Increased TGF-α as a mechanism of acquired resistance to the anti-EGFR inhibitor cetuximab through EGFR-MET interaction and activation of MET signaling in colon cancer cells.

PURPOSE: Although cetuximab, an anti-EGF receptor (EGFR) monoclonal antibody, is an effective treatment for patients with KRAS wild-type metastatic colorectal cancer (mCRC), its clinical use is limited by onset of resistance. EXPERIMENTAL DESIGN: We characterized two colorectal cancer models to study the mechanisms of acquired resistance to cetuximab. RESULTS: Following chronic treatment of nude mice bearing cetuximab-sensitive human GEO colon xenografts, cetuximab-resistant GEO (GEO-CR) cells were obtained. In GEO-CR cells, proliferation and survival signals were constitutively active despite EGFR inhibition by cetuximab treatment. Whole gene expression profiling identified a series of genes involved in the hepatocyte growth factor (HGF)-MET-dependent pathways, which were upregulated in GEO-CR cells. Furthermore, activated, phosphorylated MET was detected in GEO-CR cells. A second colorectal cancer cell line with acquired resistance to cetuximab was obtained (SW48-CR). Inhibition of MET expression by siRNA restored cetuximab sensitivity in GEO-CR and SW48-CR cells, whereas exogenous activation of MET by HGF stimulation in cetuximab-sensitive GEO and SW48 cells induced resistance to cetuximab. Treatment of GEO-CR and SW48-CR cells with PHA665752, a selective MET inhibitor, inhibited cell growth, proliferation, and survival signals and impaired cancer cell migration. Overexpression of TGF-α, a specific EGFR ligand, was involved in the acquisition of cetuximab resistance in GEO-CR and SW48-CR cells. In fact, TGF-α overexpression induced the EGFR-MET interaction, with subsequent MET phosphorylation and activation of MET downstream effectors in GEO-CR and SW48-CR cells. CONCLUSIONS: These results suggest that overexpression of TGF-α through induction of EGFR-MET interaction contributes to cetuximab resistance in colorectal cancer cells. The combined inhibition of EGFR and MET receptor could represent a strategy for preventing and/or overcoming cetuximab resistance in patients with colorectal cancer.

Antitumor activity of pimasertib, a selective MEK 1/2 inhibitor, in combination with PI3K/mTOR inhibitors or with multi-targeted kinase inhibitors in pimasertib-resistant human lung and colorectal cancer cells.

The RAS/RAF/MEK/MAPK and the PTEN/PI3K/AKT/mTOR pathways are key regulators of proliferation and survival in human cancer cells. Selective inhibitors of different transducer molecules in these pathways have been developed as molecular targeted anti-cancer therapies. The in vitro and in vivo anti-tumor activity of pimasertib, a selective MEK 1/2 inhibitor, alone or in combination with a PI3K inhibitor (PI3Ki), a mTOR inhibitor (everolimus), or with multi-targeted kinase inhibitors (sorafenib and regorafenib), that block also BRAF and CRAF, were tested in a panel of eight human lung and colon cancer cell lines. Following pimasertib treatment, cancer cell lines were classified as pimasertib-sensitive (IC50 for cell growth inhibition of 0.001 µM) or pimasertib-resistant. Evaluation of basal gene expression profiles by microarrays identified several genes that were up-regulated in pimasertib-resistant cancer cells and that were involved in both RAS/RAF/MEK/MAPK and PTEN/PI3K/AKT/mTOR pathways. Therefore, a series of combination experiments with pimasertib and either PI3Ki, everolimus, sorafenib or regorafenib were conducted, demonstrating a synergistic effect in cell growth inhibition and induction of apoptosis with sustained blockade in MAPK- and AKT-dependent signaling pathways in pimasertib-resistant human colon carcinoma (HCT15) and lung adenocarcinoma (H1975) cells. Finally, in nude mice bearing established HCT15 and H1975 subcutaneous tumor xenografts, the combined treatment with pimasertib and BEZ235 (a dual PI3K/mTOR inhibitor) or with sorafenib caused significant tumor growth delays and increase in mice survival as compared to single agent treatment. These results suggest that dual blockade of MAPK and PI3K pathways could overcome intrinsic resistance to MEK inhibition.

Synergistic effects of metformin treatment in combination with gefitinib, a selective EGFR tyrosine kinase inhibitor, in LKB1 wild-type NSCLC cell lines.

PURPOSE: EGF receptor (EGFR) tyrosine kinase inhibitors (TKI) have been found to be effective against lung cancer, but clinical resistance to these agents has developed as their usage has increased. Metformin is a widely used antidiabetic drug and also displays significant growth-inhibitory and proapoptotic effects in several cancer models, alone or in combination with chemotherapeutic drugs. EXPERIMENTAL DESIGN: The effects of gefitinib, a selective EGFR-TKI, and metformin on a panel of non-small cell lung cancer (NSCLC) cell lines were assessed by using MTT, bromide assay, flow cytometry, anchorage-independent growth, coimmunoprecipitation, and Western blot analysis. RESULTS: The combination of metformin with gefitinib induced a strong antiproliferative and proapoptotic effect in NSCLC cell lines that harbored wild-type LKB1 gene. Treatment with metformin as single agent, however, induced an activation and phosphorylation of mitogen-activated protein kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. The inhibition of EGFR phosphorylation and of downstream signaling by adding gefitinib to metformin treatment abrogated this phenomenon and induced a strong apoptotic effect in vitro and in vivo. CONCLUSIONS: Metformin and gefitinib are synergistic in LKB1 wild-type NSCLC cells. However, further studies are required to investigate better the effect of metformin action on the RAS/RAF/MAPK pathway and the best context in which to use metformin in combination with molecular targeted agents.

Rarity of human T helper 17 cells is due to retinoic acid orphan receptor-dependent mechanisms that limit their expansion.

The reason why CD4(+) T helper 17 (Th17) cells, despite their well-known pathogenic role in chronic inflammatory disorders, are very rare in the inflammatory sites remains unclear. We demonstrate that human Th17 cells exhibit low ability to proliferate and to produce the T cell growth factor interleukin-2 (IL-2), in response to combined CD3 and CD28 stimulation. This was due to the upregulated expression of IL-4-induced gene 1 (IL4I1) mRNA, a secreted L-phenylalanine oxidase, which associated with a decrease in CD3ζ chain expression and consequent abnormalities in the molecular pathway that allows IL-2 production and cell proliferation. High IL4I1 mRNA expression was detectable in Th17 cell precursors and was strictly dependent on Th17 cell master gene, the retinoid acid related orphan receptor (RORC). Th17 cells also exhibited RORC-dependent CD28 hyperexpression and the ability to produce IL-17A after CD28 stimulation without CD3 triggering. Our findings suggest that the rarity of human Th17 cells in inflamed tissues results from RORC-dependent mechanisms limiting their expansion.

Endogenous urotensin II selectively modulates erectile function through eNOS.

BACKGROUND: Urotensin II (U-II) is a cyclic peptide originally isolated from the neurosecretory system of the teleost fish and subsequently found in other species, including man. U-II was identified as the natural ligand of a G-protein coupled receptor, namely UT receptor. U-II and UT receptor are expressed in a variety of peripheral organs and especially in cardiovascular tissue. Recent evidence indicates the involvement of U-II/UT pathway in penile function in human, but the molecular mechanism is still unclear. On these bases the aim of this study is to investigate the mechanism(s) of U-II-induced relaxation in human corpus cavernosum and its relationship with L-arginine/Nitric oxide (NO) pathway. METHODOLOGY/PRINCIPAL FINDINGS: Human corpus cavernosum tissue was obtained following in male-to-female transsexuals undergoing surgical procedure for sex reassignment. Quantitative RT-PCR clearly demonstrated the U-II expression in human corpus cavernosum. U-II (0.1 nM-10 µM) challenge in human corpus cavernosum induced a significant increase in NO production as revealed by fluorometric analysis. NO generation was coupled to a marked increase in the ratio eNOS phosphorilated/eNOS as determined by western blot analysis. A functional study in human corpus cavernosum strips was performed to asses eNOS involvement in U-II-induced relaxation by using a pharmacological modulation. Pre-treatment with both wortmannin or geldanamycinin (inhibitors of eNOS phosphorylation and heath shock protein 90 recruitment, respectively) significantly reduced U-II-induced relaxation (0.1 nM-10 µM) in human corpus cavernosum strips. Finally, a co-immunoprecipitation study demonstrated that UT receptor and eNOS co-immunoprecipitate following U-II challenge of human corpus cavernosum tissue. CONCLUSION/SIGNIFICANCE: U-II is endogenously synthesized and locally released in human corpus cavernosum. U-II elicited penile erection through eNOS activation. Thus, U-II/UT pathway may represent a novel therapeutical target in erectile dysfunction.

Antitumor activity of bortezomib in human cancer cells with acquired resistance to anti-epidermal growth factor receptor tyrosine kinase inhibitors.

The understanding of the molecular mechanisms which regulate cancer cell sensitivity to epidermal growth factor receptor (EGFR) inhibitors is necessary for the optimal use of these drugs in cancer treatment. We developed an in vitro model of acquired resistance to two EGFR tyrosine kinase inhibitors (TKI), erlotinib and gefitinib, by continuously treating the human non-small cell lung cancer (NSCLC) cell line CALU-3 with escalating doses of each drug. In these two EGFR inhibitor-resistant cancer cell lines a significant increase in the expression of activated, phosphorylated AKT and of survivin compared to parental cells was observed. Treatment with several agents known to target directly or indirectly the AKT signalling pathway did not affect significantly EGFR inhibitor-resistant cancer cell proliferation. In contrast, bortezomib, a proteasome inhibitor, induced a significant inhibition of cancer cell growth and an increase in apoptosis in EGFR inhibitor-resistant cancer cells as compared to treatment with LY294002, a PI3K inhibitor, suggesting that, in addition to interference with AKT signalling, other mechanisms are involved in the pro-apoptotic effects of bortezomib. Bortezomib treatment activated endoplasmic reticulum (ER) stress-mediated apoptosis, as demonstrated by the induction of GADD153, an ER stress-inducible transcription factor, and of the death receptor DR5, in EGFR inhibitor-resistant cells, but not in parental cells. This effect resulted in the activation of the extrinsic apoptotic pathway, as shown by caspase 8 dependent-PARP and bid cleavage. Bortezomib significantly inhibited the growth of EGFR inhibitor-resistant CALU-3 cells which were established as subcutaneous tumor xenografts in athymic nude mice. These results suggest that bortezomib treatment could be a useful approach to overcome resistance to anti-EGFR therapies.

Fat: a matter of disturbance for the immune system.

Obesity is increasingly being recognized as a risk factor for a number of benign and malignant gastrointestinal conditions. However, literature on the underlying pathophysiological mechanisms is sparse and ambiguous. There is compelling evidence that both overnutrition and undernutrition negatively interfere with the immune system. Overnutrition has been found to increase susceptibility to the development of inflammatory diseases, autoimmune diseases and cancer. In the regulation of immune and inflammatory processes, white adipose tissue plays a critical role, not only as an energy store but also as an important endocrine organ. The obese state is characterised by a low-grade systemic inflammation, mainly as a result of increased adipocytes as well as fat resident- and recruited-macrophage activity. In the past few years, various products of adipose tissue including adipokines and cytokines have been characterised and a number of pathways linking adipose tissue metabolism with the immune system have been identified. Activation of the innate immune system plays a major role in hepatic steatosis. Non-alcoholic fatty liver disease includes a wide spectrum of diseases, from pure steatosis to non-alcoholic steatohepatitis in the absence of significant alcohol consumption. Although steatosis is considered a non-progressive disease, non-alcoholic steatohepatitis may deteriorate in advanced chronic liver diseases, cirrhosis, and hepatocellular carcinoma. An important parallel between obesity-related pathology of adipose tissue and liver pertains to the emerging role of macrophages, and growing evidence suggests that Kupffer cells critically contribute to progression of non-alcoholic fatty liver disease. Moreover, a close link between specific immune activation and atherosclerosis has been well established, suggesting that fat can directly trigger immune responses. This review discusses the role of fat as "a matter of disturbance for the immune system" with a focus on hepatic steatosis.

Peripheral T cells from patients with early systemic sclerosis kill autologous fibroblasts in co-culture: is T-cell response aimed to play a protective role?

OBJECTIVES: Oligoclonal T-cell infiltrates have been detected in the skin of patients with early dcSSc. Peripheral T cells from patients with early dcSSc co-cultured with autologous fibroblasts have been found to expand the same T-cell clonotypes found in the affected skin. Here, we characterize oligoclonally expanded T lymphocytes and investigate functional changes occurring in early-dcSSc co-cultured T lymphocytes and fibroblasts. METHODS: Peripheral T lymphocytes from five patients with early (<3-year duration) dcSSc were co-cultured with the autologous fibroblasts obtained by punch biopsy of involved skin. RESULTS: T-cell clonotypes expanded in co-cultures were found to be alphabeta(+) and HLA-DR(+), and to promote the apoptosis of autologous fibroblasts. Fibroblasts up-regulated Fas and underwent apoptosis that paired with the expression of Fas ligand (Fasl) on CD4(+) T cells. Finally, the addition of a blocking anti-Fas antibody to the co-cultures resulted in a marked reduction of fibroblast apoptosis, suggesting a critical role of Fas/Fasl engagement in mediating apoptosis in co-cultured fibroblasts. In the co-culture supernatants, we found TGF-beta, IL-1beta, IL-6 and IL-8, cytokines that are known to promote fibrosis in SSc. The same results were registered in each co-culture. CONCLUSIONS: Taken together, these data suggest that T-cell response in SSc may also represent an attempt of the immune system to kill fibroblasts, cells likely expressing (auto)antigens, although the overall outcome of the T-cell response contributes to sustain inflammatory loops leading to fibrosis.

Systemic administration of sphingosine-1-phosphate increases bronchial hyperresponsiveness in the mouse.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that plays important roles in allergic responses, including asthma. S1P acts on many cell types, such as mast cells, the airway epithelium, airway smooth muscle, and many immune cells. In this study we have evaluated whether a systemic administration of S1P to Balb/c mice modifies airway reactivity. Our data show that S1P (0.1-10 ng) given subcutaneously to Balb/c mice causes a specific and dose-dependent increase in cholinergic reactivity of bronchial tissues in vitro. This effect is (1) dose dependent, with a maximal effect of the dose of 10 ng of S1P; and (2) time dependent, reaching a maximal effect 21 days after S1P administration. Similarly, in the whole lung assay there is a dose- and time-dependent increase in lung resistance. Lungs isolated from S1P-treated mice displayed an increase in mast cell number. Furthermore, there is an increase of IL-4, IL-13, and IL-17 production. In conclusion, our data demonstrate that S1P signaling is involved in the complex pathway underlying airway hyperresponsiveness.