KAI1 mRNA Research Articles

Abstract 1879: Transcriptional regulation of KAI1 by p73 in colorectal cancer metastasis

Abstract Background: p73 is a member of the p53 family transcription factors and known tumor suppressor. However, its transcriptional activity is complex owing to its multiple isoforms, and limited information is available regarding the transcriptional and functional regulation of the p73 gene in colorectal cancer. In this study, we examined the role of p73 in colorectal cancer. Methods: We established a TAp73 overexpressing cell line using the Tet-on inducible expression system and analyzed global RNA expression by RNA-sequencing (RNA-seq). We studied the effects of TAp73 expression on the migration, invasiveness, and EMT of colon cancer cells. We conducted experiments in animals with liver metastasis of colorectal cancer. To investigate the roles of p73 in colon cancer, we examined the samples from patients, who had undergone surgical resection for colorectal cancer between 2009 and 2013. mRNA expression of TAp73 and KAI1 was analyzed in 108 colorectal cancer specimen and adjacent non-cancerous tissues by real-time RT-PCR. Results: RNA-seq showed the association between TAp73 and KAI1 expression. Migration and invasiveness of colon cancer cells were reduced by TAp73 expression, but induced by KAI1 knockdown. TAp73 regulates KAI1 expression in colon cancer cell lines. TAp73-induced decrease in invasiveness and epithelial-mesenchymal transition (EMT) was abrogated by KAI1 knockdown in TAp73 overexpressing cells. Migration ability of TAp73 overexpressing cells was recovered by KAI1 knockdown. Hepatic metastases were significantly reduced in mice injected with TAp73 overexpressing cells and increased in those injected with TAp73 overexpressing cells with KAI1 knockdown. mRNA expression of TAp73 and KAI1 was higher in colorectal cancer tissues than in adjacent non-cancerous tissues. Significant correlation between TAp73 and KAI1 mRNA expression was detected in early stage primary colorectal cancer, but not in the advanced stage. Conclusions: This study suggests that p73 (TAp73) acts as a tumor suppressor in the progression of colorectal cancer and regulates the expression of KAI1, which is a key target of p73 in colorectal cancer liver metastases. Note: This abstract was not presented at the meeting. Citation Format: Woo Kyun Bae, Kyung Hyun Yoo, Keunsoo Kang, Lothar Hennighausen, Ik-Joo Chung. Transcriptional regulation of KAI1 by p73 in colorectal cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1879. doi:10.1158/1538-7445.AM2017-1879

Identification of a novel metastasis inducing lncRNA which suppresses the KAI1/CD82 metastasis suppressor gene and is upregulated in triple-negative breast cancer.

Inactivation of tumor/metastasis suppressor genes via epigenetic silencing is a frequent event in human cancers. KAI1/CD82 is a metastasis suppressor gene whose normal protecting activity is deficient in twelve different solid malignancies. Here we have identified and characterized a primarily nuclear non-polyadenylated, antisense (as)-lncRNA, initiating upstream of the KAI1 human metastasis suppressor gene transcription start site; and elongating in the opposite direction to KAI1 mRNA. We show that the KAI1 promoter is bi-directional giving rise to KAI1 mRNA and its as-lncRNA. Moreover, expression of this lncRNA transcript emerges to be inversely related to the KAI1 mRNA expression, and in direct relationship to the invasiveness level of human breast cancer derived cell lines. Importantly, knockdown of the KAI1 as-lncRNA in the triple-negative breast cancer cell line MDA-MB-231 have led to increased KAI1 mRNA and protein expression, manifested in stronger adhesion to fibronectin, retardation of cell migration and reduced cell invasion in vitro. Accordingly we have named this lncRNA, SKAI1BC, standing for “Suppressor of KAI1 in Breast Cancer”. These results uncover a potential way to harness tumor metastasis via targeting SKAI1BC in human breast cancer, and perhaps also in other KAI1-deficient human malignancies.

The expression and significance of kang ai 1 protein in colorectal adenoma and colorectal carcinoma

Objective To observe the expression and clinical significance of kang ai 1 (Kai1) in the process from the progression from colorectal polyps to cancer, invasion and metastasis. Methods Immunohistochemistry was used to examine the expression of Kai1 in the colorectal tissues, colorectal adenoma tissues and colorectal carcinoma tissues. Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect Kai1 mRNA expression. Results The expression of Kai1 protein was positive in 18 cases of colorectal carcinoma (45.00%), significantly lower than that in colorectal adenoma (91.25%) and colorectal tissues (100.00%), respectively, with significant differences among them (P=0.031). FQ-PCR showed that the expression of Kai1 mRNA in colorectal carcinoma, colorectal adenoma and normal colorectal mucosa was incresed by 2.038±0.933, 9.983±4.337 and 12.260±2.182 respectively (F=11.960, P=0.015). The expression of Kai1 protein and mRNA had a significant correlation with the lymph node metastasis of patients with colorectal carcinoma (P=0.011). Conclusion The abnormal expression of Kai1 protein and mRNA is related with the carcinogenesis and the development of colorectal carcinoma. Key words: Kang ai 1; Colorectal carcinoma; Gene expression

Promoter CpG-Site Methylation of the KAI1 Metastasis Suppressor Gene Contributes to Its Epigenetic Repression in Prostate Cancer.

Repression of the KAI1 metastasis suppressor gene is closely associated with malignancy and poor prognosis in many human cancer types including prostate cancer. Since gene repression in human cancers frequently results from epigenetic alterations by DNA methylation and histone modifications, we examined whether the KAI1 gene becomes silenced through these epigenetic mechanisms in prostate cancer. KAI1 mRNA and protein levels were determined by RT-PCR and immunoblotting analyses, respectively. Methylation status of the KAI1 promoter DNA in prostate cancer cell lines and tissues was evaluated by methylation-specific PCR analysis of bisulfite-modified genomic DNAs. Methylated CpG sites in the KAI1 promoter were identified by sequencing the PCR clones of the bisulfite-modified KAI1 promoter DNA. KAI1 protein levels in human prostate cancer tissue samples were examined by immunofluorescence staining of the tissues with an anti-KAI1 antibody. Among the three human prostate cancer cell lines examined, PC3 and DU145 cells exhibited markedly decreased levels of KAI1 mRNA and protein as compared to LNCaP cells, even though the exogenous KAI1 promoter not being methylated was normally functional in all these cell lines. Treatment of the low KAI1-expressing cell lines with a demethylating agent, 5'-aza-2'-deoxycytidine, significantly elevated KAI1 expression levels, implicating the involvement of DNA methylation in KAI1 downregulation. Methylation of CpG islands within the KAI1 promoter region was observed in the low KAI1-expressing cells, but not in the high KAI1-expressing cells. Also, methyl CpG-binding proteins such as MBD2 and MeCP2 were complexed to the KAI1 promoter in the low KAI1-expressing cells. Bisulfite sequencing analysis identified the intensively methylated CpG residues in the KAI1 promoter clones derived from prostate cancer cells and tissues with no or low KAI1 expression. As in prostate cancer cell lines, prostate cancer tissues from patients also displayed a negative association between KAI1 expression levels and methylation status of the KAI1 promoter. The present data suggest that the KAI1 gene might be repressed by epigenetic alterations through the promoter CpG-site methylation during prostate cancer progression. This epigenetic mechanism could provide a clue for understanding how the KAI1 gene was silenced in metastatic prostate cancers. Prostate 77: 350-360, 2017. © 2016 Wiley Periodicals, Inc.

Elevated KAI1 Protein Expression Identified in Malignant Melanoma

Dear Editor: KAI1, also known as CD82, is a member of the TM4SF protein family. KAI1 was first discovered during a study on T-cell activation. While recent studies have proposed that TM4SF affects the behavior of cancer cell growth, the exact mechanism has not been described. KAI1/CD82 is found in high levels in the spleen, lung, liver, placenta, kidney and prostate, in moderate levels in the pancreas, skeletal muscle, and thymus, and in low levels in the brain, heart, ovary, stomach, and uterus. However, It was not reported that KAI1/CD82 protein was expressed in normal skin tissue. Although there have been many studies on the role in the regulation of cell growth, cell-to-cell adhesion and motility, KAI1 and the TM4 family function remains unclear. An association between KAI1/CD82 and cancer progression was discovered during a study conducted to identify metastasis suppressor genes. KAI1 expression is reduced in a large number of cancers. Dong et al.1 reported reduced RNA expression of KAI1 during prostate cancer metastasis by northern blot analysis. Transfecting metastatic prostate cancer cells into nude mice resulted in suppression of lung metastasis by KAI1. Tests using reverse transcriptase-polymerase chain reaction and immunohistochemistry have reported reduced levels of CD82 mRNA and protein in thyroid cancer2. A study by Hinoda et al.3 used immunohistochemical tests to assess the association between KAI1 and the progression of gastric cancer. Northern blot analysis conducted by Guo et al.4 showed similar levels of KAI1 mRNA expression in both normal and cancerous esophagus and similar levels in tissue samples with or without metastasis. Therefore, metastasis in esophageal and gastric cancer is independent of KAI14. However, a recent study has revealed increased expression of KAI1 protein in chromophobe renal cell carcinoma5. In light of this, we explored the expression of KAI1 in skin cancers in order to determine whether it downregulates or upregulates the expression of skin tumors. The Institutional Review Board of Seoul Soonchunhyang University Hospital reviewed and approved this research protocol, which involved the use of tissue samples. We experimentally assessed whether loss of KAl1 protein expression in metastatic cancer is a common event in malignant skin cancers or if it occurs only in some tumor types. Based on the aforementioned finding, we chose basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and malignant melanoma (MM) and used western blot analysis in order to determine if KAI1 expression suppresses all cancer types or certain cancer types. We evaluated the expression of KAI1 protein in MM, SCC and BCC using western blot and immunohistochemistry. In this study, KAI1, which is a marker of metastatic potentiality in breast cancer, was measured in BCC, SCC and MM using western blot and the immunohistologic method. The results show that KAI1 was hardly expressed in 6 cases of BCC. In 6 cases of SCC, KAI1 was hardly expressed. Loss of KAI1 protein expression including SCC, BCC in our study corresponded to previous studies results6-8. In the 6 cases of MM, the expression of KAI1 was strongly positive (Fig. 1A). The amount of expression in western blotting is displayed graphically (Fig. 1B). The expression patterns of KAI1 protein in MM were similar with those by Yusenko and Kovacs5. However, different patterns were shown in other types of cancer including prostate, gastric, colon, cervix, breast, bladder, lung, pancreas, liver, and thyroid cancers. An immunohistochemical study showed that the staining pattern of KAI1 in normal human skin tissues mirrored those of western blot analysis (Fig. 2). Fig. 1 (A) KAI1/CD82 protein was expressed on malignant melanoma by western blot analysis. (B) The score of relative KAI1/CD82 protein expression. Normal: normal skin, MM: malignant melanoma, SCC: squamous cell carcinoma, BCC: basal cell carcinoma. Fig. 2 Representative immunohistochemistry staining for KAI1/CD82 protein expression in paraffin-embedded malignant melanoma (A: strongly positive immunostaining, ×200), squamous cell carcinoma (B: very weakly positive immunostaining, ×200), ... To the best of our knowledge, KAI1 expression in cutaneous BCCs, SCCs and MMs has not yet been studied. KAI1 protein expression has been known to be associated with a higher grade of malignant skin tissue. However, the exact function and mechanism of KAI1 during tumor genesis have not been confirmed. While KAI1 is suppressed in specific types of cancer, KAI1 is over expressed in MM. Interestingly, KAI1 was not expressed in cutaneous BCCs, SCCs and normal tissues. More work is needed to characterize the pathway through which KAI1 is overexpressed in MM skin tumor.

Expression changes of tumor metastasis-related genes after overexpression of KAI1 in retinoblastoma Y79 cells

To investigate the expression changes of tumor metastasis-related genes after overexpression of KAI1 in retinoblastoma Y79 cells. Experimental study. Y79 cells were transfected with a lentivirus vector containing KAI1 and enhanced green fluorescent protein (EGFP) fusion gene, or a control lentivirus vector containing EGFP. Positive transfectants stably expressing high levels of KAI1 were named Y79-KAI1 and control transfectants were named Y79-KAI1/zero. These transfectants were selected by puromycin resistance and analysis with fluorescent microscopy. The expression of KAI1 mRNA and its protein among Y79, Y79-KAI1 and Y79-KAI1/zero were detected by fluorescent quantitative RT-PCR and Western blot. Differential expression of tumor metastasis-related genes in Y79-KAI1 and Y79-KAI1/zero was analyzed with human tumor metastasis PCR array. One-way ANOVA was used to analyze the differences of KAI1 mRNA and protein expression among the three groups. The stably transfected cell lines of Y79-KAI1 and Y79-KAI1/zero were established. The result of fluorescent quantitative real-time PCR showed that the relative quantification of mRNA level of KAI1 gene in the three kinds of cells above was 183.67 ± 21.20, 1.42 ± 0.55, 1.00 ± 0.00, respectively. And the expression level of KAI1 mRNA in Y79-KAI1 cells was significantly higher than those in Y79-KAI1/zero and Y79 cells (F = 108.74, P = 0.000). The results of Western blot showed that the expression level of the KAI1 protein in Y79-KAI1 cells was significantly higher than those in Y79-KAI1/zero and Y79 cells (F = 34.36, P = 0.001). Immunofluorescent staining showed that Y79 and Y79-KAI1/zero cells had no detectable KAI1 expression, while Y79-KAI1 cells expressed KAI1 in the cytoplasm surrounding the nuclei. Among the 84 tumor metastasis-related genes examined, 7 genes were up-regulated more than 2 folds and 6 genes were down-regulated over 50%. Over-expression of KAI1 may result in differential expression of tumor metastasis-related genes in Y79 cells, which may be related to the inhibitory effect on the tumor metastasis of retinoblastoma.

KAI1 Gene Is Engaged in NDRG1 Gene-mediated Metastasis Suppression through the ATF3-NFκB Complex in Human Prostate Cancer

KAI1 Gene Is Engaged in NDRG1 Gene-mediated Metastasis Suppression through the ATF3-NFκB Complex in Human Prostate Cancer

Expression of KAI1/CD82 in distant metastases from estrogen receptor‐negative breast cancer

The tetraspanin protein superfamily member KAI1 suppresses tumor growth and metastasis in animal models and is downregulated in various human malignancies. In breast cancer, KAI1 is preferentially lost in estrogen receptor (ER)-positive tumors. Interestingly, most ER-negative primary breast cancers retain KAI1 expression. This study aimed to evaluate whether or not KAI1 is downregulated during progression to metastasis of these carcinomas. Expression of KAI1, ER, progesterone receptor, c-ErbB2, and Ki67 was analyzed in tissue microarrays comprising a large collection of distant organ metastases from human breast cancers (n = 92) by immunohistochemistry. Results were compared with a previously characterized set of primary breast tumors (n = 209). Immunoreactivity for KAI1 was observed in one-third of the metastases and was associated with lack of ER expression (P = 0.005). The high frequency of KAI1-positive cases in ER-negative primary tumors was maintained in ER-negative metastases. Expression of KAI1 was also observed in MDA-MB-468 and SK-BR-3, two ER-negative breast cancer cell lines of metastatic origin. Moreover, a reanalysis of independent microarray gene expression data indicated maintenance of KAI1 mRNA expression in metastases from ER-negative breast cancers. Furthermore, in a series of matched pairs of mammary carcinomas and metachronous distant metastases, all metastases from KAI1-positive/ER-negative primary tumors were KAI1-positive as well. Collectively, these findings demonstrate that the expression of KAI1 is maintained during progression to metastasis in a large proportion of ER-negative mammary carcinomas. This has significant implications for the use of KAI1 as a clinical marker and the understanding of the metastatic process in human breast cancer.

Expression of KAI1 gene in non-small cell lung cancer and its clinical significance.

Objective To investigate the expression of KAI1,as a suppressor of metastasis,in the tissues of NSCLC and its relationship with the clinicopathological indexes.Methods The expressions of KAI1/CD82 protein were detected with Western blot in 48 cases of NSCLC from patients with lung cancer and benign lung tissue of patients with benign lung disease undergoing surgery in our hospital.The RT-PCR method was also used to assess KAI1 mRNA in the same tissue samples,and the results were statistically analyzed.Results The positive rates of KAI1mRNA in NSCLC and benign lung tissues were 52%,90% respectively;the positive rates of KAI1/CD82 in NSCLC and benign lung tissues were 48%,85% respectively(P0.01).The expression level of KAI1mRNA and KAI1/CD82 protein in lung cancer were lower than those in benign lung tissue(P0.01).The expression level of KAI1/CD82 were correlated with tumor stage,grade of tumor differentiation and metastasis status(P0.05),among which the metastasis status in particular(P0.01).The expression of KAI1/CD82 had relations with its mRNA(P0.01).Conclusion The down-regulation of KAI1 may be correlated with carcinogenesis,development and metastasis in NSCLC.Its reduction may be mainly at transcriptional level,and it could be used as a marker for predicting the potential metastasis and prognosis of patients with NSCLC.

Differential Expression of KAI1 Metastasis Suppressor Protein in Renal Cell Tumor Histological Subtypes

The similar appearance of renal tumor histological subtypes can complicate differential diagnoses. This problem is most notable for the chromophobe subtype of renal cell carcinoma, which can be histologically indistinguishable from oncocytoma with investigational molecular markers failing to provide reliable differentiation. KAI1 is a metastasis suppressor gene whose expression correlates inversely with the metastatic potential of most solid tumor cancer types. We tested the hypothesis that KAI1 is differentially expressed among renal tumor histological subtypes. Immunohistochemical staining for KAI1 protein was performed in 152 nephrectomy specimens, including 48 clear cell, 35 papillary and 31 chromophobe renal cell carcinoma samples, 28 oncocytomas and 10 tumor-free kidneys. Staining was scored as none/minimal, low, moderate or high. KAI1 mRNA levels were compared by quantitative reverse transcriptase-polymerase chain reaction in an additional 22 chromophobe renal cell carcinoma and oncocytoma samples. In all 10 tumor-free kidneys KAI1 protein was detected exclusively in distal tubule cell membranes. Of the tumor specimens KAI1 protein was absent in all papillary renal cell carcinoma specimens. It was present in only 1 of 48 clear cell renal cell carcinomas (2%) and 2 of 28 oncocytomas (7%) but only at low levels. In contrast, 27 of 31 chromophobe renal cell carcinoma specimens (87%) expressed KAI1 protein, most at moderate or high levels. The diagnostic accuracy of KAI1 immunostaining for discerning chromophobe renal cell carcinoma from oncocytoma was 90% with similar results observed at the RNA level. KAI1 is an accurate biomarker for chromophobe renal cell carcinoma that may aid in the diagnostic differentiation of chromophobe renal cell carcinoma from oncocytoma. It remains to be determined whether KAI1 expression contributes to the low metastatic potential of chromophobe renal cell carcinoma.

Phorbol Ester Enhances KAI1 Transcription by Recruiting Tip60/Pontin Complexes

Down-regulation of the KAI1 (CD82) metastasis suppressor is common in advanced human cancer, but underlying mechanism(s) regulating KAI1 expression are only now being elucidated. Recent data provide evidence that low levels of KAI1 mRNA in LNCaP cells are caused by binding of beta-catenin/Reptin complexes to a specific motif in the proximal promoter, which prevents binding of Tip60/Pontin activator complexes to the same motif, thus inhibiting transcription. Here, we explored a pathway by which phorbol 12-myristate 13-acetate (PMA) up-regulates KAI1 transcription in LNCaP prostate cancer cells. Pretreatment with specific inhibitors showed that induction of KAI1 by PMA uses classic isoforms of protein kinase C (cPKC), is independent of Ras and Raf, and requires activation of MEK1/2 and ERK1/2, but does not involve p38MAPK. Induction of KAI1 transcription by PMA was associated with enhanced overall acetylation of histones H3 and H4, but only acetylation of H3 was blocked by a PKC inhibitor. Chromatin immunoprecipitation showed that PMA induces recruitment of Tip60/Pontin activator complexes to NFkappaB-p50 motifs in the proximal promoter, and this was blocked by a PKC inhibitor. These changes were not associated with differences in overall levels of Tip60, Pontin, beta-catenin, or Reptin protein expression but with PMA-induced nuclear translocation of Tip60.

KAI1/CD82 is a novel target of estrogen receptor‐mediated gene repression and downregulated in primary human breast cancer

The cell-surface glycoprotein KAI1 suppresses tumor growth and metastasis in various animal models. Downregulation of KAI1 has been implicated in the progression of cancer. However, the mechanisms of KAI1 inactivation are poorly understood. This is the first study that investigates expression and regulation of KAI1 in human breast cancer. KAI1 expression was analyzed on custom-made tissue microarrays comprising 209 well-characterized breast cancers and normal mammary gland tissue. Strong KAI1 immunoreactivity was observed throughout the normal mammary gland epithelium. In breast cancer tissue, KAI1 immunoreactivity was lost in 161/209 (77%) cases. Strikingly, KAI1 was preferentially lost in estrogen receptor (ER)-positive breast cancers (p < 0.001). This was validated by real-time RT-PCR analyses showing a 7.5-fold downregulation of KAI1 mRNA in ER-positive relative to ER-negative tumors (p = 0.028). Notably, this was also corroborated by Affymetrix microarray expression data of an independent cohort of 49 breast cancers. Class comparison analysis identified KAI1 as downregulated in ER-positive tumors. Subsequently, human breast cancer cell lines were employed to test a potential role of ER-activity in the downregulation of KAI1, as suggested by our expression analyses. Exposure of ER-positive breast cancer cells to fulvestrant, a clinically approved ER-antagonist that reverses ER-mediated gene repression, induced a significant upregulation of KAI1 and inhibited cell proliferation as well as migration. In summary, we demonstrate for the first time that KAI1 is a target of ER-mediated gene-repression, and thus, it is downregulated in ER-positive breast cancer. Importantly, KAI1 might be reinducible by endocrine therapy with ER-antagonists in patients suffering from ER-positive breast cancer.

An alternatively spliced KAI1 mRNA is expressed at low levels in human bladder cancers and bladder cancer cell lines and is not associated with invasive behaviour

Levels of the KAI1 metastasis suppressor are reduced in advanced stages of many human cancers, leading to the loss of KAI1 function. A recent study has suggested that the loss of KAI1 function may also occur via alternative splicing of KAI1 mRNA which deletes an exon encoding a critical 28 amino acids from the protein. Using PCR, 20 bladder tumours at differing stage and grade, a non-tumourigenic urothelial cell line (SV-HUC-1) and 17 bladder cancer cell lines were examined for expression of this alternatively spliced (AS) KAI1 mRNA. Full-length KAI1 mRNA was present in all tumour samples and low levels of AS KAI1 mRNA in 15/20 samples. There was no association between the presence or absence of AS mRNA and clinicopathological characteristics of these tumours. Low levels of AS KAI1 mRNA were present in SV-HUC-1 and 14/17 bladder cancer cell lines. There was no association between the presence or absence of AS KAI1 mRNA and tumourigenicity, or in vivo invasive abilities of these cell lines. In all cell lines expressing AS KAI1 mRNA, levels were 3- to 5-fold lower than levels of wild-type mRNA, irrespective of wild-type mRNA levels. Low levels of an alternatively spliced form of KAI1 mRNA are present in most bladder cancer tumours and tumour cell lines, but are not associated with invasive behaviour.

P53 dominant-negative mutant R273H promotes invasion and migration of human endometrial cancer HHUA cells

Dominant negative (DN) mutations of tumor suppressor p53 (TP53) are clinically associated with cancer progression and metastasis of endometrial malignancy. To investigate the DN effect on tumor migration and invasion, we generated cells that stably co-expressed wild-type (wt) and R273H DN mutant TP53 (273H cells), and wt and R213Q recessive mutant TP53 (213Q cells), by transfection in endometrial cancer cells HHUA that expressed wt p53. R273H, but not R213Q, repressed wt p53-stimulated transcription of p21, Bax, and MDM2. 273H cells also showed markedly increased in vitro invasion and migration potentials, and displayed reduced Maspin, PAI-1, and KAI1 mRNA expressions as compared with 213Q and wt cells. The induction of wt p53 function by use of Adriamycin resulted in the inhibition of the invasion/migration capacity in association with the up-regulation of p53 target genes to a far greater degree in 213Q and wt cells than in 273H cells. R273H expression in p53-null cancer cell SK-OV-3 and Saos-2 did not significantly affect cell invasion and migration activities. Taken together, these results suggest that transdominance of R273H mutant over wt p53 rather than a gain-of-function promotes tumor metastasis by increasing invasion and migration in HHUA cells.

Expression of KAI1 gene in non-small cell lung cancer and its relationship with P53

Recent researches have manifested that down-regulation of KAI1 relates to metastasis in various tumors, but its association with non-small cell lung cancer (NSCLC) and the mechanism for its down-regulation are not clear. The aim of this study is to investigate the expression of KAI1 in NSCLC and its relationship with clinicopathological characteristics and mutant P53 protein. The expression of KAI1/CD82 and mutant P53 protein was detected in 48 cases of NSCLC tissues by Western blot, and KAI1 mRNA was detected by RT-PCR method, with 20 cases of pulmonary benign disease tissues and normal lung tissues as control.. The positive rate of KAI1 mRNA was 52% in lung cancer group and 90% in control group, respectively (P < 0.01), KAI1/CD82 was 48% and 85% respectively (P < 0.01), and mutant P53 protein was 65% and 5% respectively (P < 0.01). The positive rate of KAI1 mRNA, KAI1/CD82 and mutant P53 protein closely related to the tumor stages, cell differentiation and lymph node metastasis status (P < 0.05 or P < 0.01). The expression of KAI1/CD82 highly related to KAI1 mRNA (P < 0.01) and mutant P53 protein (P < 0.05), while expression of KAI1 mRNA did not relate to mutant P53 protein expression (P > 0.05). The down-regulation of KAI1 may relate to carcinogenesis, development and metastasis of NSCLC. Its reduction may occur mainly at transcriptional level and correlate with p53 in NSCLC. KAI1 and p53 might be helpful to predict the potential metastasis of NSCLC.

KAI1 promoter activity is dependent on p53, junB and AP2: evidence for a possible mechanism underlying loss of KAI1 expression in cancer cells

A molecular mechanism to explain reduced KAI1 expression in invasive and metastatic tumour cells remains elusive. In this report, we extend an earlier study in bladder cells to confirm that a 76 bp region of the KAI1 promoter (residues -922 to -847), with binding motifs for p53, AP1 and AP2, is required for high level activity of a KAI1 reporter in prostate cancer cell lines. Gel shift and supershift experiments supported binding of p53, junB and heterodimers of AP2alpha/AP2gamma or AP2beta/AP2gamma to this sequence. Introduction of mutations into specific motifs demonstrated an essential requirement for p53 and junB to reporter activity, and that functional synergy between these two factors enhanced activity. A further elevation of reporter activity required AP2. Roles of individual p53, junB and AP2 proteins, as well as functional synergy between p53 and junB, were confirmed in transfection experiments. Western blotting analysis showed that an absence of wild-type p53, and/or a loss of junB and AP2 protein expression, correlated with downregulation of KAI1 mRNA levels in a series of prostate cancer cell lines. A loss of p53 function and/or expression of junB, combined with reduced expression of specific AP2 proteins may underly downregulated KAI1 expression in tumour cells.

KAI1 COOH-Terminal Interacting Tetraspanin (KITENIN), a Member of the Tetraspanin Family, Interacts with KAI1, a Tumor Metastasis Suppressor, and Enhances Metastasis of Cancer

We cloned recently an alternatively spliced variant of KAI1 mRNA that lacked exon 7 at the COOH-terminal region and showed differences in metastasis suppression when compared with the wild-type KAI1. These findings indicated that the COOH-terminal region of KAI1 is critical for its metastasis suppressor function. In this study, we isolated a cDNA clone of VANGL1, a member of the tetraspanin protein family, which interacted specifically with the COOH-terminal cytoplasmic domain of KAI1 in the yeast two-hybrid system. We renamed it KAI1 COOH-terminal interacting tetraspanin (KITENIN). We found that KITENIN-overexpressing CT-26 mouse colon cancer cells showed increased tumorigenicity and early hepatic metastasis in vivo, as well as increased invasiveness and adhesion to fibronectin in vitro compared with parental cells. Moreover, increased levels of KITENIN were observed in a human gastric tumor and its metastatic tissues, compared with the normal adjacent mucosa. Our results indicate that KITENIN promotes adhesion and invasion of cancer cells in vitro and in vivo, and suggest that KITENIN participates in the regulation of the tumor formation and metastasis by interacting with KAI1, a metastasis suppressor and antisense KITENIN strategy that can be used to inhibit metastasis in various cancers.

Role of tumor metastasis suppressor gene KAI1 in digestive tract carcinomas and cancer cells.

The KAI1 gene is identified as a tumor metastasis suppressor gene in many types of cancer. We examined KAI1 gene and its protein KAI1/CD82 expression by in situ hybridization and immunohistochemical analysis, and found that KAI1 mRNA and protein expression were inversely correlated with lymph node and distant metastasis in digestive tract carcinomas, but not with age and gender of the patient, or with tumor differentiation. Moreover, KAI1/CD82 protein expression positively reflected the survival outcome of patients. Western blot analysis showed that VP-16 increased KAI1/CD82 protein expression obviously in various cancer cell lines, especially in those that were highly metastatic. This increased KAI1/CD82 expression was associated with its translocation from the cytomembrane to the nucleus, in which it interacted with nuclear p53 protein, forming a strong complex, observed by confocal microscopy and co-immunoprecipitation, respectively. In nude mice, after feeding with VP-16, the number of tumors metastasized from spleen to liver was obviously reduced, and KAI1/CD82 protein expression became stronger in those metastatic tumors. Accordingly, this demonstrated that KAI1 might be used as an indicator for predicting the clinical outcome, and VP-16 may be clinically considered as a promising candidate for anti-metastasis with regard to its potential to upregulate KAI1 expression.

Downregulation of KAI1 mRNA in localised prostate cancer and its bony metastases does not correlate with p53 overexpression.

Recent data have proposed that transcription of the KAI1 metastasis suppressor gene is directly mediated by p53 and that loss of KAI1 expression in advanced prostate cancer is simply due to loss of p53 function after mutation. To investigate this possibility, we have examined KAI1 mRNA (by in situ hybridisation) and p53 protein expression (by immunohistochemistry) as an indicator of wildtype or mutant p53, in a series of 77 paraffin-embedded prostate tissue samples, including post-mortem normal prostates (2), benign prostatic hyperplasia (10), localised cancer (grades 4-6, 25; grades 7-9, 21) and prostate-derived bony metastases (19). Overall, we confirmed that expression of KAI1 mRNA decreased from normal tissue, through localised cancer to bony metastases (P=0.055, tending to significance), while levels of p53 staining significantly increased with cancer progression (P=0.046). These were consistent with the possibility that loss of p53 function might be responsible for loss of KAI1 mRNA. However, by close examination of KAI1 and p53 in adjacent tissue sections, we found no correlation between decreased levels of KAI1 mRNA and overexpression of p53 protein (P=0.497). In addition, high levels of KAI1 mRNA could be identified in samples irrespective of p53 staining. Our data suggest that mutation of p53 is independent of the loss of KAI1 mRNA, and do not support a role for p53 in regulating the expression of KAI1.

Identification of regulatory regions within the KAI1 promoter: a role for binding of AP1, AP2 and p53

The mechanism underlying loss of KAI1 gene expression in invasive and metastatic tumour cells is unknown. A possible scenario could involve altered expression or function of protein factors normally involved in regulating KAI1 transcription. To explore this possibility, we have initiated a study to characterise regulatory elements of the KAI1 promoter, using as a model, two bladder cancer cell lines (BL13 and HT1376) expressing high levels of endogenous KAI1 messenger RNA (mRNA). Transfection experiments using reporter plasmids with progressive KAI1 promoter deletions, identified a 76 bp region upstream of the transcription initiation site which contained putative binding motifs for AP2, p53 and AP1, as essential for reporter activity. DNA-binding studies using nuclear extracts from both cell lines, showed that AP1 and AP2 formed specific complexes with oligonucleotides containing KAI1 promoter motifs. Mutation of either motif abrogated reporter activity and abolished specific complex formation. In BL13 cells (endogenous wildtype p53), but not in HT1376 cells (endogenous mutant p53), mutation of the p53-binding motif also abrogated reporter activity and abolished specific complex formation in gel shift assays. These data suggested that a combination of AP2, p53 and AP1 binding to specific motifs within the KAI1 promoter might be required for high level promoter activity and that loss of expression or function of these factors might contribute to loss of KAI1 expression in invasive tumours and tumour cell lines. To explore this possibility, we examined levels of these proteins in nuclear extracts of BL13 and HT1376, as well as three bladder cancer cell lines which expressed little or no KAI1 mRNA. Our data suggested that a loss of KAI1 mRNA was not simply due to absence of AP2, AP1 or p53 expression.