EGFR signaling leads to downregulation of PTP-LAR via TACE-mediated proteolytic processing.

Proteolytic processing and ectodomain shedding have been described for a broad spectrum of transmembrane proteins under both normal and pathophysiological conditions and has been suggested as one mechanism to regulate a protein's function. It has also been documented for the receptor-like protein tyrosine phosphatase PTP-LAR, induced by treating cells with the tumor promoter TPA or the calcium ionophor A23187. Here we identified the epidermal growth factor receptor (EGFR) as both an association partner of PTP-LAR, that mediates phosphorylation of the latter, as well as an inducer of LAR-cleavage. Both overexpression of this kinase and stimulation of endogenous EGFR in various tumor cell lines were shown to induce proteolytic processing of the catalytic LAR-P-subunit. In contrast to TPA-induced shedding of PTP-LAR, EGFR-mediated cleavage did not require PKC-activity. For both stimuli, however, processing of the P-subunit turned out to be dependent on the activation of the MAP kinases ERK1 and ERK2, and was completely abrogated upon pre-treating cells with Batimastat, indicating the involvement of a metalloproteinase in this pathway. Being strongly impaired in fibroblasts derived from ADAM-17/TACE-knockout-mice or tumor cells that express a dominant negative mutant of ADAM-17/TACE, cleavage of PTP-LAR is suggested to be mediated by this metalloproteinase. Paralleled by rapid reduction of cell surface-localized LAR-E-subunit, EGFR-induced cleavage could be shown to lead to degradation of the catalytic LAR-P-subunit, thereby resulting in a significantly reduced overall cellular phosphatase activity of PTP-LAR. These results for the first time identify a protein tyrosine phosphatase as a potential substrate of TACE and describe proteolytic processing of PTP-LAR as a means of regulating phosphatase activity downstream and thus under the control of EGFR-mediated signaling pathways.

PTP-PEST phosphatase variations in human cancer.

Signal transduction via tyrosine phosphorylation, normally fine-tuned by the concerted action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is a key mechanism in tumorigenesis. PTP-PEST, a ubiquitously expressed cytoplasmic tyrosine phosphatase, is thought to play an important role in cell adhesion and motility, and may be involved in metastasis. A search for sequence variations within the gene PTPN12 (alias PTP-PEST) was performed in breast cancer cell lines, leading to the identification of three amino acid substitutions at positions 322, 573, and 709. These alterations were also found in squamous cell carcinoma cell lines and could be verified in primary human breast and kidney tumor samples. Analysis of peripheral blood samples confirmed the germline origin of these alterations. Furthermore, functional characterization of the Ile322 and Ala573 PTP-PEST mutants revealed an enhancement of in vitro phosphatase activity, whereas the Lys709 variant showed reduced catalytic activity. These data demonstrate the existence of PTP-PEST variants that might be meaningful for human cancer and underscore the need for further characterizing PTP-PEST and its signaling pathways in context of this disease.

Clinical Translation and Validation of a Predictive Biomarker for Patritumab, an Anti-human Epidermal Growth Factor Receptor 3 (HER3) Monoclonal Antibody, in Patients With Advanced Non-small Cell Lung Cancer.

BACKGROUND: During early clinical development, prospective identification of a predictive biomarker and validation of an assay method may not always be feasible. Dichotomizing a continuous biomarker measure to classify responders also leads to challenges. We present a case study of a prospective-retrospective approach for a continuous biomarker identified after patient enrollment but defined prospectively before the unblinding of data. An analysis of the strengths and weaknesses of this approach and the challenges encountered in its practical application are also provided. METHODS: HERALD (NCT02134015) was a double-blind, phase 2 study in patients with non-small cell lung cancer (NSCLC) randomized to erlotinib with placebo or with high or low doses of patritumab, a monoclonal antibody targeted against human epidermal growth factor receptor 3 (HER3). While the primary objective was to assess safety and progression-free survival (PFS), a secondary objective was to determine a single predictive biomarker hypothesis to identify subjects most likely to benefit from the addition of patritumab. Although not identified as the primary biomarker in the study protocol, on the basis of preclinical results from 2 independent laboratories, expression levels of the HER3 ligand heregulin (HRG) were prospectively declared the predictive biomarker before data unblinding but after subject enrollment. An assay to measure HRG mRNA was developed and validated. Other biomarkers, such as epidermal growth factor receptor (EGFR) mutation status, were also evaluated in an exploratory fashion. The cutoff value for high vs. low HRG mRNA levels was set at the median delta threshold cycle. A maximum likelihood analysis was performed to evaluate the provisional cutoff. The relationship of HRG values to PFS hazard ratios (HRs) was assessed as a measure of internal validation. Additional NSCLC samples were analyzed to characterize HRG mRNA distribution. RESULTS: The subgroup of patients with high HRG mRNA levels ("HRG-high") demonstrated clinical benefit from patritumab treatment with HRs of 0.37 (P = 0.0283) and 0.29 (P = 0.0027) in the high- and low-dose patritumab arms, respectively. However, only 102 of the 215 randomized patients (47.4%) had sufficient tumor samples for HRG mRNA measurement. Maximum likelihood analysis showed that the provisional cutoff was within the optimal range. In the placebo arm, the HRG-high subgroup demonstrated worse prognosis compared with HRG-low. A continuous relationship was observed between increased HRG mRNA levels and lower HR. Additional NSCLC samples (N = 300) demonstrated a similar unimodal distribution to that observed in this study, suggesting that the defined cutoff may be applicable to future NSCLC studies. CONCLUSIONS: The prospective-retrospective approach was successful in clinically validating a probable predictive biomarker. Post hoc in vitro studies and statistical analyses permitted further testing of the underlying assumptions. However, limitations of this analysis include the incomplete collection of adequate tumor tissue and a lack of stratification. In a phase 3 study, findings are being confirmed, and the HRG cutoff value is being further refined. CLINICALTRIALSGOV NUMBER: NCT02134015.

Fibroblast growth factor receptor 4 regulates proliferation, anti-apoptosis and alpha-fetoprotein secretion during hepatocellular carcinoma progression and represents a potential target for therapeutic intervention.

BACKGROUND/AIMS: FGFR4, a member of the fibroblast growth factor receptor family, has been recently associated with progression of melanoma, breast and head and neck carcinoma. Given its uniquely high expression in the liver, we investigated its contributory role to hepatocellular carcinoma (HCC). METHODS: We performed a comprehensive sequencing of full-length FGFR4 transcript in 57 tumor/normal HCC tissue pairs, and quantified their mRNA expressions. Notable mutations and expression patterns were correlated with patient data. Clinically significant trends were examined in in vitro models. RESULTS: We found eight genetic alterations including two highly frequent polymorphisms (V10I and G338R). Secretion of alpha-fetoprotein (AFP), a HCC biomarker, was increased among patients bearing homozygous Arg388 alleles. One-third of these patients exhibited increased FGFR4 mRNA expression in the matched tumor/normal tissue. Subsequent in vitro perturbation of FGFR4 signaling through both FGF19-stimulation and FGFR4 silencing confirmed a mechanistic link between FGFR4 activities and tumor aggressiveness. More importantly, inhibition of FGFR activity with PD173074 exquisitely blocked HuH7 (high FGFR4 expression) proliferation as compared to control cell lines. CONCLUSIONS: FGFR4 contributes significantly to HCC progression by modulating AFP secretion, proliferation and anti-apoptosis. Its frequent overexpression in patients renders its inhibition a novel and much needed pharmacological approach against HCC.

Genetic alterations in the tyrosine kinase transcriptome of human cancer cell lines.

Protein tyrosine kinases (PTKs) play a critical role in the manifestation of cancer cell properties, and respective signaling mechanisms have been studied extensively on immortalized tumor cells. To characterize and analyze commonly used cancer cell lines with regard to variations in the primary structure of all expressed PTKs, we conducted a cDNA-based sequence analysis of the entire tyrosine kinase transcriptome of 254 established tumor cell lines. The profiles of cell line intrinsic PTK transcript alterations and the evaluation of 155 identified polymorphisms and 234 somatic mutations are made available in a database designated "Tykiva" (tyrosine kinome variant). Tissue distribution analysis and/or the localization within defined protein domains indicate functional relevance of several genetic alterations. The cysteine replacement of the highly conserved Y367 residue in fibroblast growth factor receptor 4 or the Q26X nonsense mutation in the tumor-suppressor kinase CSK are examples, and may contribute to cell line-specific signaling characteristics and tumor progression. Moreover, known variants, such as epidermal growth factor receptor G719S, that were shown to mediate anticancer drug sensitivity could be detected in other than the previously reported tumor types. Our data therefore provide extensive system information for the design and interpretation of cell line-based cancer research, and may stimulate further investigations into broader clinical applications of current cancer therapeutics.