Increased Midkine Gene Expression in Human Gastrointestinal Cancers

Midkine (MK) is a product of a retinoic acid-responsive gene, and is a novel growth differentiation factor. We examined the expression of the MK gene in specimens of 47 surgically removed human carcinomas of the gastrointestinal organs, namely, gastric, colorectal, hepatocellular, pancreatic, esophageal, ampullary duodenal and bile duct carcinomas. In most cases, the MK mRNA level was higher in cancer specimens than in the corresponding non-cancerous tissues. Furthermore, MK mRNA was more highly expressed in the colon adenocarcinoma lesion than in the adenoma lesions, in the two familial polyposis cases. While MK mRNA was not detected in the normal liver, it became detectable in cirrhotic tissues in 2 of 4 cases, and its expression was increased in the cancerous tissues. Thus, the increase of MK mRNA level is a phenomenon seen in many human gastrointestinal carcinomas. The increased expression of the MK gene in gastric carcinoma was significantly more prominent in well and moderately differentiated adenocarcinomas than in poorly differentiated adenocarcinomas and signet ring cell carcinomas.     

Molecular cloning, expression and purification of truncated midkine and its growth stimulatory activity on Wilms' tumor (G401) cells

Midkine (MK) is a heparin binding growth factor identified as a product of a retinoic acid-responsive gene; it is frequently expressed at high levels in many human carcinomas. Although the expression of the mRNA encoding truncated MK (tMK) in unique human cancer cells has been reported, the tMK polypeptide itself has not yet been identified. In order to clarify the biological role of tMK, recombinant tMK was expressed in Escherichia coli and purified. Recombinant tMK was purified as a single band in SDS-PAGE under reducing conditions showing an apparent molecular mass of 10 kDa. Purified recombinant tMK showed the same extent of proliferative activity towards Wilms' tumor (G401) cells as full length human MK. These results suggest that the structure of this recombinant tMK is same as the native polypeptide.     

Expression of glycodelin protein and mRNA in human ductal breast cancer carcinoma in situ, invasive ductal carcinomas, their lymph node and distant metastases, and ductal carcinomas with recurrence

Glycodelin, previously known as PP14, has been localized in endometrial, ovarian and cervical carcinoma cells. Recently, glycodelin was demonstrated to be expressed in cancerous human breast tissue. In this study, paraffin-embedded slides of carcinoma in situ, invasive carcinomas without metastases, invasive carcinomas with corresponding lymph node metastases, invasive carcinomas with corresponding recurrence and invasive carcinomas with corresponding distant metastases were investigated for glycodelin protein and mRNA expression. Protein expression was found in all cases of carcinoma in situ, in invasive carcinoma without lymph node metastases in 90% of cases, in breast cancer with lymph node metastases in 50% of cases, in breast cancer with recurrence in 38% of cases and in breast cancer with distant metastases in 40% of cases. Results were confirmed by in situ hybridization showing reduced glycodelin expression as lymph node metastasis progressed, compared to carcinoma in situ. Glycodelin mRNA expression is not further reduced in carcinomas with distant metastasis and recurrence compared to carcinoma in situ. Results demonstrate that invasive breast carcinomas without metastases are more likely to express glycodelin. In contrast, cases of breast cancer with metastatic infiltration and recurrence show weak expression of glycodelin. On the basis of these results, we speculate that glycodelin could be used as a prognostic marker for breast cancer.     

Correlation of elevated plasma levels of two structurally related growth factors, heparin affin regulatory peptide and midkine, in advanced solid tumor patients

Heparin affin regulatory peptide (HARP) and midkine (MK) are growth factors, expressed in carcinomas, neuroblastomas and gliomas. In this study, we measured the levels of HARP and MK in plasma samples from 77 cancer patients. The patients had advanced tumors with loco-regional (n=18) or metastatic (n=49) diseases and 10 patients have their diseases limited to the primary site. HARP and MK plasma concentrations were significantly higher in all of these different subgroups of cancer patients (P<0.05 in all cases), when compared to healthy controls (n=30). Neither HARP nor MK levels were significantly different between patients with loco-regional and metastatic tumors (P=0.203 and 0.242, respectively). Moreover, a strong correlation between the elevations of the plasma levels of these two proteins (r2=0.546) in these cancer patients was found. Measurements of these secreted angiogenic growth factors may be useful for evaluation of cancer diagnosis.     

胃肠道恶性肿瘤患者外周血微转移诊断及预后研究

目的：检测胃肠道恶性肿瘤患者外周血中细胞角蛋白２０（ＣＫ２０）ｍＲＮＡ和人端粒酶逆转录酶（ｈＴＥＲＴ）ｍＲＮＡ的表达,探讨其能否作为判断胃肠道恶性肿瘤患者外周血微转移及预后的分子生物学指标。方法：采用ＲＴ-ＰＣＲ技术检测７２例胃肠道恶性肿瘤患者、３０例肠道良性病变患者和３０例健康体检者外周血中ＣＫ２０ｍＲＮＡ和ｈＴＥＲＴｍＲＮＡ的定性和半定量表达。以人胃腺癌细胞株ＳＧＣ-７９０１作阳性对照。结果：（１）ＣＫ２０ｍＲＮＡ和ｈＴＥＲＴｍＲＮＡ在胃肠道恶性肿瘤患者外周血中的阳性率分别为５１.４０％和６１.１０％,相对系数分别为０.８４±０.１８和０８２±０.１７;在肠道良性病变患者外周血中阳性率分别为６.６７％和３.３３％,相对系数分别为０.１２±０.０１和０.１１;在健康体检者外周血中阳性率及相对系数均为０。胃肠道恶性肿瘤患者与胃肠道良性病变患者、健康体检者相比,ＣＫ２０ｍＲＮＡ和ｈＴＥＲＴｍＲＮＡ的表达均有统计学差异（Ｐ＜０.０１）。（２）ＲＴ-ＰＣＲ法联合检测胃肠道恶性肿瘤患者外周血中ＣＫ２０ｍＲＮＡ和ｈＴＥＲＴｍＲＮＡ微转移的灵敏度为７２.２２％ ～８３.３３％,特异度为６８.５７％ ～８５.７１％,准确度为７３.５８％ ～８１.１３％。（３）ＣＫ２０ｍＲＮＡ、ｈＴＥＲＴｍＲＮＡ阳性者根治术后复发转移率高于阴性者,生存率低于阴性者（Ｐ＜０.０５）。（４）ＣＫ２０ｍＲＮＡ与ｈＴＥＲＴｍＲＮＡ在胃肠道恶性肿瘤患者外周血中的表达呈正相关（Ｐ＜０.０１）。结论：胃肠道恶性肿瘤患者外周血中存在ＣＫ２０ｍＲＮＡ和ｈＴＥＲＴｍＲＮＡ的表达,两项指标联合检测可作为诊断胃肠道恶性肿瘤患者外周血微转移和判断预后的预测指标。     

Expression of midkine in the early stage of carcinogenesis in human colorectal cancer.

It has been suggested that a heparin-binding growth factor, midkine (MK), plays an important role in carcinogenesis because of its frequent overexpression in various malignant tumours. To clarify whether or not MK contributes to the early stage of carcinogenesis, we examined the status of MK mRNA in 20 adenomas with moderate- and severe-grade dysplasia, 28 carcinomas and 28 corresponding normal tissues, by means of Northern blotting. The MK expression level was significantly more elevated in adenomas than in normal tissues (P < 0.001, unpaired Student's t-test). A difference was also observed between carcinomas and the corresponding normal tissues (P < 0.04, paired Student's t-test). Moreover, MK immunostaining was positive in the adenomas with moderate- and severe-grade dysplasia and in the carcinomas, but not in mild-grade dysplasia or in normal tissues. These findings were in line with those on Western blotting. In three patients with both adenomas with moderate- or severe-grade dysplasia and carcinomas, elevated MK expression was observed in the neoplastic lesions. This is the first report of the association of elevated MK expression with the early stage of carcinogenesis in humans.     

Expression of survivin and its splice variants survivin-2B and survivin-DeltaEx3 in breast cancer

Alternative splicing of survivin mRNA gives rise to multiple isoforms, that is, survivin and 3 splice variants, survivin-2B, survivin-3B and survivin-DeltaEx3. The aim of this study was to compare the expression of survivin, survivin-2B and survivin-DeltaEx3 in normal breast tissue, fibroadenomas, primary breast cancer and axillary nodal metastases. Survivin, survivin-2B and survivin-DeltaEx3 mRNA were measured using semiquantitative RT-PCR. In the primary carcinomas, we related mRNA for each form of survivin to both survivin protein and apoptosis. For each type of breast tissue, survivin was the predominant form detected, being present in 146 out of 156 (93.6%) primary breast carcinomas, 11 out of 11 (100%) axillary nodal metastases, 21 out of 31 (67.7%) fibroadenomas and five out of 22 (22.7%) specimens of normal breast tissue. Levels of the three forms of survivin were significantly higher in the carcinomas compared to normal breast tissue (P < 0.0001). Levels of both survivin-2B and survivin-DeltaEx3 but not survivin were significantly higher in nodal metastases than primary carcinomas. Survivin mRNA levels correlated significantly with survivin protein. Finally, both survivin and survivin-DeltaEx3 but not survivin-2B correlated positively with apoptosis. Although survivin, survivin-2B and survivin-DeltaEx3 were all detected in both malignant and nonmalignant breast tissue, the predominant form was survivin. Our results suggest that the different forms of survivin may have different roles in apoptosis in breast 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. ( Cancer Sci 2009; 100: 1767–1771)     

Expression of the breast cancer metastasis suppressor gene, BRMS1, in human breast carcinoma: lack of correlation with metastasis to axillary lymph nodes.

The BRMS1 (breast cancer metastasis suppressor 1) gene has been found to suppress metastasis in animal models without inhibiting primary tumor growth. The aim of this study was to measure expression of BRMS1 mRNA in a panel of human breast carcinomas and compare its expression with parameters of local dissemination such as tumor size and lymph node metastasis. We also compared expression of BRMS1 mRNA in normal breast tissue, fibroadenomas, primary breast cancers and axillary nodal metastases from primary breast cancers. BRMS1 mRNA was detected in 10/11 (90%) specimens of normal breast tissue, 12/16 (75%) fibroadenomas, 64/82 (78%) primary breast cancer and 11/15 (64%) lymph node metastases (p, NS). In the primary cancer, expression was independent of tumor size, tumor grade, metastasis to axillary nodes and hormone receptor status. Furthermore, similar levels of BRMS1 were found in normal breast tissue, primary breast carcinomas and lymph node metastases from primary breast cancer. Our results do not suggest a role for BRMS1 in suppressing metastasis to local lymph nodes in patients with breast cancer.     

Overexpression of focal adhesion kinase in primary colorectal carcinomas and colorectal liver metastases: immunohistochemistry and real-time PCR analyses.

PURPOSE: Focal adhesion kinase (FAK), a protein tyrosine kinase that functions in signaling events between cells and their extracellular matrix, is overexpressed in a variety of human solid tumors. To determine whether FAK expression is up-regulated in colorectal cancer, we analyzed FAK mRNA and protein levels in primary colorectal tumors and colorectal liver metastases. EXPERIMENTAL DESIGN: p125(FAK) expression in formalin-fixed paraffin-embedded (FFPE) tissue was studied using immunohistochemical assays on 24 matched primary colorectal carcinomas and colorectal liver metastases as well as 18 different colorectal liver metastases using monoclonal anti-FAK 4.47. FAK mRNA expression was quantitated by real-time PCR on 39 matched normal colorectal mucosa and primary colorectal carcinomas as well as on 17 separate liver metastases. RESULTS: Elevated levels of p125(FAK) expression were demonstrated in both primary colorectal tumors and colorectal liver metastases compared with normal colorectal mucosa. Immunohistochemistry experiments demonstrated equivalent FAK expression in matched samples of colorectal primary tumors and liver metastases. Using real-time PCR in 39 matched samples, FAK mRNA copy number was significantly higher in primary colorectal tumors compared with normal colorectal mucosa. FAK expression was analyzed by both real-time PCR and immunohistochemistry in a separate set of colorectal liver metastases. Immunohistochemistry demonstrated high levels of FAK expression in 89% of samples. Furthermore, FAK mRNA copies in these unmatched liver metastases were significantly higher than the primary tumor FAK mRNA copies. CONCLUSION: These experiments have shown that both primary colorectal cancers and colorectal liver metastases express high levels of FAK mRNA and p125(FAK) protein. Furthermore, the majority of colorectal liver metastases demonstrated robust FAK expression equivalent to or greater than that in the primary colorectal tumor.     

Overexpression of Midkine in Pancreatic Duct Adenocarcinomas Induced by N-Nitrosobis(2-oxopropyl)amine in Hamsters and Their Cell Lines

The expression of midkine (MK) was investigated in pancreatic ductal hyperplasias, atypical hyperplasias and adenocarcinomas induced by N -nitrosobis(2-oxopropyl)amine (BOP) in hamsters, and in hamster ductal adenocarcinoma cell lines (HPD-1NR, -2NR and -3NR). MK mRNA was clearly overexpressed in invasive pancreatic duct adenocarcinomas (PCs) and the three cell lines as assessed by northern blot analysis, and MK protein expression increased from ductal hyperplasia through atypical hyperplasias, intraductal carcinomas and invasive PCs by immunohistochemistry. The extent of overexpression of MK mRNA in PCs was almost the same as in hamster whole embryonic tissue. MK is reported to be a retinoid-responsive gene, but MK mRNA expression was not affected by treatment with all- trans retinoic acid (tRA) or N -(4-hydroxyphenyl)retinamide (4- HPR) in HPD-1NR cells. The results thus suggest that MK expression is involved in the development and progression of pancreatic ductal adenocarcinomas induced by BOP in hamsters, with loss of upregulation by retinoic acid.     

Abdominal pain with anorexia in patients with breast carcinoma

Breast cancer is the second commonest primary tumour responsible for gastrointestinal metastases after malignant melanoma. The real incidence of gastrointestinal metastases in breast cancer patients is probably underestimated owing to the non-specific presenting symptoms and death of patients caused by other more obvious metastases. The predominant histological subtype of gastrointestinal metastases of breast cancer is invasive lobular carcinoma and the median interval from diagnosis of primary breast cancer to gastrointestinal metastases is five years. We report two cases of disseminated breast cancer with gastrointestinal involvement with a rather long survival.     

Heterotopic ossification in the skeletal muscle metastases of advanced stomach cancer

The formation of heterotopic bone tissue in carcinomas of the gastrointestinal tract and/or their metastases is extremely rare. In a 74-year-old female with gastric adenocarcinoma, we observed extensive bone formation within metastases in the skeletal muscles. There are only 7 other cases of heterotopic bone formation in gastric cancer reported in the world literature. We suggest that heterotopic ossification in primary cancers and/or their metastases is probably the result of metaplasia of stromal fibroblasts into osteoblasts.     

Rare clonal karyotypic variants in primary cultures of human breast carcinoma cells

Cytogenetic analyses were performed on 40 previously untreated primary human breast carcinomas, four untreated breast metastases, nine human breast fibroadenomas, and ten normal human mammary tissues, all in primary culture. The results revealed predominantly normal diploid cells with abnormal clones in two of 40 primary carcinomas and one of four metastases. 3p deletion [del(3)(p14-21)], similar to that associated with small cell lung cancer, was found in a primary tumor from a patient with bilateral breast cancer. In addition, a clone with t(1;4) was found in another primary breast carcinoma, while a t(1;5) clone was found in a metastatic tumor.     

MT1-MMP and MMP-7 in invasion and metastasis of human cancers

Previous experimental and biochemical studies on matrix metalloproteinases (MMPs) have indicated that MMPs are implicated in cancer invasion and metastases. Studies on the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in various human cancer tissues have further demonstrated that activation of proMMP-2 mediated by a combination of TIMP-2 and MT1-MMP (the proMMP-2/TIMP-2/MT1-MMP system) correlates well with the progression of most of these cancers such as the breast carcinomas, thyroid papillary carcinomas, gastric adenocarcinomas, oral squamous cell carcinomas and gliomas, whereas MMP-7 plays an important role in the metastases of endometrial and gastrointestinal carcinomas. Although MMP-7 is a typical secreted MMP, a member of transmembrane 4 superfamily (TM4SF) captures proMMP-7 on the carcinoma cell membranes through interaction with its propeptide, leading to its pericellular activation. Thus, these results strongly suggest that proteolysis at the cell-extracellular matrix interfaces of cancer cells by the proMMP-2/TIMP-2/MT1-MMP and proMMP-7/TM4SF systems plays crucial roles in the progression of human cancers. In this article, we address the current views on the roles of these MMPs acting on the cell membranes in human cancer invasion and metastases.     

Antisense oligodeoxynucleotide targeted to Midkine, a heparin-binding growth factor, suppresses tumorigenicity of mouse rectal carcinoma cells.

Midkine (MK), a heparin-binding growth factor, is overexpressed in a wide range of human carcinomas and is believed to contribute to tumorigenesis and tumor progression. To develop an antitumor reagent, we designed a phosphorothioate antisense oligodeoxynucleotide molecule based on the secondary structure of MK mRNA. The antisense MK at the dosage of 5 microM suppressed MK production by CMT-93 mouse rectal carcinoma cells after cationic liposome-mediated transfection, to 13% of that in control cultures. The growth of CMT-93 cells and their colony formation in soft agar were inhibited by the addition of the antisense MK, whereas the control reagent, the sense MK, showed no effects. On s.c. injection into nude mice, CMT-93 cells transfected with the antisense MK formed tumors much smaller than those by control cells. Finally, untreated CMT-93 cells were inoculated to nude mice, and 7 days later the antisense MK (50 microM) with atelocollagen was directly injected into the preformed tumor region to evaluate the curative effect; the injection was repeated at the interval of 2 weeks. During the period of 10-41 days after initiation of therapy, the rate of increase of tumor volume treated with the antisense MK was found to be about 4.2-fold lower than that seen after treatment with the sense MK. On this occasion, proliferation of tumor cells as estimated by 5-bromodeoxyuridine incorporation was strongly inhibited, whereas angiogenesis was less affected. These findings strongly suggested the usefulness of MK antisense oligodeoxynucleotide as a new reagent for cancer therapy.