Chikako Fukukawa

Therapeutic potential of antibodies against FZD10, a cell-surface protein, for synovial sarcomas

Genome-wide expression profiling revealed overexpression of the gene encoding frizzled homologue 10 (FZD10), a cell-surface receptor for molecules in the Wnt pathway, as a potential contributor to synovial sarcomas (SS). Northern blotting and immunohistochemical staining confirmed that expression levels of FZD10 were very high in nearly all SS tumors and cell lines examined but absent in most normal organs or in some cancers arising in other tissues. Treatment of human SS cells with small-interfering RNA (siRNA) to FZD10 decreased the amount of its product and suppressed growth of SS cells. Moreover, a polyclonal antibody specifically recognizing the extracellular domain (ECD) of FZD10 was markedly effective in mediating ADCC against FZD10-overexpressing synovial sarcoma cells in vitro. Injection of the antibody into SS xenografts in nude mice attenuated tumor growth, and TUNEL assays revealed clusters of apoptotic cells in antibody-treated xenografts. Taken together, these findings suggest that a humanized antibody against FZD10 might be a promising treatment for patients with tumors that overexpress FZD10; minimal or no adverse reactions would be expected because FZD10 protein is not abundant in vital organs.

Radioimmunotherapy of human synovial sarcoma using a monoclonal antibody against FZD10

We previously reported Frizzled homolog 10 (FZD10), a member of the Frizzled family, to be a promising therapeutic target for synovial sarcomas. In this report, we established a murine monoclonal antibody (MAb), namely, MAb 92‐13 that had specific binding activity against native FZD10 product expressed in synovial sarcoma 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. We confirmed the specific binding activity of this MAb in vivo after injection of fluorescent‐labeled MAb i.p. or i.v. into the mice carrying synovial sarcoma xenografts by the use of the in vivo fluorescent imaging system as well as radioisotopes. Moreover, MAb 92‐13 was effectively internalized into the synovial sarcoma cells after its binding to FZD10 on the cell surface. A single i.v. injection of the Yttrium‐90 (90Y)‐MAb 92‐13 drastically suppressed tumor growth of synovial sarcoma in mice without any severe toxicity. Median time to tumor progression was 58 days for mice treated with 90Y‐MAb 92‐13 and 9 days for mice treated with non‐labeled antibody control or untreated mice (difference = 49 days; P = 7 × 10−5). This result indicates that MAb 92‐13 could be utilized as the novel treatment modality for synovial sarcoma and other FZD10‐positive tumors. (Cancer Sci 2008; 99: 432–440)

Activation of the non-canonical Dvl-Rac1-JNK pathway by Frizzled homologue 10 in human synovial sarcoma

We previously reported that Frizzled homologue 10 (FZD10), a member of the Wnt signal receptor family, was highly and specifically upregulated in synovial sarcoma and played critical roles in its cell survival and growth. We here report a possible molecular mechanism of the FZD10 signaling in synovial sarcoma cells. We found a significant enhancement of phosphorylation of the Dishevelled (Dvl)2/Dvl3 complex as well as activation of the Rac1–JNK cascade in synovial sarcoma cells in which FZD10 was overexpressed. Activation of the FZD10–Dvls–Rac1 pathway induced lamellipodia formation and enhanced anchorage-independent cell growth cells. FZD10 overexpression also caused the destruction of the actin cytoskeleton structure, probably through the downregulation of the RhoA activity. Our results have strongly implied that FZD10 transactivation causes the activation of the non-canonical Dvl–Rac1–JNK pathway and plays critical roles in the development/progression of synovial sarcomas.

Critical roles of LGN/GPSM2 phosphorylation by PBK/TOPK in cell division of breast cancer cells.

To investigate the molecular mechanism of mammary carcinogenesis and identify novel molecular targets for breast cancer therapy, we analyzed genome‐wide gene expression profiles of 81 clinical breast cancer samples. Here, we report the critical role of LGN/GPSM2 (Leu‐Gly‐Asn repeat‐enriched protein/G‐protein signaling modulator 2) in the growth of breast cancer cells. Semiquantitative RT‐PCR and Northern blot analyses confirmed upregulation of LGN/GPSM2 in a large proportion of breast cancers. Immunocytochemical staining identified LGN/GPSM2 at the spindle in cells at metaphase, and at midzone and midbody in cytokinetic cells. Western blot analysis indicated the highest expression and the phosphorylated form of LGN/GPSM2 protein in G2/M phase. Treatment with small‐interfering RNAs (siRNAs) targeting LGN/GPSM2 caused incompletion of cell division and resulted in significant growth suppression of breast cancer cells. We found that the 450th threonine (Thr450) of LGN/GPSM2 was phosphorylated by the serine/threonine kinase PBK/TOPK during mitosis. Overexpression of LGN/GPSM2‐T450A in which Thr450 was substituted with alanine induced growth suppression and aberrant chromosomal segregation. These findings imply an important role of LGN/GPSM2 in cell division of breast cancer cells and suggest that the PBK/TOPK‐LGN/GPSM2 pathway might be a promising molecular target for treatment of breast cancer.

Involvement of G-patch domain containing 2 overexpression in breast carcinogenesis

Through analysis of the detailed genome‐wide gene expression profiles of 81 breast tumors, we identified a novel gene, G‐patch domain containing 2 (GPATCH2), that was overexpressed in the great majority of breast cancer cases. Treatment of breast cancer cells MCF‐7 and T47D with siRNA against GPATCH2 effectively suppressed its expression, and resulted in the growth suppression of cancer cells, suggesting its essential role in breast cancer cell growth. We found an interaction of GPATCH2 protein with hPrp43, an RNA‐dependent ATPase. Their interaction could significantly enhance the ATPase activity of hPrp43 and induce a growth‐promoting effect on mammalian cells. Because northern blot analyses of normal human organs implied GPATCH2 to be a novel cancer/testis antigen, targeting GPATCH2 or inhibition of the interaction between GPATCH2 and hPrp43 could be a promising novel therapeutic strategy of breast cancer. (Cancer Sci 2009)

Inverse correlation of the up‐regulation of FZD10 expression and the activation of β‐catenin in synchronous colorectal tumors

We investigated the immunohistochemical expression patterns of Frizzled homolog 10 (FZD10), a cell‐surface receptor for molecules in the Wnt pathway, in tissue samples derived from 104 patients with colorectal cancers (CRCs). There was no immunoreactivity for FZD10 in normal colonic mucosa, and only tumor cells in polyps and CRC tissues showed spotted immunostaining patterns in apical sides of the cytoplasm. In metastatic liver lesions, tumor cells showed cytoplasmic immunostaining similar to primary lesions, whereas normal liver parenchyma showed almost no immunostaining. Frequencies of FZD10‐immunopositive cells in tumor tissues were significantly higher in CRCs than those in polyps (3.3 ± 10.3%vs 20.5 ± 31.7%, P = 0.0016), and almost equivalent with those in metastatic liver lesions (33.2 ± 39.7%vs 26.4 ± 33.4%, P = 0.133). Analyses of paired samples (polyps and CRCs, or CRCs and metastatic liver lesions from the same patient) suggested that a subset of CRCs possessed intrinsic genetic mechanisms causing the evolution of FZD10‐positive clones during tumor progression, making FZD10 a promising candidate for molecular imaging and a target for therapy. To our surprise, cancer cells immunopositive for FZD10 showed significantly less nuclear accumulation of β‐catenin, compared to FZD10‐immunonegative cancer cells, and there was a strong inverse correlation between nuclear immunostaining scores for β‐catenin expression and expression patterns of FZD10 (P = 0.0002), suggesting that FZD10 has a distinct role from other FZDs in canonical Wnt signal transduction. (Cancer Sci 2009; 100: 405–412)

Molecular target therapy for synovial sarcoma

Although the specific chromosomal translocation and fusion gene SYT-SSX in synovial sarcoma (SS) has been identified, the molecular mechanism of its tumorigenesis is largely unknown. Recent gene-expression profiles of soft-tissue tumors using cDNA microarray demonstrated that SS has the distinct gene-expression pattern from other sarcomas and has a similar pattern to that of malignant peripheral nerve sheath tumors, indicating that the origin of SS is likely to be the neural crest cells. Through this analysis, several genes were found to be specifically upregulated in SS and considered to play an important role in the proliferation of SS cells. Among them, Frizzled homolog 10 was identified as a good candidate molecule for the development of novel therapies to treat SS patients.