Overexpression of the Wilms' tumor gene WT1 in de novo lung cancers

Expression of the Wilms' tumor gene WT1 in de novo lung cancer was examined using quantitative real-time RT-PCR and immunohistochemistry. Overexpression of the WT1 gene was detected by RT-PCR in 54/56 (96%) de novo non-small cell lung cancers examined and confirmed by detection of WT1 protein with an anti-WT1 antibody. Overexpression of the WT1 gene was also demonstrated in 5/6 (83%) de novo small cell lung cancers by immunohistochemistry. Furthermore, when the WT1 gene was examined for mutations by direct sequencing of genomic DNA in 7 lung cancers, no mutations were found. These results suggest that the nonmutated, wild-type WT1 gene plays an important role in tumorigenesis of de novo lung cancers and may provide us with the rationale for new therapeutic strategies for lung cancer targeting the WT1 gene and its products.     

Intragenic homozygous deletion of the WT1 gene in Wilms' tumor.

One example of intragenic homozygous deletion of the WT1 gene on chromosome 11p13 was found after screening 42 samples of Wilms' tumor DNA from Japanese patients. After construction of a restriction map for the genomic sequence covering the 3' half of the gene, the deletion was analysed at the nucleotide sequence level. The deletion occurred in the patient's germline on his paternal chromosome, and most of the short arm of his maternal chromosome 11 was subsequently lost in the tumor. The size of the deletion was about 8 kb, removing exons 6 and 7 and resulting in premature termination. The deletion seemed to be created by recombination between short homologous sequences found in an Alu repeat, with a 16-bp duplication left at the junction. This case conforms to a two-hit model for the genesis of a certain group of tumors, and supports the hypothesis that WT1 is one of the recessive oncogenes responsible for Wilms' tumor.     

Overexpression of rhophilin 2 promotes pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and is the seventh leading cause of global cancer deaths. In recent years, targeted therapy has been used for pancreatic cancer; however, the drugs available for use in targeted therapy for pancreatic cancer are still very limited. Hence, identification of novel targeted molecules for PDAC is required. Rhophilin 2 (RHPN2) was proven to be a driver gene in glioblastoma. However, the function of RHPN2 in PDAC remains unknown. In the present study, the function of RHPN2 was investigated. The RHPN2 levels were overexpressed by pcDNA3.1-RHPN2 and downregulated by si-RHPN2. Cell proliferation was assessed using the MTT assay and apoptosis was assessed using flow cytometry. The results revealed that high RHPN2 levels in PDAC tissue were correlated with a low overall survival rate of patients with PDAC. Inhibition of RHPN2 reduced SW1990 and PANC1 proliferation and increased the rate of apoptosis. Network analysis demonstrated that centrosomal protein 78 expression was negatively correlated with RHPN2 expression. In conclusion, the present study demonstrated that RHPN2 may promote PDAC making it a potential candidate for targeted therapy.     

Targeting the tumor microenvironment in pancreatic ductal adenocarcinoma

Introduction: The dismally slow improvement in patient survival over the years for pancreatic cancer patients is mainly due to two factors: the late diagnosis, at which point the disease is spread to distant organs; and the fact that tumor cells are surrounded by a dense, highly immunosuppressive microenvironment. The tumor microenvironment not only shields pancreatic cancer cells from chemotherapy but also leaves it unsusceptible to various immunotherapeutic strategies that have been proven successful in other types of cancer.

Areas covered: This review highlights the main components of the pancreatic tumor microenvironment, how they cross-talk with each other to generate stroma and promote tumor growth. Additionally, we discuss the most promising treatment targets in the microenvironment whose modulation can be robustly tested in combination with standard of care chemotherapy. Currently, active clinical trials for pancreatic cancer involving components of the microenvironment are also listed.

Expert opinion: Although immunotherapeutic approaches involving checkpoint inhibition are being pursued enthusiastically, there is still more work to be done with several other emerging immune targets that could provide therapeutic benefit.     

Absence of mutations in the Wilms' tumor gene WT1 in primary breast cancer

BACKGROUND: It was recently demonstrated that the WT1 gene was overexpressed in primary breast cancer and that the high expression levels of WT1 mRNA significantly correlated with poor prognosis. However, it remained undetermined whether or not the WT1 gene expressed in breast cancer had mutations. METHODS: Breast cancer tissues were obtained from 36 patients with breast cancer. WT1 genomic DNA was PCR-amplified and examined for mutations by direct sequencing. RESULTS: The sequencing analysis showed the absence of mutations through the whole 10 exons of the WT1 gene in the 36 cases of primary breast cancer. Two different single nucleotide polymorphisms (SNP) without an amino acid change (Pro42, C to T in exon 1, and/or Arg300, A to G in exon 7) were detected in the WT1 gene in 31 (86%) of the 36 cases examined. CONCLUSION: The results indicate that the wild-type WT1 gene plays an important role in the tumorigenesis of primary breast cancer.     

Expression of the Wilms' tumor gene (WT1) in human leukemias.

Leukemic cells from seventy patients with various types of human leukemias were examined for expression of the WT1 gene, the Wilms' tumor gene located at chromosome 11p13. WT1 was expressed in 7 of 16 cases of acute lymphoblastic leukemia, 15 of 22 with acute myelogenous leukemia and 8 of 10 in blast crisis of chronic myelogenous leukemia. No detectable WT1 RNA was found in chronic leukemias, including chronic lymphocytic leukemia, plasma cell leukemia, hairy cell leukemia and chronic myelogenous leukemia in chronic phase. The expression pattern of WT1 in these human leukemia samples indicates the involvement of this gene in the early stage of hematological cell differentiation.     

Overexpression of receptor tyrosine kinase Axl promotes tumor cell invasion and survival in pancreatic ductal adenocarcinoma

BACKGROUND:

The receptor tyrosine kinase Axl has been reported to be overexpressed in a variety of human cancers. Although previous studies have identified the role of Axl in the transformation, proliferation, survival, and invasion in cancers, the expression and functions of Axl in pancreatic cancer have not been studied in detail.

METHODS:

The expression of Axl protein in 12 pancreatic cancer cell lines and 54 patient samples of stage II pancreatic ductal adenocarcinoma (PDA) and their paired non‐neoplastic pancreatic tissue samples were examined. Using univariate and multivariate analysis, Axl expression was correlated with survival and other clinicopathologic features. To examine Axl functions in PDA, the effects of Axl knockdown on the invasion ability and radiation‐induced apoptosis in PDA cell lines were measured.

RESULTS:

Axl was overexpressed in 38 of 54 (70%) stage II PDA samples and 9 of 12 (75%) PDA cell lines. Axl overexpression was associated with higher frequencies of distant metastasis and poor overall and recurrence‐free survivals (P = .03 and P = .04, respectively) independent of tumor size and stage or lymph node status in patients with stage II PDA. Knockdown of Axl expression in PDA cells abolished Gas6‐mediated Akt activation, decreased invasion, and increased radiation‐induced PARP cleavage and the percentage of apoptosis.

CONCLUSIONS:

This study showed that Gas6 and Axl are frequently overexpressed in PDA cells and are associated with a poor prognosis in patients with stage II PDA. Axl promotes the invasion and survival of PDA cells. Therefore, targeting the Axl signaling pathway may represent a new approach to the treatment of PDA. Cancer 2011. © 2010 American Cancer Society.     

Wilms' tumor gene WT1-shRNA as a potent apoptosis-inducing agent for solid tumors

Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an important role in leukemogenesis and tumorigenesis. We tested apoptosis-inducing ability of short hairpin RNAs targeting exon 5 (shWTE5), exon10 (shWTE10) and 3'UTR (shWT3U) of the WT1 gene. Among the three WT1-shRNAs, since shWTE5 most effectively induced apoptosis, its ability as an apoptosis-inducing agent was intensively examined. shWTE5 induced mitochondrial damage and resultant apoptosis in five WT1-expressing solid cancer cells originated from gastric (AZ-521), lung (LU99B), ovarian (TYKnuCPr) cancers, fibrosarcoma (HT-1080) and glioblastoma (A172). Moreover, shWTE5 significantly enhanced apoptosis induced by chemotherapeutic agents, doxorubicin (DOX) and etoposide (ETP), or by death ligand TRAIL in all of the four solid tumor cells examined (HT-1080, LU99B, TYK and A172). Transduction of one each of WT1 isoforms with exon 5 [17AA(+)KTS(+) and 17AA(+)KTS(-)] prevented mitochondrial damage induced by ETP or TRAIL and inhibited apoptosis. These results showed that shWTE5 induced apoptosis through the suppression of the WT1 isoform with exon 5. Furthermore, shWTE5 increased expression of proapoptotic Bak and Bax proteins and decreased antiapoptotic Bcl-xL and Bcl-2 proteins in WT1-expressing HT-1080 cells, indicating that WT1 isoforms with exon 5 might play an antiapoptotic role through regulation of Bcl-2 family genes in solid tumor cells. The results presented here demonstrated that WT1-shRNA targeting exon 5 should serve as a potent anti-cancer agent for various types of solid tumors.     

The Wilms' tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis.

The Wilms' tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1(GFP/+)) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (<1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFbetaR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit(+)lin(-)Sca-1(+) (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1(GFP/+) mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.