From Cancer Genomics to Cancer Treatment
Authors: Yusuke Nakamura
Journal: Clinical Cancer Research (2007)

PL07-02  cDNA microarray technologies have enabled us to obtain comprehensive data for gene expression profiles of human cancers. To isolate novel targets for diagnosis (predictive marker for the efficacy of treatment as well as tumor marker) and for treatment of cancer (molecular-targeting drug, cancer vaccine, antibody), we have been comparing expression profiles of cancer cells originated from various organs with their corresponding non-cancerous tissues using a cDNA microarray that consists of more than 30,000 cDNAs/ESTs. These experiments disclosed a number of genes that appeared to be involved in development and/or progression of cancers in those tissues. So far, we have analyzed more than 1,200 cases of clinical cancer samples of the liver, pancreas, stomach, colon, esophagus, bile duct, uterus, lung, ovary, kidney, urinary bladder, testis, prostate, breast, and soft tissues as well as acute and chronic myeloid leukemias. We have selected hundreds of candidate genes by the criteria as follows; (1) genes whose expression was transactivated in a large proportion of cancer tissues in comparison with their corresponding normal tissues and (2) genes whose expression was not observed or hardly detectable in any important vital organs. The further functional analysis identified dozens of genes that are likely to function as oncogenes in various cancers. The suppression of expression of such genes with small-interfering RNAs (siRNAs) induced cell cycle arrest, apoptosis, or suppression of anchoring-dependent cell growth.  For example, through the genome-wide expression profiles of renal cell carcinomas (RCCs), we identified that hypoxia-inducible protein-2 (HIG2) was expressed exclusively in RCCs and fetal kidney. Induction of HIG2 cDNA into mammalian cells led to secretion of the gene product into culture media and resulted in enhancement of cell growth. Small interfering RNA (siRNA) effectively inhibited expression of HIG2 in human RCC cells that endogenously expressed high levels of the protein, and significantly suppressed cell growth. Moreover, addition of polyclonal anti-HIG2 antibody into culture media induced apoptosis in RCC-derived cell lines. ELISA analysis of clinical samples identified secretion of HIG2 protein into the plasma of RCC patients even at an early stage of tumor development, whereas it was detected at significantly lower levels in healthy volunteers or patients with chronic glomerulonephritis. The combined evidence suggests that this molecule represents a promising candidate for development of molecular-targeting therapy and could serve as a prominent diagnostic tumor-marker for patients with renal carcinomas.  We also identified overexpression of Dickkopf-1 (DKK1) in the majority of human cancers in several organs and found a significant association of DKK1 expression with poor prognosis of lung and esophageal cancers. We established an ELISA system to measure serum DKK1 and found that the proportion of the DKK1-positive cases was 185 (69.8%) of 265 lung cancer, 51 (63.0%) of 81 esophageal cancer, 108 (59.3%) of 182 cervical cancer, 15 (55.6%) of 27 prostate cancer, 110 (65.1%) of 169 breast cancer, 89 (53.0%) of 168 hepatocellular carcinoma, 32 (29.9%) of 107 bile duct cancer, 14 (34.1%) of 41 pancreatic cancer, and 39 (38.6%) of 101 gastric cancer patients, while only 10 (4.8%) of 207 healthy volunteers were falsely diagnosed. In addition, the invasive or growth activity of the cancer cells which highly expressed DKK1 were suppressed by addition of anti-DKK1 antibody into their culture media. Administration of anti-DKK1 antibody to tumor-implanted mice significantly suppressed tumor growth without any obvious adverse events. The data suggest usefulness of DKK1 as a serum biomarker in clinic and as a target for the development of therapeutic antibodies.  In addition, we reported previously Frizzled homologue 10 (FZD10), a member of Frizzled family, to be a promising therapeutic target for synovial sarcomas (SS). We established a murine monoclonal antibody (MAb), namely, 92-13 for FZD10 product that had specific binding activity against native FZD10 product expressed in SS cell lines. Subsequent immunohistochemical analyses with the MAb 92-13 confirmed an absence or hardly-detectable level of FZD10 protein in any normal human organs except the placenta. Moreover, we validated the specific binding activity of this MAb in vivo afterinjection of fluorescent-labeled mAb into the mice carrying SS xenografts by the use of the in vivo fluorescent imaging system as well as radioisotopes. Since the MAb 92-13 was effectively internalized into the SS cells after its binding to FZD10 on the cell surface, we attempted radioimmunotherapy using an 90Yttrium-labeled MAb 92-13 (90Y-MAb 92-13) to a mouse SS-xenograft model. Expectedly, a single intravenous injection of 90Y-MAb 92-13 drastically inhibited tumor growth of SS in mice; in four of eleven mice treated, tumors were completely disappeared at 60 days after the treatment. This result indicates that MAb 92-13 could be utilized as the novel treatment modality for SS.  These results indicated that systematic expression analysis should be a very effective approach for identification of molecules that are potential targets for development of novel therapeutic drugs and diagnostic tools.