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Type III receptor tyrosine kinases (RTKs), FLT3 and c-Kit play important roles in a variety of cellular processes. A number of SH2-domain containing proteins interact with FLT3 and c-Kit and regulate downstream signaling. The SH2-domain containing non-receptor protein tyrosine kinase CSK is mainly studied in the context of regulating Src family kinases. Here we present an additional role of this kinase in RTK signaling. We show that CSK interacts with FLT3 and c-Kit in a phosphorylation dependent manner. This interaction is facilitated through the SH2-domain of CSK. Under basal conditions CSK is mainly localized throughout the cytosolic compartment but upon ligand stimulation it is recruited to the inner side of cell membrane. CSK association did not alter receptor ubiquitination or phosphorylation but disrupted downstream signaling. Selective depletion of CSK using siRNA, or inhibition with CSK inhibitor, led to increased phosphorylation of Akt and Erk, but not p38, upon FLT3 ligand (FL) stimulation. Stem cell factor (SCF)-mediated Akt and Erk activation was also elevated by CSK inhibition. However, siRNA mediated CSK knockdown increased SCF stimulated Akt phosphorylation but decreased Erk phosphorylation. CSK depletion also significantly increased both FL- and SCF-induced SHC, Gab2 and SHP2 phosphorylation. Furthermore, CSK depletion contributed to oncogenic FLT3- and c-Kit-mediated cell proliferation, but not to cell survival. Thus, the results indicate that CSK association with type III RTKs, FLT3 and c-Kit can have differential impact on receptor downstream signaling.

Background: The TP73 and TP63 genes are members of the p53 tumor suppressor family and are expressed in different N -terminal isoforms either with proapoptotic (transactivation domain, TA) and antiapoptotic (N-terminally truncated, ΔN) function. Unlike p53, the role of p73 and p63 in tumor is controversial. It has been recently hypothesized that altered ΔN:TA expression ratio, rather than single isoform overexpression, plays a role in the pathogenesis of many diseases, including lung cancer. Methods: Isoform-specific, real-time polymerase chain reaction and immunohistochemistry analysis on matched cancer and corresponding normal tissues from surgically resected non-small cell lung cancers (NSCLCs) have been performed aiming to explore the expression levels of each p63 and p73 N -terminal isoforms and their ΔN:TA expression ratio. Results: For both p63 and p73, a N -terminal isoform-specific modulation that alter ΔN:TA isoform balance was identified. In particular, ΔNp63 isoform was significantly up-modulated, whereas TAp63 was slightly down-modulated in NSCLC specimens. Likewise, Δ2p73 and Δ2/3p73 were up-modulated, whereas ΔNp73 and ΔN′p73 isoforms were down-modulated. Moreover, a higher TAp63 and ΔN′p73 transcripts expression, detected in the normal tissue surrounding the tumors, correlates with poor patient outcome, representing independent prognostic factors for overall survival (ΔN′p73: p = 0.049, hazard ratio = 3.091, 95% confidence interval = 1.005–9.524 and TAp63: p = 0.001, hazard ratio = 8.091, 95% confidence interval = 2.254–29.05). Conclusion: Our findings suggest that p63 and p73 altered ΔN:TA expression ratio occurs in NSCLC likely contributing to the molecular pathogenesis of this tumor.

Introduction K-Ras mutations, occurring prevalently within codons 12/13, are detected in different cancer types including colorectal carcinoma (CRC) and might be responsible for resistance to anti-EGFR monoclonal antibodies (Cetuximab®) treatment. A sensitive method for K-Ras mutations detection is therefore crucial issue for choosing the most appropriate therapy. Currently, Sanger sequencing (SS) is accepted as gold standard method for mutational analysis but it is time-consuming and cost-ineffective. In addition, since in tumor analysis mutated clone may be present at different percentage, the SS sensitivity limit of 15-20% is crucial. Pyrosequencing (PS) has 5% detection limit, but still cost-ineffective, not largely available and shown a grey area (5-10%) in which genotype remains undeterminable. Therefore, a more sensitive method for detecting k-Ras mutations represents still a challenge. In this study we developed a novel, sensitive, friendly to use and very cheap assay for K-Ras mutations in CRC, based on PNA-directed PCR-clamping. Methods PNAs are synthetic molecules acting as potent DNA mimic in terms of sequence specific hybridization. As consequence, PNA/DNA heteroduplex is thermodynamically more stable than DNA/DNA counterpart but PNA cannot be extended by polymerase to function as a primer. Based on this, we designed a PNA molecule and one of the PCR primers perfectly matching the WT K-Ras sequence (encompassing codons 12/13), thus leading to a direct competition towards complementary template genomic DNA. In WT samples, perfect matching occurs because PNA-template hybridization is favoured and DNA amplification suppressed. By contrast, in case of mutated K-ras a single base pair mismatch will strongly impair the PNA-template stability favouring PCR amplification. Sixty formalin-fixed paraffin-embedded specimens from metastatic CRC were analyzed for K-Ras mutations by using both SS and PS and results and compared with PNA-directed PCR clamping method. Sensitivity was tested performing serial dilutions with plasmids carrying either wt or mutated sequences. Results PNA-methods displayed higher sensitivity compared to both SS and PS, allowing mutations detection up to 98.5%. The results showed a concordance of 63.3% among the three methods, while the concordance between PS and PNA-method resulted significantly increased. 47 samples were found to be mutated by PNA-PCR clamping, while 30 and 26 by PS and SS, respectively. Major discrepancy (13 out of 14) regards the detection of mutations by PNA-methods, otherwise unidentified either by SS or PS. Four samples, previously fallen into grey area, were classified as mutated by PNA-method. PNA-method failed to detect mutations just in 1 out of 30 cases, thus displaying a specificity of 96.66%. Correlation between clinical outcome after Cetuximab treatment and genotyping by PNA-method, which will confirm the clinical usefulness of the new method, is ongoing. Conclusion We developed an innovative method, able to detect K-Ras mutations in tumor paraffin-embedded specimens with very high sensitivity (98.5%). This is a cheap, easy to apply, quick and sensitive technique that could represent a novel tool, useful in clinical practice to drive therapeutic decisions.