Research Background

The tropomyosin-related kinase (Trk) family of neurotrophin receptors, TrkA, TrkB, and TrkC, and their neurotrophin ligands primarily regulate growth, differentiation, and survival of neurons. Neurotrophins and their corresponding receptors have been shown to induce a variety of pleiotropic responses in malignant cells, including enhanced tumor invasiveness and chemotaxis. TrkA and TrkB were identified as major players in neuroblastoma biology. TrkC is highly expressed in favorable neuroblastomas as well as medulloblastomas. In studies of noncutaneous cancers with a perineural invasive phenotype such as prostate carcinoma, cells acquire an independent autocrine neurotrophin axis and in pancreatic carcinoma, perineural invasion is associated with high nerve growth factor/Trk presence. TrkA, TrkB, and TrkC were also found to be possible factors influencing the behavior of basal cell carcinoma and cutaneous squamous cell carcinoma. Malignant keratinocytes express TrkA, B, and C as a unique survival pathway, and higher levels of expression of TrkA, B, and C may predict perineural invasion in cutaneous squamous cell carcinoma. A recent study shows that TrkB is a potent and specific suppressor of caspase-associated anoikis of non-malignant epithelial cells. TrkB overexpression is sufficient to transform nonmalignant cells into invasive and metastatic cells, by a mechanism that requires activation of the phosphoinositide-3-kinase/protein kinase B pathway. This finding suggests that TrkB might drive one or more facets of tumor formation and metastasis.

In addition, The trkC gene is also known to be a partner of the ETV6-NTRK3 (Tel-TrkC) fusion gene that results from a reciprocal chromosomal translocation, t(12;15)-(p13;q25), in congenital fibrosarcoma, a cellular variant of congenital mesoblastic nephroma, acute myeloid leukemia, and human secretory breast carcinoma. This gene fusion encodes a unique fusion protein containing the helix-loop-helix protein dimerization domain of Tel and the catalytic domain of the protein tyrosine kinase of TrkC, which may contribute to oncogenesis by disregulation of TrkC downstream signal transduction pathways. Accumulating evidence has demonstrated that the rearranged Trk oncogene is often observed in non-neuronal neoplasms such as colon and papillary thyroid cancers. Furthermore, activation of high-affinity Trks is involved in the stimulation of clonal growth of epithelial cancer cells in vitro. Besides activation of TrkC by fusion with multimerizing proteins, the TrkC activation in human tumors seems to be through overexpression of the full-length protein. however, relatively little is known about whether TrkA, B, C and EN activity is also essential to maintain the malignant properties in human tumors. So, We will characterize the molecular actions of EN, TrkA, B, and TrkC and its involvement in human cancer pathogenesis and screen chemical libraries for compounds capable of specifically inhibiting TrkC tyrosine kinase with the intent of developing targeted therapies for these malignancies.

AIM 1. Identify a specific inhibitor of TrkC

TGF-β receptor complex is new addition to the family of human tumor suppressor genes and that inactivation of these receptors is important in multiple human malignancies. Because human cancer cells frequently demonstrate resistance to the normal growth inhibitory effects of TGF-β, it has been proposed that the development of such TGF-β resistance represents a significant step during carcinogenesis. We found that ETV6-NTRK3 chimeric tyrosine kinase (EN) suppresses TGF-β signaling by directly binding to the type II TGF-β receptor, thereby preventing it from interacting with the type I TGF-β receptor. This activity requires a functional EN protein tyrosine kinase, and TβRII appears to be a direct target of EN. This finding suggests that TrkC oncogenic tyrosine kinase can block TGF-tumor suppressor activity.
In our preliminary study, loss of TrkC expression by siTrkC restored responsiveness to TGF-β and markedly reduced lung metastases. Given our initial results, it is importance to investigate new target molecules specific for the TrkC receptor using TGF-β responsive elements.

AIM 2. The role of the EN and TrkC on post-translational regulation of the TβRII gene.
Several lines of evidence have suggested that the transcriptional repression of TβRII gene may be important in modulationg TGF-β responsiveness. Our research suggested that TβRII, a putative tumor suppressor gene, is a target of the EN and TrkC. NIH3T3 cell lines stably transfected with the TrkC and EN markedly reduced mRNA level and protein of TβRII but Skp2 level markedly increased and Skp2 involved in TβRII ubiqitination. Also, 4T1 siTrkC stable cells restore TGF-β sensitivity. We hypothesized that Skp2 stimulated production by EN and TrkC leads to degrade TβRII.

AIM 3. Fuctional roles of EN, TrkA, TrkB, and TrkC in epithelial–mesenchymal transition (EMT)
NF-kB/Rel transcription factors are central to controlling programmed cell death (PCD). Activation of NF-kB blocks PCD induced by numerous triggers, including ligand engagement of TNF-α family receptors. The protective activity of NF-kB is also crucial for oncogenesis and cancer chemoresistance. Several lines of evidence have suggested that Twist-1 is an evolutionarily conserved target of NF-kB, blocks PCD induced by chemotherapeutic drugs and TNF-α in NF-kB-deficient cells, and is essential to counter this PCD in cancer cells. Also, Up-regualtion of Twist, a highly conserved basic helix-loop-helix transcription factor, in prostatic cancer cells can promote epithelial to mesenchymal transition through down-regulation of E-cadherin. Another reported Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel. In our preliminary study, TrkC over-expressed 67NR stable cells increased Twist mRNA levels. Also, Twsit levels decreased in 4T1 siTrkC stable cells and suppression of TrkC expression in highly metastatic mammary carcinoma cells specifically inhibits their growth and their ability to metastasize from the mammary gland to the lung. Ectopic expression of TrkC suppressed anoikis of non-malignant mammary epithelial cells, which correlated with activation of the phosphatidylinositol-3-OH kinase/protein kinase B pathway. So, We will investigate how TrkC involve epithelial–mesenchymal transition (EMT).

AIM 4. Molecular mechanism of TrkA, TrkB, and TrkC in inhibition of Activin, TGF-β, and BMP signaling.
We reported ETV6-NTRK3 chimeric tyrosine kinase suppresses TGF-β signaling by inactivating the TGF-β type II receptor. Also, our preliminary date suggest that TrkA, TrkB, and TrkC interact with both of activin type II and BMP type II receptor and inhibits activin and BMP signaling. Our findings will be provide evidence for the mechanism by which oncogenic tyrosine kinases can block activin and BMP-2 mediated tumor suppressor activity.