Low TrkA Research Articles

272 Neurotrophin receptors: conflicting roles in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) is the second most frequent form of skin cancer showing a rapidly increasing incidence worldwide. NTs mediate their effects through the common neurotrophin receptor CD271 and the tyrosine kinases family of receptors (Trks). In the skin, CD271 is implicated in the switch between stem and early progenitors, while Trk receptors mediate cell survival. However, the precise role of NT receptors in cSCC is still to be defined. Gene and protein expression analysis revealed that CD271 overexpression, while reducing SCC spheroid growth and increasing apoptosis, down-regulates TrkA and Survivin, both required for keratinocyte stemness. Conversely, CD271 silencing upregulates TrkA, as well as CXCL8, which is associated with cancer invasion. Moreover, CD271 silencing increases viability and aggressiveness of cSCC, as further demonstrated by the cSCC zebrafish model By using the novel flow-based W8TM machine, we show that CD271 overexpression significantly increases mass density, reduces their weight and diameter. RNAseq analysis of CD271 overexpressing spheroids show a major fold-enrichment in cell differentiation and keratinization genes. CD271 overexpressing spheroids display low TrkA and Ki67 expression, while the expression of Keratin 1 is increased. TrkA/Fc chimera decreases cell viability and SCC spheroid invasion area, confirming the dependence of SCC cells from the autocrine NTs. K252, that blocks Trk phosphorylation, reduces spheroid invasion to the same extent as chemotherapy or 5FU, the effect being upregulated by CD271 overexpression. K252 or CD271 overexpression improves the outcome of photodynamic therapy or chemotherapy, as measured by cSCC spheroid size and viability. Our data strongly indicate the opposite functions of NT receptors in cSCC, and their modulation may be a potential treatment strategy in this cancer.

Krüppel-like factor 4 (KLF4) suppresses neuroblastoma cell growth and determines non-tumorigenic lineage differentiation

Neuroblastoma (NB) is an embryonal tumor and possesses a unique propensity to exhibit either a spontaneous regression or an unrestrained growth. However, the underlying mechanism for this paradoxical clinical outcome remains largely unclear. Quantitative RT-PCR analysis on 102 primary NB tumors revealed that lower Krüppel-like factor 4 (KLF4) expression is frequently found in the unfavorable NB (Mann-Whitney test, P=0.027). In particular with the high-risk factors such as age of patient >1 year, MYCN amplification and low TRKA expression, the decreased expression of KLF4 was significantly associated with an unfavorable NB outcome. Despite knockdown of KLF4 alone is not sufficient to increase tumorigenicity of NB cells in vivo, stable expression of KLF4 short hairpin RNA in Be(2)-C cells significantly promoted growth of NB cells and inhibited cell differentiation toward fibromuscular lineage. In concordant with these observations, overexpression of KLF4 in SH-SY-5Y cells profoundly suppressed cell proliferation by direct upregulation of cell-cycle inhibitor protein p21(WAF1/CIP1), and knocking down p21(WAF1/CIP1) could partially rescue the suppressive effect of KLF4. Importantly, KLF4 overexpressing cells have lost their neuroblastic phenotypes, they were epithelial-like, strongly substrate-adherent, expressing smooth muscle marker and became non-tumorigenic, suggesting that KLF4 expression is crucial for lineage determination of NB cells, probably, favoring spontaneous tumor regression. Subsequent global gene expression profiling further revealed that transforming growth factor beta (TGFβ) and cell-cycle pathways are highly dysregulated upon KLF4 overexpression, and myogenic modulators, MEF2A and MYOD1 were found significantly upregulated. Taken together, we have demonstrated that KLF4 contributes to the favorable disease outcome by directly mediating the growth and lineage determination of NB cells.

In vitro and in vivo effects of bicalutamide on the expression of TrkA and P75 neurotrophin receptors in prostate carcinoma

Neurotrophine tyrosine kinase receptors (NTR) are expressed in prostate carcinoma (PCa), and their distribution seems to be related to disease malignancy. In this article we analyzed the expression of NTRK1 (TrkA), NTRK2 (TrkB), NTRK3 (TrkC), and p75NTR in a 102 patient cohort with clinically localized tumors, which had been surgically treated with radical prostatectomy (RP). Among these, 61 patients received RP as sole treatment, and 41 patients received neoadjuvant hormone therapy (NHT) for 120 days with bicalutamide 150 mg/day. In addition, we analyzed the NTR expression in vitro in the androgen receptor positive, androgen-sensitive cell strains derived from CWR22R. We demonstrated that: (i) TrkA and TrkC levels were significantly upmodulated, whereas (ii) p75NTR seemed to be reduced, and (iii) TrkB expression seemed to be not affected by NHT. TrkA were constitutively activated when its levels were very high. In vitro studies showed that the dehydrotestosterone (DHT) was able to maintain low TrkA and TrkC protein levels. Conversely, DHT was able to maintain p75NTR at high levels. Bicalutamide treatment induced TrkA and TrkC and reduced p75NTR expression. Antiproliferative effects of CEP701 were dependent to TrkA levels. A therapeutical effect of CEP701 was seen in all culture conditions, and bicalutamide seemed to sensitize prostate cancer cells to the effects of a pan TrkA inhibitor CEP701, suggesting that a sequential therapy between these drugs could further increase the efficacy of Trk inhibition.

TheMYCNEnigma: Significance ofMYCNExpression in Neuroblastoma

MYCN amplification strongly predicts adverse outcome of neuroblastoma. However, the significance of MYCN expression in the clinical and biological behavior of neuroblastoma has been unclear. To address this question, we first examined the expression of MYCN in combination with TrkA (a favorable prognostic indicator of neuroblastoma) in 91 primary neuroblastoma by quantitative reverse transcription-PCR and investigated the relationship among patient survival, MYCN, and TrkA expressions. Three subsets of neuroblastoma were defined based on MYCN and TrkA expression. Neuroblastoma expressing the highest level of MYCN but little TrkA were MYCN-amplified cases, which had a 5-year survival of 9.3%. Interestingly, MYCN and TrkA expression showed a linear correlation (r = 0.5664, P < 0.00005) in neuroblastoma lacking MYCN amplification, and the 5-year survival of neuroblastoma patients with low MYCN and low TrkA expressions was 63.7%, whereas those with high expression of both had a 5-year survival of 88.1% (P < 0.00005). This nonlinear distribution of disease outcome relative to MYCN expression in neuroblastoma explains why MYCN expression is not predictive of neuroblastoma disease outcome by dichotomous division of the neuroblastoma cohort. However, high-level MYCN expression is associated with favorable outcome in neuroblastoma lacking MYCN amplification. Furthermore, forced expression of MYCN significantly suppresses growth of neuroblastoma cells lacking MYCN amplification by inducing apoptosis and enhancing favorable neuroblastoma gene expression. Collectively, these data suggest that high-level MYCN expression in neuroblastoma lacking MYCN amplification results in a benign phenotype. Thus, the high MYCN expression confers the opposite biological consequence in neuroblastoma, depending on whether or not MYCN is amplified.

Correlation of elevated level of blood midkine with poor prognostic factors of human neuroblastomas.

The heparin-binding growth factor midkine (MK) is the product of a retinoic acid-responsive gene, and is implicated in neuronal survival and differentiation, and carcinogenesis. We previously reported that MK mRNA expression is elevated in neuroblastoma specimens at all stages, whereas pleiotrophin, the other member of the MK family, is expressed at high levels in favourable neuroblastomas. As MK is a secretory protein, it can be detected in the blood. Here, we show a significant correlation of the plasma MK level with prognostic factors of neuroblastomas. The plasma MK level was determined in 220 patients with neuroblastomas, and compared with that in children without malignant tumors (n=17, <500 pg ml−1). The plasma MK level became significantly elevated with advancing stages (stage 1: 445 pg ml−1 (median), n=73; stage 2: 589, n=39; stage 3: 864, n=40; stage 4: 1445, n=56; and stage 4S: 2439, n=12). More importantly, a higher MK level was strongly correlated with poor prognostic factors: over 1 year of age (P=0.0299), MYCN amplification (P<0.0001), low TrkA expression (P=0.0005), nonmass screening, sporadic neuroblastomas (P<0.0001), and diploidy/tetraploidy (P=0.0007). Thus, these results demonstrate that the plasma MK level is a good marker for evaluating the progression of neuroblastomas. Moreover, considering the ability of antisense MK oligodeoxyribonucleotide to suppress tumour growth of colorectal carcinoma cells in nude mice, as recently reported, the present study suggests that MK is a possible candidate molecular target for therapy for neuroblastomas.

Prognostic impact of telomerase activity in patients with neuroblastoma.

Neuroblastoma is one of the most common malignant neoplasms occurring among children. The prognosis for this disease is strongly associated with age, disease stage, histology, and some biologic features. It has been reported that telomerase, a ribonucleoprotein enzyme, which maintains the telomere length in immortal cells, is related to disease stage and other biologic features. The purpose of this study was to evaluate the prognostic value of telomerase activity compared to TrkA expression in 65 patients with neuroblastoma. Telomerase activity and TrkA expression were examined in tissue samples collected between 1980 and 1994 from 65 patients by polymerase chain reaction-based telomerase activity. TrkA expression was examined by immunoblotting using a rabbit anti-gp140 proto-trk polyclonal antibody. Low telomerase activity was found in 22 of 30 (73.3%) patients with Stages 1, 2, or 4S neuroblastomas; 7 of 13 (53.8%) with Stage 3; and 8 of 22 (36.3%) with stage 4; no telomerase activity was detected in 7 of 22 (31.8%) patients with Stage 4 neuroblastoma. The 5-year event-free survival (EFS) rate was 86.5% for patients with low telomerase activity, while it was 53.8% for patients with high telomerase activity. By the combination of telomerase activity and TrkA expression, the 5-year EFS rate was highest among patients with a high TrkA expression and a low or non-existent telomerase activity (91.7%), and it was lowest among patients with a low TrkA expression and a high telomerase activity (29.6%). Thus, it appears that telomerase activity would be a useful prognostic factor for neuroblastoma, especially when used in combination with the TrkA expression.

Gene expression of neuropeptide Y and neuropeptide-Y1 receptor in relation to proto-oncogene tyrosine kinase A, nerve-growth-factor low-affinity-receptor and the transcription factor N-myc in human neuroblastomas

The expression of neuropeptide Y (NPY) and NPY-Y1 receptor (NPY-Y1R) in relation to that of tyrosine kinase A (trkA), nerve-growth-factor low-affinity-receptor (LNGFR) and the transcription factor N-myc was studied in 26 neuroblastomas and one ganglioneuroma by quantitative Northern-blot analysis. A correlation of NPY-Y1R with LNGFR (r = 0.85, p < 0.01) and trkA (r = 0.38, p < 0.05), respectively, could be shown, while no correlation between NPY and its receptor NPY-Y1R was observed. Comparison of a high and a low level NPY expressing group revealed that the high NPY expressing group also had high LNGFR and high trkA levels while the low NPY expressing group had low LNGFR and low trkA levels which were significantly different (NPY: p = 0.035, trkA: p = 0.008, LNGFR p = 0.004). Dividing the tumors in a high and a low N-myc expressing group showed that the low N-myc expressing group contained both high and low trkA expressing tumors while the high N-myc expressing group exclusively were low level trkA expressing tumors. The frequency distribution in both groups concerning trkA expression showed a significant difference (p < 0.01). In conclusion the coexpression of NPY-Y1R and LNGFR or trkA may indicate a similar gene regulation during ontogenesis of the peripheral nervous system.