Introdução: O Erlotinibe é um inibidor oral da tirosina quinase localizada no domínio intracelular do receptor do fator de crescimento epidérmico (EGFR). É uma droga ativa contra o carcinoma epidermóide de cabeça e pescoço (CECCP) que apresenta alta expressão deste receptor, demonstrando desta forma possível sinergismo com a quimioterapia e a radioterapia. Objetivo: Avaliar a expressão do EGFR e da proteína Akt fosforilada por imuno-histoquímica como fator preditivo a resposta terapêutica ao Erlotinibe em um estudo fase II que incluiu 32 pacientes com CECCP localmente avançado; também foram analisados mutações do gene EGFR nos éxons 18,19,20 e 21. Pacientes e métodos: Neste estudo pacientes portadores de CECCP localmente avançado foram tratados com uma combinação de Cisplatina 100mg/m2 intravenoso, administrada nos dias 8, 29 e 50 do tratamento; e radioterapia na dose de 70 Gy administrada em 39 frações a partir do dia 8. O Erlotinibe foi iniciado uma semana antes da radioterapia e mantido até o último dia da radioterapia. Biópsias pré-tratamento, extraídas dos blocos de parafina, foram analisadas por imunohistoquímica para avaliar a expressão do EGFR e da Akt fosforilada. O resultado dessas amostras foi quantificado por um programa de análise digital de imagem. O status mutacional do gene EGFR (nos éxons 18, 19,20 e 21) foi analisado utilizando PCR convencional e sequenciamento. Resultados: A resposta completa ao tratamento ocorreu em vinte pacientes (62,5%), sendo que dois foram tratados com Laringectomia de resgate e ficaram sem evidência de doença. A análise de sobrevida com relação ao estadiamento e com o sítio anatômico evidenciou diferença estatisticamente significativa (p= 0.05). A análise das proteínas EGFR e p-Akt por imuno-histoquímica, quando os sítios estavam agrupados não apresentou valor preditivo de resposta ao tratamento; no entanto ao avaliarmos os sítios anatômicos separadamente, apenas a quantificação de EGFR em hipofaringe foi uma variável preditiva de resposta ao tratamento com erlotinibe (p=0.05). Em relação às análises moleculares nenhuma mutação foi detectada no seqüenciamento dos éxons estudados da proteína EGFR. Conclusão: A expressão do EGFR parece ser um fator preditivo à reposta terapêutica, no entanto outros estudos com identificação de outros biomarcadores e amostras maiores são necessários para elucidar quais pacientes com CECCP podem ser beneficiados com este tratamento / Purpose: Erlotinib, an oral tyrosine-kinase inhibitor, is active against squamous cell carcinoma of the head and neck (HNSCC) and possibly has a synergistic interaction with chemotherapy and radiotherapy. We investigated the expression of EGFR and phosphorylated AKT by immunohistochemistry as predictors of response to Erlotinib in a cohort of 32 locally advanced HNSCC, enrolled in a Phase II trial. In addition, we assessed mutation on hotspots of EGFR gene (exons18,19,20,21). Patients and Methods: This study was conducted in a Phase I/II trial of cisplatin 100 mg/m2 on days 8, 29 and 50; and radiotherapy 70 Gy starting on day 8. Erlotinib was started orally 1 week before chemo radiation and continued daily just to the last day of chemo radiation. Pretreatment archival tumor specimens were evaluated for EGFR and phosphorylated-Akt (p-Akt) by immunohistochemistry. These immunostains were quantified by digital image analysis. EGFR gene mutational status was also assessed using conventional PCR and sequencing. Results: Complete response to treatment occurred in twenty patients (62.5%), and two were treated with salvage laryngectomy and were without evidence of disease. Survival analysis in relation to the staging and the tumor site showed a statistically significant difference (p = 0.05). Analysis of EGFR protein and p-Akt by immunohistochemistry, when sites were grouped showed no predictive value for treatment response, however when evaluating the anatomical sites separately, only the quantification of EGFR in the hypopharynx was a significant predictor of response to treatment with erlotinib (p = 0.05). Regarding the molecular analysis no mutations were detected in the sequencing of the exons studied EGFR protein. Conclusion: The expression of EGFR seems to be a predictive factor for response to therapy, although other studies with identification of other biomarkers and larger samples are needed to elucidate which patients may benefit HNSCC with this treatment

<p>Midgut carcinoid tumours are rare with an incidence of 0.5-2.1/100 000. The primary tumour is usually small and grows slowly but has almost always set metastases at diagnosis. When radically operated, most patients will eventually recur in their disease. </p><p>We evaluated different methods in detecting recurrent disease in 61 malignant midgut carcinoid tumours that had been radically operated. Thirty-eight patients have been diagnosed with a recurrence. In 32/38 of these patients P-Chromogranin A was the first method to indicate a recurrence. We therefore recommend using P-CgA in the work up in these patients.</p><p>We investigated characteristics, survival and independent factors that could be of bad prognostic value. We found that in our 284 malignant midgut carcinoid tumours, 208/284 (73%) had distant metastases and 30/284 (11%) had carcinoid heart disease. Median survival was 115.5 months and five-year survival was 77%. In a multivariate analysis liver metastases and carcinoid heart disease were poor prognostic factors.</p><p>We performed a phase II study with octreotide pamoate investigating the clinical effect in 12 malignant midgut carcinoid tumours in a progressive phase. We found that 9/12 (75%) were stabilised for a median duration of 12 months. We think that this is a good effect considering the advanced stage.</p><p>We investigated the frequency of four different tyrosine kinase receptors, platelet derived growth factor receptor (PDGR) α and β, epidermal growth factor receptor (EGFR) and c-kit, in 36 malignant midgut carcinoid tumours with immunohistochemistry. We found that 13/34 (38%) tumour samples expressed PDGFRα, 29/33 (88%) PDGFRβ, 24/33 (73%) EGFR, whereas none expressed c-kit. This implicates that midgut carcinoid tumours might be susceptible to treatment with tyrosine kinase receptor inhibitors. </p>

The membrane mucin Muc4 is a heterodimer, bi-functional glycoprotein complex that is normally expressed in epithelial tissue. Functional studies on the extracellular mucin subunit of Muc4 have shown that it acts to promote anti-adhesion properties by sterically interfering with cell-cell and cell-matrix interactions and that the extent of this effect is directly associated with the number of tandem repeats on this subunit. Functional studies on the transmembrane subunit of Muc4 have shown that this subunit participates in intracellular signaling through interaction with the receptor tyrosine kinase ErbB2. This role of Muc4 was shown to be mediated by stabilizing the heregulin ligand-induced ErbB2-ErbB3 heterodimer through interference with the internalization process of these receptors, thus potentiating the PI3K, a survival-signaling pathway that is mediated by this heterodimer. However, Muc4 was also shown to potentiate ErbB2 phosphorylation in the absence of heregulin by an unknown mechanism. The aim of this work was to examine the role of Muc4 in intracellular signaling by evaluating the ligand-independent Muc4-ErbB2 interaction. Biochemical analyses of A375 human melanoma cells expressing Muc4 under different cell treatments, and probed with phospho-specific antibodies, were used to understand the mechanism. An antibody microarray screen was used to decipher the intracellular activated signaling pathways. The results of the mechanistic analysis indicated that Muc4 potentiates ErbB2 signaling significantly by interacting with ErbB2 and ErbB3 and by stabilizing the kinase active ErbB2 receptor, thus increasing its phosphorylation signal half-life and resulting in sustained ErbB2 signaling. The signaling pathway analysis suggests that through Muc4 direct interaction with ErbB2, signaling pathways that promote loss of cell polarity are activated. Loss of cell-cell adhesion is mediated by interference with the cadherin-catenin complex stability, and loss of cell-matrix adhesion is mediated by facilitating focal adhesion turnover. Together, these results suggest that Muc4 is a potent oncogenic factor, and further enhance our understanding of the role that Muc4 plays in ligand-independent intracellular signaling.

Midgut carcinoid tumours are rare with an incidence of 0.5-2.1/100 000. The primary tumour is usually small and grows slowly but has almost always set metastases at diagnosis. When radically operated, most patients will eventually recur in their disease. We evaluated different methods in detecting recurrent disease in 61 malignant midgut carcinoid tumours that had been radically operated. Thirty-eight patients have been diagnosed with a recurrence. In 32/38 of these patients P-Chromogranin A was the first method to indicate a recurrence. We therefore recommend using P-CgA in the work up in these patients. We investigated characteristics, survival and independent factors that could be of bad prognostic value. We found that in our 284 malignant midgut carcinoid tumours, 208/284 (73%) had distant metastases and 30/284 (11%) had carcinoid heart disease. Median survival was 115.5 months and five-year survival was 77%. In a multivariate analysis liver metastases and carcinoid heart disease were poor prognostic factors. We performed a phase II study with octreotide pamoate investigating the clinical effect in 12 malignant midgut carcinoid tumours in a progressive phase. We found that 9/12 (75%) were stabilised for a median duration of 12 months. We think that this is a good effect considering the advanced stage. We investigated the frequency of four different tyrosine kinase receptors, platelet derived growth factor receptor (PDGR) α and β, epidermal growth factor receptor (EGFR) and c-kit, in 36 malignant midgut carcinoid tumours with immunohistochemistry. We found that 13/34 (38%) tumour samples expressed PDGFRα, 29/33 (88%) PDGFRβ, 24/33 (73%) EGFR, whereas none expressed c-kit. This implicates that midgut carcinoid tumours might be susceptible to treatment with tyrosine kinase receptor inhibitors.

In this work we report that potato carboxypeptidase inhibitor suppresses the growth of several human and mouse pancreatic adenocarcinoma cell lines. The inhibitor also reduces the growth of solid tumors obtained by subcutaneous injection of human adenocarcinoma cell line in nude mice and the appearance of metastasis in mice injected intraesplenically with B16H10 melanoma cells. A transgenic line has been characterized, mice develop choroid plexus carcicomas and insulinomas. Lifespan of animals immunized with PCI I s significantly higher than control mice. To further characterize the effects of PCI on tumor cell growth, analyses of cell cycle phase distribution have been performed. A significant increase in the G0/G1 phase is also detected. This finding is correlated with an increase in the mRNA expression of p53. Moreover, we have demonstrated that PCI is easily internalized by the cells and the inhibitor remain altered after the internalization / En el presente trabajo se ha demostrado que el inhibidor de carboxipeptidasa de patata (PCI) tiene un efecto inhibitorio sobre el crecimiento tumoral tanto in vitro como in vivo, y se ha estudiado su mecanismo de acción, lo que ha llevado a descubrir que el PCI es un antagonista del EGF. Con dicho fin se han utilizado técnicas de cultivo celular en conjunción con técnicas bioquímicas, de biología molecular, de análisis computacional y ensayos con modelos animales / En el present treball s’ha demostrat que l’inhibidor de carboxipeptidasa de patata (PCI) té un efecte inhibitori sobre el creixement tumoral tant in vitro com in vivo i s’ha estudiat el seu mecanisme d’acció, que ha permès descobrir que el PCI és un antagonista de l’EGF. Amb aquest fi s’han utilitzat tècniques de cultiu cel·lular amb tècniques bioquímiques, de biologia molecular, d’anàlisi computacional i assajos amb models animals

The incidence of breast cancer became the most common female cancer, and the fourth cause of female cancer death. In this study, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) have been combined with multivariate statistics to investigate breast cancer tissues and cell lines. Core needle biopsy and fine needle aspiration (FNA) are techniques largely applied in the diagnosis of breast cancer. In this study, we have established an efficient protocol for detecting breast tissue and FNA samples with MALDI-TOF/MS. With the help of statistical analysis software, we can find the lipid-derived ion signals which can be use to distinguish breast cancer tumor tissues from non-tumor parts. This strategy can differentiate normal and tumor tissue, which is potential to apply in clinical diagnoses. The analysis of breast cancer tissue is challenging as the complexity of the tissue sample. Direct tissue analyses by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) allows us to investigate the molecular structure and their distribution while maintaining the integrity of the tissue and avoiding the loss of signals from extraction steps. Combined MALDI-IMS with statistic software, tissues can be analyzed and classified based on their molecular content which is helpful to distinguish tumor regions from non-tumor regions of breast cancer tissue. Our result shows the differences in the distribution and content of lipids between tumor and non-tumor tissue which can be supplements of current pathological analysis in tumor margins. In this study, MALDI-TOF/MS combined with multivariate statistics were used to rapidly differentiate breast cancer cell lines with different estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status. The protocol for efficiently detecting peptides and proteins in breast cancer cells with MALDI-TOF/MS was established, two multivariate statistics including principle component analysis (PCA) and hierarchical clustering analysis were used to process the obtaining MALDI mass spectra of six different breast cancer cell lines and one normal breast cell lines. Based on the difference of the peptide and protein profiles, breast cancer cell lines with same ER and HER-2 status were grouped in nearby region on the PCA score plot. The results of hierarchical cluster analysis also revealed high conformity between breast cancer cell protein profiles and respective hormone receptor types.

Oral cancer is the sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates that have changed little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination alone are insufficient for detecting early tumor progression and molecular transformation. Development of new diagnostic tools incorporating tumor biomarkers could enhance early detection by providing molecular-level insight into the biochemical and cellular changes associated with oral carcinogenesis. The work presented in this doctoral dissertation aims to address this clinical need through the development of new automated cellular analysis methods, incorporating lab-on-a-chip sensor techniques, for examination of molecular and morphological biomarkers associated with oral carcinogenesis. Using the epidermal growth factor receptor (EGFR) as a proof-of-principle biomarker, the sensor system demonstrated capacity to support rapid biomarker analysis in less than one-tenth the time of traditional methods and effectively characterized EGFR biomarker over-expression in oral tumor-derived cell lines. Successful extension from in vitro tumor cell lines to clinically relevant exfoliative brush cytology was demonstrated, providing a non-invasive method for sampling abnormal oral epithelium. Incorporation of exfoliative cytology further helped to define the important assay and imaging parameters necessary for dual molecular and morphological analysis in adherent epithelium. Next, this new sensor assay and method was applied in a small pilot study in order to secure an initial understanding of the diagnostic utility of such biosensor systems in clinical settings. Four cellular features were identified as useful indicators of cancerous or pre-cancerous conditions including, the nuclear area and diameter, nuclear-to-cytoplasm ratio, and EGFR biomarker expression. Further examination using linear regression and ROC curve analysis identified the morphological features as the best predictors of disease while a combination of all features may be ideal for classification of OSCC and pre-malignancy with high sensitivity and specificity. Further testing in a larger sample size is necessary to validate this regression model and the LOC sensor technique, but shows strong promise as a new diagnostic tool for early detection of oral cancer. / text

Darbo tikslas nustatyti skirstymo baltymo GAB1 ryšį su anti-apoptoziniu PI3K/Akt bei mitogeniniu Ras/MAPK signalo perdavimo keliais ir įvertinti GAB1 įtaką šių kelių sąveikai EGFR signalo perdavimo tinkle. Darbo uždaviniai: įvertinti epitelinių ląstelių endogeninio GAB1 veiksmingumą EGF signalo perdavimo metu; nustatyti sąveikos pobūdį tarp PI3K/Akt ir Ras/MAPK kelių EGF signalo metu; kiekybiškai įvertinti GAB1 svarbą EGF signalo perdavimui per PI3K/Akt ir Ras/MAPK kelių in vivo, rezultatus lyginant su matematinio modelio prognozėmis in silico; nustatyti GAB1 veiksmingumo ir jo reguliacijos grįžtamaisiais ryšiais įtaką PI3K-MAPK sąveikos stiprumui priklausomai nuo EGF dozės ir laiko; ištirti GAB1 svarbą EGFR ir insulino receptoriaus signalo perdavimo tinklų sąveikai; modifikuoti Westerno pernašos metodą palyginamajai kiekybinei ir kokybinei baltymų analizei. Darbo išvados: stimuliavus EGFR, skirstymo baltymas GAB1 tampa veiksmingu; EGF lemia reciprokinę PI3K/Akt ir Ras/MAPK signalo perdavimo kelių sąveiką per GAB1; GAB1 yra pagrindinis teigiamo atgalinio ryšio elementas PI3K kelyje, padedąs pagreitinti, stiprinti ir išlaikyti MEK/ERK kinazių atsaką; PI3K-MAPK sąveikos stiprumas kinta laike ir yra atvirkščiai proporcingas EGF signalo stiprumui; GAB1 reikalingas sinergistiškai stiprinti insulinu ir mažomis EGF dozėmis stimuliuojamų ląstelių Ras/MAPK atsaką; sukurtas „Multi-juostelių“ imunopernašos metodas yra tinkamas palyginamajai kiekybinei ir kokybinei baltymų analizei... [toliau žr. visą tekstą] / The aim of the thesis was to determine a connection of endogenous docking protein GAB1 with anti-apoptotic PI3/Akt and Ras/MAPK signaling pathways and to estimate GAB1 contribution to their crosstalk in EGFR signaling network. The tasks: to evaluate GAB1 efficacy upon EGFR stimulation; to examine the nature of crosstalk between PI3K/Akt and Ras/MAPK pathways; to evaluate the contribution of GAB1 to EGF signaling via PI3K/Akt and Ras/MAPK pathways in vivo comparing the results with prognosis in silico; to estimate the EGF dose- and time-dependent impact of GAB1 efficacy and its feedback regulation on the strength of PI3K-MAPK interaction; to investigate the role of GAB1 for crosstalk of EGFR and insulin receptor signaling networks; to modify Western blotting procedure for comparative quantitative and qualitative protein analysis. The conclusions: the docking protein GAB1 is functional upon EGFR stimulation; PI3K/Akt and Ras/MAPK signaling pathways crosstalk reciprocally via GAB1 in response to EGF; GAB1 is major positive feedback element in PI3K pathway amplifying and sustaining MEK/ERK response to EGF; the strength of PI3K-MAPK interaction depends on time and is inversely proportional to EGF signal strength; GAB1 is required to synergistically potentate the Ras/MAPK response to tandem cell treatment with insulin and low EGF doses; the developed Multistrip immunoblotting method is suitable for comparative quantitative and qualitative protein analysis. In comparison with a... [to full text]

The altered protein expression and activity of receptor tyrosine kinases (TK) are implicated in the progression of various types of cancers. One such dysfunction is the overexpression of the epidermal growth factor receptor (EGFR) that correlates with aggressive tumor progression and poor prognosis. On the other hand, c-Src non-receptor tyrosine kinase is overexpressed and activated in a large number of human malignancies and has been strongly linked to progression to distant metastases. c-Src-induced phosphorylation of EGFR is required for EGF-mediated mitogenesis, tumorigenesis and tumour invasiveness. Thus we surmised that molecules termed "combi-molecules" designed to block both EGFR and c-Src should not only possess significant growth inhibitory potency but also strong anti-invasive properties. In this thesis, we utilized molecular modeling to design molecules containing two moieties: one that straddles the structure of the known Src inhibitor PP2 and the other that mimics the backbone of Iressa, a potent EGFR inhibitor. Of all the molecules synthesized, only SB163 containing the longest spacer between the two moieties was capable of inducing a dose dependent inhibition of both Src and EGFR. More importantly, SB163 blocked cell motility in the wound healing assay and showed significantly greater anti-invasive activity than a PP2+Iressa combination. The observation that SB163 was a less potent EGFR or Src inhibitor than Iressa and PP2 suggests that its superior potency when compared with the PP2+ Iressa combination may be at least partially attributed to mechanisms other than EGFR or Src blockade. This was also corroborated by the fact that SB163, despite its significant bulkiness (>700) could induce dose dependent inhibition of other kinase such PDFGR and Abl. The results in toto suggest that conferring multiple kinase targeting properties to single molecules can lead to highly anti-proliferative and anti-invasive agents. Traditionally, multi-kinase targeted molecules were discovered serendipitously through multi-kinase testing. Here we initiated a more rational approach to the design of single multi-targeted molecules. Cancer being a complex disease driven by tumours characterized by multiple disordered signaling pathways, this approach may well represent a novel avenue in the therapy of refractory malignancies.

Overall survival of the metastatic forms of epithelial derived cancers, especially head and neck squamous cell carcinomas (HNSCC), has not significantly improved even with the application of aggressive combined modality approaches incorporating radiation and chemotherapy. Cumulative evidence implicates the epidermal growth factor receptor (EGFR) as an important therapeutic target in HNSCC. We have previously demonstrated that the combination of lovastatin, a potent inhibitor of the mevalonate pathway, with EGFR tyrosine kinase inhibitors induced robust synergistic cytotoxicity. However, the use of high dose statins in our clinical trial was associated with significant toxicities including higher than anticipated rate of muscle pathologies. Our goal was to uncover novel downstream targets of the mevalonate pathway that may enhance the efficacy or limit toxicities of this novel combination therapeutic approach. In this study we have demonstrated that GGTI-298, an inhibitor of protein geranylgeranylation, through its ability to disrupt the actin cytoskeleton, inhibits EGFR dimerization and cellular trafficking. This novel mechanism targeting the EGFR has clinical implications as GGTI-298 in combination with tarceva, a clinically relevant EGFR inhibitor, showed enhanced cytotoxicity and inhibitory effects on EGFR activation and its downstream signaling.