KAI1 protein is down-regulated during the progression of human breast cancer.

The KAI1 gene was identified as a metastasis suppressor gene for human prostate cancer. Recently, we showed that KAI1 mRNA levels were higher in an immortal, normal-like breast epithelial cell line and nonmetastatic breast cancer cell lines but lower substantially in highly metastatic breast cancer cell lines. In this study, we examined KAI1 protein expression in breast cancer cell lines by Western blot and immunohistochemical study. KAI1 protein levels paralleled KAI1 mRNA levels and were inversely correlated with the metastatic potential of breast cancer cells. Furthermore, we examined KAI1 protein expression immunohistochemically in specimens from 81 patients with breast cancer and then correlated the findings with the clinical and histopathological parameters of the patients. High levels of KAI1 protein expression were found in normal breast tissues and noninvasive breast cancer (ductal carcinoma in situ). In contrast, KAI1 expression was reduced in most of the infiltrating breast tumors. We found that, in general, more malignant tumors demonstrated significantly lower KAI1 expression (P = 0.004). Additionally, among 29 specimens demonstrating multiple stages of malignancy within a single specimen, 23 demonstrated significant differences in KAI1 expression between benign breast tissue, ductal carcinoma in situ, and invasive carcinoma. The higher the incidence for malignancy within a given specimen, the lower the KAI1 expression (P < 0.001). These data suggest that in advanced breast cancer, KAI1 expression is down-regulated. Therefore, KAI1 may be a potentially useful indicator of human breast cancer progression.     

Overexpression of KAI1 suppresses in vitro invasiveness and in vivo metastasis in breast cancer cells.

KAI1 is a metastasis suppressor gene for human prostate cancer and is also involved in the progression of a variety of other human cancers. Previously, we have demonstrated that KAI1 expression was down-regulated in metastatic breast cancer cell lines as well as in highly aggressive breast cancer specimens. To determine whether KAI1 expression is responsible for the metastasis suppression in breast cancer, we transfected the human KAI1 cDNA into two highly malignant breast cancer cell lines, LCC6 and MDA-MB-231, which both have low levels of endogenous KAI1 expression. Parental, vector-only transfectants and KAI1 transfectant clones were injected into the mammary fat pads and tail veins, respectively, of athymic nude mice and assessed for both spontaneous and experimental lung metastasis. High KAI1 expression significantly suppressed the metastatic potential of KAI1-transfected LCC6 cells. Metastasis suppression correlated with the reduced rate of tumor growth and a decreased clonogenicity in soft agar. Furthermore, KAI1 expression significantly suppressed the in vitro cell invasion in KAI1-transfected MDA-MB-231 cells. Our results suggested that KAI1 may function as a negative regulator of breast cancer metastasis.     

KAI1 metastasis suppressor protein is down-regulated during the progression of human endometrial cancer.

PURPOSE: KAI1 is a metastasis suppressor gene located on human chromosome 11p11.2. It is a member of the structurally distinct family of cell surface glycoprotein, transmembrane 4 protein superfamily. KAI1 was initially isolated as a gene that suppressed metastasis of rat prostate tumor cells. Decreased KAI1 expression has been observed recently in various human cancers, including pancreatic, lung, hepatic, colorectal, breast, ovarian, esophageal, and cervical cancers. Frequent down-regulation of the KAI1 protein was also observed in endometrial cancer cell lines. The aim of this study was to determine whether this gene is altered in human endometrial carcinoma. In addition, its prognostic significance in this tumor was also evaluated. EXPERIMENTAL DESIGN: Tumor specimens from 18 cases with various degrees of endometrial hyperplasia, 97 primary endometrial carcinomas with various stages, and 28 metastatic lesions of this cancer were examined in this study. Using the method of immunohistochemistry, we characterized the KAI1 protein expression in the 143 endometrial tumors. Expression of KAI1 at RNA level was also examined in 35 of the 143 samples using a real-time quantitative PCR method. The data from immunohistochemical analysis were correlated with various clinicopathological factors. RESULTS: High levels of KAI1 protein expression were detected in almost all of the specimens with endometrial hyperplasia (17 of 18). In contrast, loss of KAI1 expression occurred in an increasing frequency (27.8-71.4%) from early stages of primary endometrial carcinomas to metastatic tumors (P < 0.001). In addition, more poorly differentiated tumors demonstrated significantly lower KAI1 expression as compared with the well-differentiated tumors (P < 0.001). It was also found that patients with KAI1-negative tumors had a lower survival rate than those with KAI1-decreased or positive tumors (P = 0.0042 and 0.0286, respectively). However, in multivariate analysis, the prognostic significance of KAI1 expression was inferior to tumor stage. CONCLUSION: These data suggest that KAI1 expression is down-regulated in advanced endometrial cancer. Clinically it may be a useful indicator of the tumor progression and may provide prognostic information on the outcome of this disease.     

Reduced KAI1 expression in pancreatic cancer is associated with lymph node and distant metastases

KAI1 belongs to a structurally distinct family of membrane glycoproteins, which function via cell-cell and cell-extracellular matrix interactions, thereby potentially influencing the ability of cancer cells to invade tissues and to metastasize into lymph nodes and distant organs. In the present study, we examined KAI1 expression in lymph node and liver metastases in comparison with primary pancreatic cancer to evaluate its influence on metastasis. KAI1 mRNA analysis was performed by Northern blot analysis and in situ hybridization. In addition, the respective protein was studied by immunostaining. Fourteen primary pancreatic cancer samples in which no lymph node metastases were present and 25 primary pancreatic cancer samples in which lymph node metastases were present at the time of tumor resection were included. In 20 of these cases, primary pancreatic cancer tissues and corresponding lymph node metastases from the same patient were studied. Furthermore, 11 liver metastases were available for KAI1 analysis. Increased steady-state levels of KAI1 mRNA were found in 33/39 (85%) primary pancreatic cancers in comparison with normal controls. Statistical analysis of KAI1 mRNA levels and clinical parameters of the patients revealed that KAI1 mRNA levels were significantly higher in non-metastasized tumors compared with tumors in which lymph node or distant metastases were present. In lymph node metastases KAI1 mRNA expression was lower than in the corresponding primary tumors: In 14 of 20 lymph node metastases no KAI1 mRNA expression and in 6 of 20 lymph node metastases only weak KAI1 mRNA levels were present in some cancer cells. Cancer cells of distant metastases were devoid of or exhibited low KAI1 mRNA levels compared with those of primary pancreatic cancers. A similar pattern was observed by immunostaining. These data support the hypothesis that KAI1 gene expression might influence the metastatic ability of pancreatic cancer cells in vivo. Reduction of KAI1 appears to promote cancer cell spread in lymph nodes and distant organs. Int. J. Cancer (Pred. Oncol.) 79:349–355, 1998. © 1998 Wiley-Liss, Inc.     

Loss of KAI1 expression in the progression of colorectal cancer

The transmembrane 4 superfamily member KAI1 (CD82) has been shown to inhibit pulmonary metastases in experimental metastasis models of prostate cancer and melanoma. KAI1 expression is decreased in the progression of common solid epithelial tumors of adulthood, including lung, prostate, breast, esophageal, gastric, pancreatic, and bladder cancers. The purpose of our study was to investigate KAI1 expression in the progression of human colorectal cancer. We first analyzed 20 colorectal cancer cell lines by immunoblot techniques. KAI1 was expressed heterogeneously, with the tumor cell lines having a more complex degree of glycosylation compared with that of the normal colonic tissue. KAI1 was highly expressed in the primary SW480 colon cancer cell line but was down-regulated 15-fold in the matched metastatic SW620 cell line. We also investigated KAI1 protein expression by immunohistochemistry in tissues from 84 patients with colorectal cancer. Each tissue section was assigned a KAI1 mean score (KMS) from 0 to 300 based on the product of the percentage of cells that stained for KAI1 and the intensity of the stain (1, 2, or 3). In 84 patients with colorectal cancer, KAI1 was expressed at high levels in normal colonic mucosa (KMS 226) but was expressed at lower levels in the primary tumors (KMS 65; P < 0.0001). In a subset of 12 patients with stage IV metastatic disease, we observed a progressive down-regulation of KAI1, from the normal adjacent colonic mucosa (KMS 193) to the primary tumor (KMS 72; P = 0.0001) to the liver metastasis (KMS 25; tumor compared with metastasis, P = 0.0135). We found no correlation between loss of KAI1 expression and stage of disease. In 10 patients, we also noted loss of KAI1 expression in the transition from normal colonic mucosa (KMS 237) to adenoma (KMS 174) to carcinoma (KMS 62; P < 0.0167 for all three comparisons). We conclude that the down-regulation of KAI1 occurs early in the progression of colorectal cancer.     

Suppression of telomerase, reexpression of KAI1, and abrogation of tumorigenicity by nerve growth factor in prostate cancer cell lines.

Nerve growth factor (NGF) is expressed in the prostate, where it appears to be involved in the control of epithelial cell growth and differentiation. NGF production is decreased in prostate tumors. However, the role of this neurotrophin in the control of proliferation and progression of prostate cancers is still a matter of investigation. Prostate adenocarcinomas are telomerase-positive tumors. Chronic exposure of DU145 and PC3 prostate tumor cell lines to NGF resulted in a dramatic down-regulation of telomerase activity. This effect was correlated in terms of concentrations and time with a remarkable down-regulation of cell proliferation both in vitro and in vivo but was not secondary to NGF-induced quiescence. No down-regulation of telomerase activity was, in fact, detectable during serum starvation-induced quiescence. LNCaP cells, which do not express NGF receptors, appear to be insensitive to the actions of NGF. DU145 and PC3 cells do not express the KAI1 metastasis suppressor gene, which is present in the prostate and is progressively lost during the progression of prostate cancers. Chronic NGF treatment strongly induced the reexpression of this gene in these cell lines, and this effect was correlated with the suppression of their invasive potential in vitro. The data presented here suggest that NGF reverts two metastatic prostate cancer cell lines to slowly proliferating, noninvasive phenotypes characterized by a very low telomerase activity and by the expression of the KAI1 metastasis suppressor gene.     

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. ( Cancer Sci 2009; 100: 1767–1771)     

Biological Significance and the Related Molecular Mechanism of Etsl mRNA Expression in Lung Cancer by Tissue Microarray （TMA）

Objective： To investigate the expressions and proteins in the pathogenesis, progression of lung molecular mechanism of Ets-1 mRNA, and TGFβ1 and c-Met cancer by tissue microarray （TMA） method. Methods： The expressions of Ets-1 mRNA, and TGFβ1 and c-Met proteins were detected in 89 primary lung cancers, 12 lung cancer with lymph-node metastasis and 12 precancerous lesions by FISH（fluorescence in situ hybridization） and immunohistochemical method, and 10 normal lung tissues were used as controls. Results： The expressions of Ets-1 rnRNA, and TGFβ1 and c-Met proteins were significantly higher in 89 primary lung cancer than in the control group （P〈0.05）. The expressions of Ets-1 mRNA, and TGFβ1 and c-Met proteins were related to lymph node metastasis and clinical stages. There was a positive correlation between the Ets-1 mRNA expression and TGFβ1 and c-Met proteins （P〈0.05）. Conclusion： Ets-1 mRNA, TGFβ1 and c-Met proteins may be related to the pathogenesis, progression and malignant behavior of lung cancer. They may play an important role in prognosis assessment of lung cancer.     

Expression of collagenase-3 (matrix metalloproteinase-13) in human gastric cancer

Background: Collagenase-3 (matrix metalloproteinase-13; MMP-13) is a recently identified member of the matrix metalloproteinases (MMPs) with broad substrate specificity, and a potential role in tumor metastasis and invasion has been proposed for this enzyme. To date, in gastrointestinal tract tumors, collagenase-3 expression has been reported only in esophageal carcinoma; the presence and possible implications of this enzyme in the progression of gastric cancer are unknown. Methods: In this study, MMP-13 mRNA expression was analyzed in a series of 110 matched gastric adenocarcinomas and the corresponding adjacent normal mucosae as well as in nine gastric cancer cell lines. In addition, the mRNA expression of gelatinase A (MMP-2) and membrane type-1 matrix metalloproteinase (MT1-MMP), two MMPs which have the ability to activate MMP-13 in vitro, was also examined in the same cases and cell lines. The production and localization of MMP-13, MMP-2, and MT1-MMP were investigated by immunohistochemistry, immunofluorescence, Western blot analysis, and zymography. Results: MMP-13 mRNA was expressed in 23 of the 110 carcinomas (21%), and MT1-MMP mRNA was expressed in 45 (40%), but no MMP-13 or MT1-MMP mRNA was detected in any of the normal mucosae. Also, eight of the nine gastric cancer cell lines expressed mRNA of MMP-13, and in each cell line there was coordinate expression with either MT1-MMP or MMP-2 mRNA. MMP-13 and MT1-MMP were detected at the bases of invadopodia of the cultured cancer cells as well as in the invasive front of the tumors, as shown by immunofluorescence and immunohistochemistry, respectively. Western blot analysis revealed the presence of MMP-13 protein in those cell lines and carcinomas that expressed its mRNA. On zymography, almost all cell lines that expressed MMP-13 showed gelatinolytic bands corresponding to the active form of MMP-13 or one of its intermediate forms. Also, zymographic analysis of the tumor specimens revealed strong gelatinolytic bands of MMP-13 and MMP-2, whereas these bands in normal mucosa were weak. There was no significant relationship between MMP-13 mRNA expression and histologic type, lymph node metastasis, wall invasion, or distant metastasis. However, patients with MMP-13 mRNA-positive tumors had a poorer prognosis than those with MMP-13-mRNA-negative cancer. Furthermore, patients with simultaneous expression of MMP-13 and MT1-MMP mRNA showed the poorest prognosis, as compared with those having tumors expressing either MMP-13 or MT1-MMP, or neither MMP-13 nor MT1-MMP mRNA. Conclusion: These findings suggest that MMP-13 expression may contribute to the progression of gastric cancer, and its coordinate overexpression with MT1-MMP and/or MMP-2 may have a cooperative effect in the progression of gastric cancer. Received: May 22, 2002 / Accepted: October 2, 2002 Offprint requests to: Y. Yonemura, Present address: Department of Gastroenterological Surgery, 1007 Shimo-nagakubo, Nagaizumi-machi, Shizuoka 411-8777, Japan     

Reduced Invasive and Metastatic Potentials of KAI1-transfected Melanoma Cells

KAI1 is a metastasis suppressor gene for human prostate cancer. To reveal the effect of KAI1 on the in vivo metastasis of tumors other than prostatic cancer, we transfected a human KAI1 cDNA into highly metastatic B16-BL6 murine melanoma cells and established stable transfectant clones with different expression levels of KAI1 message. The following results were obtained with the use of those transfectants. (1) Cell aggregation assay revealed a significantly enhanced Ca²⁺-independent aggregation of B16-BL6 cells by KAI1 cDNA transfection compared with mock transfectants ( P <0.01). (2) The in vivo phagokinetic activity and invasive ability of KAI1 transfectants were clearly decreased as compared with those of mock transfectants ( P <0.01). There was no significant effect of KAI1 expression on the in vitro or in vivo proliferation of B16-BL6 cells. (3) Lung colony formation of intravenously injected KAI1 transfectants in nude mice was significantly reduced as compared with mock transfectants or parental B16-BL6 cells ( P <0.01). These data suggest that KAI1 expression gives rise to the suppression of invasive and metastatic potentials of B16-BL6 cells.     

KAI1基因在非小细胞肺癌中的表达及意义

 目的 探讨肿瘤转移抑制基因KAI1基因在非小细胞肺癌组织中的表达及其与患者临床病理指标的关系。 方法 采用RT-PCR和Western blot法,检测48例肺癌患者手术切除的新鲜肺癌组织标本和20例同期手术切除的肺部良性病变周围正常组织中KAI1 mRNA、KAI1/CD82,并结合患者的临床病理资料对其结果进行统计分析。 结果 肺癌组织和肺部良性病变组织中KAI1 mRNA的阳性率分别为52%和90%,KAI1/CD82蛋白的阳性率分别为48%和85%,肺癌组KAI1mRNA及KAI1/CD82蛋白表达均低于肺部良性病变组（P<0.01）;KAI1mRNA、KAI1/CD82表达水平与肺癌患者的临床分期、组织分化程度、淋巴结转移有关（P<0.05）,其中KAI1/CD82表达与淋巴结转移状况密切相关(P<0.01),肺癌组织中KAI1 mRNA与KAI1/CD82表达有相关性（P<0.01）。 结论 KAI1基因的低表达可能与非小细胞肺癌的发生、发展和转移有关;其下调的机制可能主要发生在转录水平;KAI1基因的表达可作为一项评估肺癌患者转移潜能的指标。     

Nuclear factor-kappaB-dependent expression of metastasis suppressor KAI1/CD82 gene in lung cancer cell lines expressing mutant p53

KAI1/CD82 has been shown to be a metastasis suppressor for several human cancers, and a recent study revealed that wild-type tumor suppressor p53 can directly activate KAI1/CD82 gene expression. However, the response of KAI1/CD82 expression in cancer cells to exogenous stimulants has not been investigated. The present study examined whether tumor necrosis factor (TNF), which mediates many of the cellular responses associated with inflammatory reactions or cancer progression, can affect the KAI1/CD82 expression in lung cancer cells and, if so, whether nuclear factor (NF)-kappaB, a key molecule in TNF-mediated gene expression, is involved in the mechanism of KAI1/CD82 induction. Our results demonstrated that expression of KAI1/CD82 in PC-14 cells expressing mutant p53 could be augmented by TNF-alpha, and that transfer of the gene for a specific inhibitor of NF-kappaB, IkappaB alphaSR (mutant IkappaB alpha; NF-kappaB super-repressor), into PC-14 cells could inhibit this augmentation. The amount of NF-kappaB in the nucleus of PC-14/IkappaB alphaSR cells correlated well with KAI1/CD82 mRNA and protein expression. In addition, IkappaB alphaSR gene transfer inhibited the spontaneous expression of KAI1/CD82 protein in KAI1/CD82-high-expressing RERF-LC-OK cells, which contain a mutant-type p53. These observations indicate that NF-kappaB activation may play a role in the regulation of KAI1/CD82 expression in lung cancer cells independently of wild-type p53, and suggest that KAI1/CD82 expression may be regulated by interaction with the host microenvironment.     

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.