Wnt pathway reprogramming during human embryonal carcinoma differentiation and potential for therapeutic targeting

<p>Abstract</p> <p>Background</p> <p>Testicular germ cell tumors (TGCTs) are classified as seminonas or non-seminomas of which a major subset is embryonal carcinoma (EC) that can differentiate into diverse tissues. The pluripotent nature of human ECs resembles that of embryonic stem (ES) cells. Many Wnt signalling species are regulated during differentiation of TGCT-derived EC cells. This study comprehensively investigated expression profiles of Wnt signalling components regulated during induced differentiation of EC cells and explored the role of key components in maintaining pluripotency.</p> <p>Methods</p> <p>Human embryonal carcinoma cells were stably infected with a lentiviral construct carrying a canonical Wnt responsive reporter to assess Wnt signalling activity following induced differentiation. Cells were differentiated with all-<it>trans </it>retinoic acid (RA) or by targeted repression of pluripotency factor, POU5F1. A Wnt pathway real-time-PCR array was used to evaluate changes in gene expression as cells differentiated. Highlighted Wnt pathway genes were then specifically repressed using siRNA or stable shRNA and transfected EC cells were assessed for proliferation, differentiation status and levels of core pluripotency genes.</p> <p>Results</p> <p>Canonical Wnt signalling activity was low basally in undifferentiated EC cells, but substantially increased with induced differentiation. Wnt pathway gene expression levels were compared during induced differentiation and many components were altered including ligands (WNT2B), receptors (FZD5, FZD6, FZD10), secreted inhibitors (SFRP4, SFRP1), and other effectors of Wnt signalling (FRAT2, DAAM1, PITX2, Porcupine). Independent repression of FZD5, FZD7 and WNT5A using transient as well as stable methods of RNA interference (RNAi) inhibited cell growth of pluripotent NT2/D1 human EC cells, but did not appreciably induce differentiation or repress key pluripotency genes. Silencing of FZD7 gave the greatest growth suppression in all human EC cell lines tested including NT2/D1, NT2/D1-R1, Tera-1 and 833K cells.</p> <p>Conclusion</p> <p>During induced differentiation of human EC cells, the Wnt signalling pathway is reprogrammed and canonical Wnt signalling induced. Specific species regulating non-canonical Wnt signalling conferred growth inhibition when targeted for repression in these EC cells. Notably, FZD7 repression significantly inhibited growth of human EC cells and is a promising therapeutic target for TGCTs.</p

Comparing the Epidermal Growth Factor Interaction with Four Different Cell Lines: Intriguing Effects Imply Strong Dependency of Cellular Context

The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of 125I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®

Targeting HER proteins in cancer therapy and the role of the non-target HER3

Members of the human epidermal growth factor receptor (HER) family have been of considerable interest in the cancer arena due to their potential to induce tumorigenesis when their signalling functions are deregulated. The constitutive activation of these proteins is seen in a number of different common cancer subtypes, and in particular EGFR and HER2 have become highly pursued targets for anti-cancer drug development. Clinical studies in a number of different cancers known to be driven by EGFR or HER2 show mixed results, and further mechanistic understanding of drug sensitivity and resistance is needed to realise the full potential of this treatment modality. Signalling in trans is a key feature of HER family signalling, and the activation of the PI3K/Akt pathway, so critically important in tumorigenesis, is driven predominantly through phosphorylation in trans of the kinase inactive member HER3. An increasing body of evidence shows that HER3 plays a critical role in EGFR- and HER2-driven tumours. In particular, HER3 lies upstream of a critically important tumorigenic signalling pathway with extensive ability for feedback and cross-talk signalling, and targeting approaches that fail to account for this important trans-target of EGFR and HER2 can be undermined by its resiliency and resourcefulness. Since HER3 is kinase inactive, it is not a direct target of kinase inhibitors and not presently an easily drugable target. This review presents the current evidence highlighting the role of HER3 in tumorigenesis and its role in mediating resistance to inhibitors of EGFR and HER2

A phase II study of the histone deacetylase inhibitor vorinostat combined with tamoxifen for the treatment of patients with hormone therapy-resistant breast cancer

BackgroundHistone deacetylases (HDACs) are crucial components of the oestrogen receptor (ER) transcriptional complex. Preclinically, HDAC inhibitors can reverse tamoxifen/aromatase inhibitor resistance in hormone receptor-positive breast cancer. This concept was examined in a phase II combination trial with correlative end points.MethodsPatients with ER-positive metastatic breast cancer progressing on endocrine therapy were treated with 400 mg of vorinostat daily for 3 of 4 weeks and 20 mg tamoxifen daily, continuously. Histone acetylation and HDAC2 expression in peripheral blood mononuclear cells were also evaluated.ResultsIn all, 43 patients (median age 56 years (31-71)) were treated, 25 (58%) received prior adjuvant tamoxifen, 29 (67%) failed one prior chemotherapy regimen, 42 (98%) progressed after one, and 23 (54%) after two aromatase inhibitors. The objective response rate by Response Evaluation Criteria in Solid Tumours criteria was 19% and the clinical benefit rate (response or stable disease &gt;24 weeks) was 40%. The median response duration was 10.3 months (confidence interval: 8.1-12.4). Histone hyperacetylation and higher baseline HDAC2 levels correlated with response.ConclusionThe combination of vorinostat and tamoxifen is well tolerated and exhibits encouraging activity in reversing hormone resistance. Correlative studies suggest that HDAC2 expression is a predictive marker and histone hyperacetylation is a useful pharmacodynamic marker for the efficacy of this combination

Elevated c-Src is linked to altered cell–matrix adhesion rather than proliferation in KM12C human colorectal cancer cells

Elevated expression and/or activity of c-Src, the prototype of the Src family of protein tyrosine kinases, is associated with the development of human colon cancer. However, despite the known pleiotropic effects of these kinases in promoting (a) cell growth downstream of growth factor receptors, and (b) the dynamic regulation of integrin adhesions in fibroblast model systems, their precise role in epithelial cancer cells is unknown. Here we addressed whether elevated expression and activity of cellular Src alters cell proliferation and/or cell–matrix adhesion in cancer cells from the Fidler model of colorectal metastasis. Although elevated Src correlates with ability to metastasise to the liver after intrasplenic injection, we found that this was not linked to enhanced growth, either in vitro or in vivo as sub-cutaneous tumours. However, elevated Src was associated with enhanced attachment to extracellular matrix. In addition, adhesion to fibronectin, was suppressed by agents that inhibited Src activity, while enforced elevation of Src in non-metastatic cells was sufficient to stimulate adhesion to fibronectin and enhanced assembly of adhesion complexes, without influencing cell growth. Thus, we conclude that one role of elevated Src in human colon cancer cells is to modulate integrin-dependent cell–matrix attachment and formation of adhesion structures, which may, in turn, influence cell motility and integrin-dependent cellular responses

Combined assessment of EGFR pathway-related molecular markers and prognosis of NSCLC patients

The purpose of this study is to evaluate the prognostic value of the combined assessment of multiple molecular markers related to the epidermal growth factor receptor (EGFR) pathway in resected non-small cell lung cancer (NSCLC) patients. Tumour specimens of 178 NSCLC patients were collected and analysed for EGFR and KRAS mutation status by DNA sequencing, and for EGFR copy number by fluorescent in situ hybridisation. Tissue microarrays were generated and used to determine the expression of multiple EGFR pathway-related proteins by immunohistochemistry. We analysed the association between each marker and patient prognosis. Univariate analyses for each clinical variable and each molecular marker were performed using Kaplan–Meier curves and log-rank tests. From these results, we selected the variables KRAS mutations and expression of cytoplasmic EGFR, granular pERK, nuclear pSTAT3, cytoplasmic E-cadherin and cytoplasmic pCMET to enter into a Cox proportional hazards model, along with stage as the strongest clinical variable related with prognosis. Of the EGFR-related markers evaluated here, the markers EGFR, pERK, pSTAT3, E-cadherin, pCMET and mutations in KRAS were associated with survival when analysed in combination in our patient cohort, with P=0.00015 as the P-value for a test of the additional impact of markers on prognosis, after taking stage into consideration. Confirmation of the impact of these markers in independent studies will be necessary

New targets for therapy in breast cancer: Farnesyltransferase inhibitors

Current systemic therapies for breast cancer are often limited by their nonspecific mechanism of action, unwanted toxicities on normal tissues, and short-term efficacy due to the emergence of drug resistance. However, identification of the molecular abnormalities in cancer, in particular the key proteins involved in abnormal cell growth, has resulted in development of various signal transduction inhibitor drugs as new treatment strategies against the disease. Protein farnesyltransferase inhibitors (FTIs) were originally designed to target the Ras signal transduction pathway, although it is now clear that several other intracellular proteins are dependent on post-translational farnesylation for their function. Preclinical data revealed that although FTIs inhibit the growth of ras-transformed cells, they are also potent inhibitors of a wide range of cancer cell lines that contain wild-type ras, including breast cancer cells. Additive or synergistic effects were observed when FTIs were combined with cytotoxic agents (in particular the taxanes) or endocrine therapies (tamoxifen). Phase I trials with FTIs have explored different schedules for prolonged administration, and dose-limiting toxicities included myelosuppression, gastrointestinal toxicity and neuropathy. Clinical efficacy against breast cancer was seen for the FTI tipifarnib in a phase II study. Based on promising preclinical data that suggest synergy with taxanes or endocrine therapy, combination clinical studies are now in progress to determine whether FTIs can add further to the efficacy of conventional breast cancer therapies

Molecular basis for sensitivity and acquired resistance to gefitinib in HER2-overexpressing human gastric cancer cell lines derived from liver metastasis

Gastric cancer metastasised to the liver was found to overexpress HER2 at a significantly higher incidence than primary gastric cancers. The purpose of the present study was to investigate the possibility of molecular therapy targeting HER2 overexpression in gastric cancer liver metastasis. We developed three new HER2-overexpressing gastric cancer cell lines (GLM-1, GLM-2, GLM-4) without epidermal growth factor receptor (EGFR) mutations derived from such liver metastasis, two of which had HER2 gene amplifications. All these GLM series of cell lines were highly sensitive to gefitinib in vitro, a specific inhibitor of EGFR tyrosine kinase (Iressa) rather than anti-HER2 antibody trastuzumab (Herceptin), whereas most of the HER2 low-expressing counterparts were not. In these HER2-overexpressing GLM series, protein kinase B (Akt), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was constitutively phosphorylated, and gefitinib efficiently inhibited this Akt phosphorylation, induced strong apoptosis in vitro and exhibited antitumour activity in tumour xenografts in nude mice. This gefitinib-mediated antitumour effect in xenograft was significantly potentiated by trastuzumab treatment. On the other hand, gefitinib-resistant cells (GLM-1R) exhibited increased EGFR expression, followed by constitutive activation of mitogen-activated protein kinase (MAPK) pathway. These results suggest that the antitumour effect of gefitinib is due to the effective inhibition of HER2-driven constitutive activation of phosphatidylinositol-3-kinase (PI3K)/Akt pathway, and that the acquired resistance to gefitinib is due to the constitutive activation of Ras/MAPK pathway in compensation for PI3K/Akt pathway. Gastric cancer liver metastasis with HER2 overexpression would be a potential molecular target for gefitinib and trastuzumab

Gefitinib Induces Epidermal Growth Factor Receptor Dimers Which Alters the Interaction Characteristics with 125I-EGF

The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer® Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of 125I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0–3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8–2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF – EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with 125I-EGF