The role of cooperativity with Src in oncogenic transformation mediated by non-small cell lung cancer-associated EGF receptor mutants.

Non-small cell lung cancer (NSCLC)-associated epidermal growth factor receptor (EGFR) mutants are constitutively active and induce ligand-independent transformation in non-malignant cell lines. We investigated the possibility that the ability of mutant EGFRs to transform cells reflects a constitutive cooperativity with Src using a system in which the overexpression of mutant, but not wild-type, EGFR induced anchorage-independent cell growth. Src was constitutively activated and showed enhanced interaction with mutant EGFRs, suggesting that constitutive EGFR-Src cooperativity may contribute to mutant EGFR-mediated oncogenesis. Indeed, the mutant EGFR-mediated cell transformation was inhibited by Src- as well as EGFR-directed inhibitors. Importantly, a tyrosine to phenylalanine mutation of the major Src phosphorylation site on EGFR, Y845, reduced the constitutive phosphorylation of NSCLC-EGFR mutants, as well as that of STAT3, Akt, Erk and Src, and reduced the mutant EGFR-Src association as well as proliferation, migration and anchorage-independent growth. Reduced anchorage-independent growth and migration were also observed when dominant-negative-Src was expressed in mutant EGFR-expressing cells. Overall, our findings show that mutant EGFR-Src interaction and cooperativity play critical roles in constitutive engagement of the downstream signaling pathways that allow NSCLC-associated EGFR mutants to mediate oncogenesis, and support the rationale to target Src-dependent signaling pathways in mutant EGFR-mediated malignancies.

The non-receptor tyrosine kinase Syk is a target of Cbl-mediated ubiquitylation upon B-cell receptor stimulation.

The negative regulator Cbl functions as a ubiquitin ligase towards activated receptor tyrosine kinases and facilitates their transport to lysosomes. Whether Cbl ubiquitin ligase activity mediates its negative regulatory effects on cytoplasmic tyrosine kinases of the Syk/ZAP-70 family has not been addressed, nor is it known whether these kinases are regulated via ubiquitylation during lymphocyte B-cell receptor engagement. Here we show that B-cell receptor stimulation in Ramos cells induces the ubiquitylation of Syk tyrosine kinase which is inhibited by a dominant-negative mutant of Cbl. Intact tyrosine kinase-binding and RING finger domains of Cbl were found to be essential for Syk ubiquitylation in 293T cells and for in vitro Syk ubiquitylation. These same domains were also essential for Cbl-mediated negative regulation of Syk as measured using an NFAT-luciferase reporter in a lymphoid cell. Association with Cbl did not alter the kinase activity of Syk. Altogether, our results support an essential role for Cbl ubiquitin ligase activity in the negative regulation of Syk, and establish that ubiquitylation provides a mechanism of Cbl-mediated negative regulation of cytoplasmic targets.

Serum alpha-N-acetylgalactosaminidase is associated with diagnosis/prognosis of patients with squamous cell carcinoma of the uterine cervix.

Serum alpha-N-acetylgalactosaminidase (NaGalase) is responsible for the deglycosylation of vitamin D(3)-binding protein (Gc protein). The deglycosylated Gc protein cannot be converted into major macrophage-activating factor (MAF), leading to immunosuppression. NaGalase is universally detected in a variety of cancer patients, but not in healthy individuals (Cancer Res. 56 (1997) 2827-2831). However, the diagnostic/prognostic utility of NaGalase in squamous cell carcinoma (SCC) of the uterine cervix is not known. To address this issue, the serum NaGalase was quantitatively determined in 210 patients with different stages of SCC of the uterine cervix. NaGalase levels were increased with the progression of the cancer. After radiotherapy, the increased levels returned toward or to normal levels in early stages (FIGO stage I-IIB) but not in advanced stages (FIGO stage III-IV). The present study revealed that the amount of NaGalase in the patient's bloodstream reflects the tumor burden and aggressiveness of the disease. We conclude that NaGalase is an independent predictor of diagnosis/prognosis in SCC of the uterine cervix, and therefore suggest that quantitative NaGalase alteration may reflect important differences in the immunological functions of these neoplasms.

Enzyme-linked PNA lectin-binding assay of serum T-antigen in patients with SCC of the uterine cervix.

The expression of Thomsen-Friendenreich antigen (T-Ag) is associated with enhanced metastatic potential, poor prognosis and decreased survival rate in a variety of malignancies, and their detection and quantification can be used in serologic diagnosis. T-antigen expressions were measured by the enzyme-linked lectin assay (ELLA) with peanut agglutinin (PNA) in the sera of patients with squamous cell carcinoma (SCC) of the uterine cervix from 286 patients. This study has a sensitivity of 80%, specificity of 82% and a positive predictive value of 93%. Quantification of the T-antigen may provide useful biochemical indices for clinical assessment of the tumor spread and invasiveness of disease in SCC of the uterine cervix. Moreover, the ELLA assay is cheap, easy to perform and reproducible in the prognosis and diagnosis of SCC of the uterine cervix.

The effect of antigen presenting cells on the cytokine profiles of stable and reactional lepromatous leprosy patients.

In view of varied reports on the Th1/Th2 paradigm in leprosy, we used a novel real time (RT) fluorogenic reverse transcriptase based PCR (RT-PCR) to measure cytokine expression in peripheral blood cells from lepromatous leprosy patients with stable disease and those suffering from erythema nodosum leprosum (ENL/Type II) reactions. To evaluate the role of accessory cells in Th cell differentiation, co-expression of Th cytokines interferon gamma (IFNgamma) and interleukin (IL) 4 and regulatory cytokines IL 10 and IL 12 was compared in antigen stimulated peripheral blood mononuclear cells (PBMC), cultures containing T cells reconstituted with autologous monocytes (MO) and cultures containing T cells reconstituted with autologous dendritic cells (DC). 7/8 stable lepromatous leprosy patients showed co-expression of both IFNgamma and IL 4, suggesting a Th0 or a combination of Th1 + Th2 subsets in PBMC. The RT-PCR demonstrated that stable lepromatous patients and patients in ENL had significantly higher levels of IFNgamma mRNA molecules compared to IL 4. In fact, 5/8 ENL patients had undetectable levels of IL 4 mRNA, with a skewing of the cytokine response towards a Th1-like profile. Consistent with this. IL 12p40 mRNA molecules were significantly higher in the PBMC of ENL patients compared to stable lepromatous patients (P < 0.01). Reconstitution of purified T cells with autologous DC and MO from the stable lepromatous group resulted in down regulation of IL 4 (P < 0.03 for DC and P < 0.02 for MO) and IL 10 (P < 0. 01 for DC and P < 0.02 for MO), and a consequent skewing towards a Th1 profile similar to that seen in ENL patients. The fact that accessory cells could alter the cytokine profile in the reconstituted cultures suggests that they may play a role in determining Th subset differentiation in chronic diseases, and may influence the immunological stability of such diseases.

Dysregulation of IL-4 expression in lepromatous leprosy patients with and without erythema nodosum leprosum.

In order to increase our understanding of the immunological basis of erythema nodosum leprosum (ENL), we studied Th-like cytokine profiles in 130 leprosy patients, employing both the conventional and a novel, real-time, fluorogenic reverse transcriptase-based PCR (RT-PCR). The concomitant expression of both Th-like cytokines, interferon-gamma and IL-4, and the regulatory cytokines, IL-10 and IL-12, was studied in the peripheral blood cells of leprosy patients with and without ENL. In the conventional RT-PCR, varied cytokine profiles were observed in individual patients of all clinical types. Fifty-three percent of lepromatous patients without ENL and 59% of tuberculoid leprosy patients showed co-expression of IFN gamma and IL-4, indicating a non-polarized Th 0 pattern. Of the 36 patients with ENL, 58% demonstrated a polarized Th 1 pattern, with only 30% expressing both cytokines. Semiquantitative RT-PCR indicated a lower expression of IL-4 compared to that of IFN gamma in the lepromatous patients without ENL; the difference was even greater among those with ENL. The sensitive, real-time PCR confirmed the down-regulation of IL-4 and IL-10, with absence of IL-4 in half of the patients, resulting in skewing of the cytokine response toward a Th 1-like profile.