexpressed in OSCC. Both AIM2 and IFI16 are cytoplasmic double-stranded DNA sensors for innate immunity and act as tumor suppressors in several human cancers. Knockdown of AIM2 or IFI16 in OSCC cells results in the suppression of cell growth and apoptosis, accompanied by the downregulation of nuclear factor kappa-light-chain-enhancer of activated B cells activation. Because all OSCC cell lines have reduced p53 activity, wild-type p53 was introduced in p53-deficient OSCC cells. The expression of wild-type p53 suppressed cell growth and induced apoptosis via suppression of nuclear factor kappa-light-chain-enhancer of activated B cells activity. Finally, the co-expression of AIM2 and IFI16 significantly enhanced cell growth in p53-deficient cells; in contrast, the expression of AIM2 and/or IFI16 in cells bearing wild-type p53 suppressed cell growth. Moreover, AIM2 and IFI16 synergistically enhanced nuclear factor kappa-light-chain-enhancer of activated B cells signaling in p53-deficient cells. Thus, expression of AIM2 and IFI16 may have oncogenic activities in the OSCC cells that have inactivated the p53 system.

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Figures

Figure 1
Figure 1
Overexpression of IFI16 and AIM2 mRNA in OSCC. (a) Recurrent genetic changes are depicted based on the copy number analyzer for GeneChip (CNAG) output of the single nucleotide polymorphism array analysis of 28 oral squamous cell carcinoma (OSCC) samples, which include gains at the IFI16 and AIM2 loci on 1q23. Regions showing copy number gains are indicated by horizontal lines. AIM2, absent in melanoma 2; APCS, amyloid P component, serum; CADM3, cell adhesion molecule 3; CCDC19, coiled‐coil domain containing 19; CRP, C‐reactive protein, pentraxin‐related; DARC, Duffy blood group, chemokine receptor; DUSP23, dual specificity phosphatase 23; FCER1A, Fc fragment of IgE, high affinity I, receptor for: alpha polypeptide; FCRL6, Fc receptor‐like 6; IFI16, interferon, gamma‐inducible protein 16; IGSF9, immunoglobulin superfamily, member 9; KCNJ9, potassium inwardly‐rectifying channel, subfamily J, member 9; KCNJ10, potassium inwardly‐rectifying channel, subfamily J, member 10; MNDA, myeloid cell nuclear differentiation antigen; OR10J1, olfactory receptor, family 10, subfamily J, member 1; OR10J3, olfactory receptor, family 10, subfamily J, member 3; OR10J5, olfactory receptor, family 10, subfamily J, member 5; PIGM, phosphatidylinositol glycan anchor biosynthesis, class M; PYHIN1, pyrin and HIN domain family, member 1; SLAMF8, SLAM family member 8; SLAMF9, SLAM family member 9; TAGLN2, transgelin 2; VSIG8, V‐set and immunoglobulin domain containing 8. (b,d) Expression of the AIM2 and (c,e) IFI16 mRNA were examined in 11 primary OSCC and eight OSCC cell lines (Ca9‐22, Ho1u1, HSC2, HSC3, HSC4, HSQ89, SAS and Sa3) by quantitative real‐time PCR. Normal gingival tissues from normal volunteers were used as controls. (f) Scatter plot of DNA copy number versus mRNA expression for AIM2 (left) and IFI16 (right). Correlations were quantified using the Spearman's rank correlation coefficient. AIM2, absent in melanoma; HIN‐200, Hematopoietic interferon‐inducible nuclear proteins with a 200‐amino‐acid repeat; IFI16, interferon‐inducible 16; N0, without metastasis; N1, with metastasis.
Figure 2
Figure 2
Effects of knockdown of AIM2 and IFI16 expression on the growth of OSCC cells. (a) Retroviral constructs containing shRNA against AIM2 and/or IFI16 or luciferase (Luc) as a control were transfected into SAS cells. Forty‐eight hours after transfection, ZsGreen‐positive cells were sorted, and total RNA was extracted to analyze expression levels of AIM2 and IFI16 by RT‐PCR. (b) The growth of sorted cells was analyzed with MTT. The data are shown as the mean ± SD of triplicate samples. The statistical analysis was performed using the Student's t‐test (*P < 0.01). (c) Cell cycle phase distribution was analyzed by FACS with PIstaining. Each cycle phase distribution was analyzed by the cell recruited into the cell cycle. (d) The percentage of sorted cells undergoing apoptosis was quantitated by Annexin V staining and FACS. The lower bar graph shows the mean ± SD from three independent experiments. An asterisk indicates a statistically significant difference (P < 0.01). AIM2, absent in melanoma; IFI16, interferon‐inducible 16.
Figure 3
Figure 3
Activation of NF‐κB signaling in oral squamous cell carcinoma (OSCC). (a) The levels of total and phosphorylated (Ser32/36) inhibitor of kappa B alpha (IκBα) were examined in two normal gingival tissue samples, eight OSCC cell lines (Ca9‐22, Ho1u1, HSC2, HSC3, HSC4, HSQ89, SAS and Sa3) and 10 OSCC primary tumors by Western blot analysis. (b) Four OSCC cell lines were treated with various concentrations of Bay 11‐7082 for 48 h, and viable cell numbers were counted by MTT assay. The results are shown as percentages of the values obtained from the control Bay 11‐7082‐free culture. The data are shown as the means ± SD of triplicate samples. (c) The percentage of apoptotic cells was measured by FACS after Annexin V/PI staining in untreated and 10‐μM Bay 11‐7082‐treated SAS and HSC3 cell lines at 48 h. (d) Levels of phosphorylated IκBα and total IκBα in the HSC4 cell line treated with the indicated concentration of Bay 11‐7082 for 48 h were examined by Western blot analysis. (e) The SAS cell transfectants described in Figure 2 were examined for the levels of phosphorylated IκBα and total IκBα by Western blot analysis. (f) The SAS cell transfectants were co‐transfected with the NF‐κB–Luc reporter and an internal control Renilla luciferase (pRL‐TK) plasmid. After 36 h, luciferase activities were measured with a dual‐luciferase reporter assay system. The data are shown as mean ± SD of triplicate transfections, and statistical analysis used the Student's t‐test. NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells.
Figure 4
Figure 4
Function of p53 is inhibited in oral squamous cell carcinoma (OSCC) cells. (a) Detection of p53 protein levels in two normal gingival tissues and in eight OSCC cell lines (Ca9‐22, Ho1u1, HSC2, HSC3, HSC4, HSQ89, SAS and Sa3) by Western blot analysis. (b) SAS cell lines stably transfected with either empty vector or wild‐type p53 expression vector (FLAG‐p53wt) were examined for expression of p53 by Western blot using an anti‐p53 antibody (Ab‐6). (c) Transfectants were subjected to cell growth analysis using the MTT assay. The data are shown as the mean ± SD of triplicate samples. The statistical analysis was done using the Student's t‐test. (d) The percentage of apoptotic cells was quantified by Annexin‐V/PI staining. (e) Control vector and p53 transfectants were examined for levels of phosphorylated inhibitor of kappa B alpha (IκBα) and total IκBα by Western blot analysis. (f) The cells were co‐transfected with NF‐κB–Luc and pRL‐TK. After 36 h, luciferase activities were measured. The data are presented as in Figure 3(f). NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells.
Figure 5
Figure 5
Expression of AIM2 and IFI16 confers growth‐stimulating effects on transformed cells in the absence of wild‐type p53. (a) The growth of human lung cancer H1299 cells after transfection with various combinations of AIM2, IFI16 and wild‐type p53 (SN3‐p53wt) expression vectors was examined by MTT assay. The data are shown as the mean ± SD of triplicate samples. The statistical analysis was performed using the Student's t‐test (*P < 0.05; **P < 0.01). (b) Western blot analysis of extracts from untransfected or transfected H1299 cells described in panel (a) at 48 h. (c) The growth of human mammary tumor MCF7 cells after transfection with various combinations of the AIM2, IFI16 and shp53 expression vectors was examined by MTT assay. The data are shown as the mean ± SD of triplicate samples. The statistical analysis was performed using the Student's t‐test (*P < 0.05; **P < 0.01). (d) Western blot analysis of extracts from untransfected or transfected MCF7 cells described in panel (c) at 48 h. (e) The growth of HSQ89 cells after transfection with AIM2 and/or IFI16. (f) Western blot analysis of extracts from untransfected or transfected HSQ89 cells described in panel (e) at 48 h. AIM2, absent in melanoma; IκBα, inhibitor of kappa B alpha; IFI16, interferon‐inducible 16.
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