Teresita Muñoz-Antonia

Participation of both Gab1 and Gab2 in the activation of the ERK/MAPK pathway by epidermal growth factor

Three members of Gab family docking proteins, Gab1, Gab2 and Gab3, have been identified in humans. Previous studies have found that the hepatocyte growth factor preferentially utilizes Gab1 for signalling, whereas Bcr-Abl selectively signals through Gab2. Gab1-SHP2 interaction has been shown to mediate ERK (extracellular-signal-regulated kinase) activation by EGF (epidermal growth factor). However, it was unclear whether EGF selectively utilizes Gab1 for signalling to ERK and whether Gab2 is dispensable in cells where Gab1 and Gab2 are co-expressed. Using T47D and MCF-7 human breast carcinoma cells that express endogenous Gab1 and Gab2, we examined the role of these docking proteins in EGF-induced ERK activation. It was found that EGF induced a similar amount of SHP2-Gab1 and SHP2-Gab2 complexes. Expression of either SHP2-binding defective Gab1 or Gab2 mutant blocked EGF-induced ERK activation. Down-regulation of either Gab1 or Gab2 by siRNAs (small interfering RNAs) effectively inhibited the EGF-stimulated ERK activation pathway and cell migration. Interestingly, the inhibitory effect of Gab1 siRNA could be rescued not only by expression of an exogenous mouse Gab1 but also by an exogenous human Gab2 and vice versa, but not by IRS1 (insulin receptor substrate 1). These results reveal that Gab2 plays a pivotal role in the EGF-induced ERK activation pathway and that it can complement the function of Gab1 in the EGF signalling pathway. Furthermore, Gab1 and Gab2 are critical signalling threshold proteins for ERK activation by EGF.

ZEB1 Mediates Acquired Resistance to the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer.

Epithelial-mesenchymal transition (EMT) is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs) in non-small cell lung cancer (NSCLC). The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER) by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-β/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8), a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin), ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors.

Inhibition of farnesyltransferase increases TGFβ type II receptor expression and enhances the responsiveness of human cancer cells to TGFβ

Several small GTPases of the Ras superfamily have been shown to antagonize TGFβ signaling in human tumor cell lines. Some of these GTPases are post-translationally modified by farnesylation, a lipid modification catalyzed by farnesyltransferase and required for the proteins to attach to membranes and to function. In this study, we investigated the effect of the farnesyltransferase inhibitor FTI-277 on TGFβ-regulated cell growth and transcription. Treatment of the human pancreatic tumor cell line, Panc-1, with FTI-277 enhanced the ability of TGFβ to inhibit both anchorage-dependent and -independent tumor cell growth. FTI-277 also enhanced the ability of TGFβ to induce transcription, as measured by p3TP-lux reporter activity and collagen synthesis. The enhancement of TGFβ responses by FTI-277 correlated with the stimulation of transcription and protein expression of type II TGFβ receptor (TβRII). Consequently, FTI-277-treated cells exhibited a higher level of TGFβ binding to its receptor. Thus, inhibition of protein farnesylation stimulates TβRII expression, which leads to increased TGFβ receptor binding and signaling as well as inhibition of tumor cell growth and transformation.

Regulation of the human transforming growth factor β type II receptor gene promoter by novel Sp1 sites

The transforming growth factor-β (TGFβ) type II receptor (TβR-II) is responsible for transducing the growth inhibitory signals of TGFβ. The TβR-II gene promoter lacks both a TATA box and a CAAT box near the transcription initiation site, and has been shown to contain binding sequences for several transcription factors (Sp1, AP1, NF-Y, Cut and ERT) which are important for TβR-II gene promoter activity in vitro. However, it is still not clear which interactions are important for the regulation of TβR-II gene promoter activity in vivo. Using in vivo genomic DNA footprinting of normal human epithelial cells (HaCaT), we have identified two novel identical and strongly protected sites (ggggctgg) at positions −59 and −102 of the TβR-II gene promoter. Mutation of either site significantly reduced promoter activity in transient transfections. Protein binding to these sites, as determined by electrophoretic mobility shift assays (EMSA), was specifically competed with consensus Sp1 oligonucleotides. Furthermore, anti-Sp1/3 antibodies produced band shifts when incubated with the TβR-II −59 and −102 DNA probes. Importantly, Sp1 protein binding was influenced by the presence of an intact NF-Y binding site at position −83. Our data suggests that both Sp1 and NF-Y may play an important role in regulating TβR-II gene promoter basal activity in vivo.

Identification of genetic alterations in the TGFβ type II receptor gene promoter

Modifications in the control sequences of tumor suppressor genes have been found to play a role in the activation or inactivation of these genes and may play an important role in tumorigenesis. For example, hypermethylation of CpG islands and promoter polymorphisms have been found to be involved in transcriptional repression. A decrease in the levels of expression of one such tumor suppressor gene, the TGFbeta type II receptor (TbetaR-II), has been associated with increased tumorigenicity in a number of human tumors. Genetic alterations have been described in several tumor types in the coding region of this gene. However, no comprehensive search for genetic alterations in the TbetaR-II promoter has been reported. Genetic alterations in the promoter of the TbetaR-II gene could inhibit binding of putative regulatory factors. For example, we have reported a A-364-G alteration in the TbetaR-II promoter, which results in decreased transcriptional activity. In this study, we analyzed the 1.0kb region upstream of the TbetaR-II transcriptional start site and found genetic alterations in 46% of the head and neck squamous cell carcinoma (SqCC) samples examined. The most frequent alteration was a G-875-A alteration, present in 41.6% of the samples. Analysis of normal healthy individuals showed a similar frequency of this alteration, suggesting that alterations within the TbetaR-II promoter are unlikely to account for the decreased expression of TbetaR-II in head and neck SqCC.

Trim28 contributes to EMT via regulation of E-cadherin and N-cadherin in lung cancer cell lines.

In previous work, we demonstrated that transcription factor Trim28 (Tripartite motif containing 28) plays a tumor-suppressor role in early-staged adenocarcinoma of the lung due to its ability to restrain transcription of cell cycle-regulating genes. Herein we examine Trim28s role in the epithelial-to-mesenchymal transition (EMT) which is strongly implicated in cancer metastasis. We found that Trim28 plays a role in TGF-β-induced EMT in non-small cell lung cancer cells. Silencing Trim28 with inhibitory RNAs alters the expression of numerous EMT markers, such as E-cadherin and N-cadherin, whereas overexpression of Trim28 has an opposite effect. Trim28 expression is induced following TGF-β treatment at both protein and mRNA levels. Trim28 deficiency impairs TGF-β-induced EMT and decreases cell migration and invasion. Finally, we demonstrate that the expression of Trim28 affects the acetylation and methylation of histones on E-cadherin and N-cadherin promoters. These results suggest that Trim28 contributes to EMT and might be important for tumor metastasis in lung cancer. Taken together with our previous work these results suggest a model in which Trim28 is a tumor suppressor early in the transformation process in lung cancer, but in later stages it functions as an oncogene.

Transforming Growth Factor-β Type II Receptors and Smad Proteins in Follicular Thyroid Tumors †

Objective Resistance to transforming growth factor (TGF)‐β–mediated cell growth inhibition is a well‐known pathogenic mechanism in epithelial neoplasia. TGF‐β signaling requires normal function of downstream mediators such as TGF‐β receptors (TβRs) and Smad proteins. The goal of this study is to investigate the expression of components of the TGF‐β signaling pathway in follicular tumors of the thyroid.

Differences in Smad4 Expression in Human Papillomavirus Type 16–Positive and Human Papillomavirus Type 16–Negative Head and Neck Squamous Cell Carcinoma

The SMADs are a group of interrelated proteins that mediate transforming growth factor β (TGF-β) signaling. Upon TGF-β binding the TGF-β type I receptor phosphorylates Smad2 and Smad3, which then complex with Smad4 and translocate to the nucleus, with subsequent activation of target genes. Disruption of TGF-β signaling is thought to contribute to the development of head and neck squamous cell carcinomas (HNSCC). Alterations in the function of the DPC4/Smad4 tumor suppressor gene have been found to inactivate TGF-β signaling in several tumor types. For example, DPC4/Smad4 is lost or mutated in colorectal, pancreatic, and esophageal cancers. In addition, DPC4/Smad4 transcriptional activity and TGF-β ability to inhibit DNA synthesis is blocked by the E7 protein of the human papillomavirus type 16 (HPV16) in cervical carcinoma cell lines. HPV16 infection is a risk factor for the development of a subset of HNSCC. This study was undertaken to investigate a potential correlation between expression of components of the TGF-β signaling pathway and HPV16 status in HNSCC tumors. We examined the expression of TGF-β signaling proteins Smad2, Smad2-P, and Smad4 by immunohistochemistry in 27 HPV16-negative and 16 HPV16-positive HNSCCs. We compared the expression patterns and assessed their relationship to HPV16 status. No significant differences were detected between HPV16-positive and HPV16-negative tumors in the expression of Smad2 and Smad2-P. Smad4 expression, however, was decreased in 56% of the HPV16-positive tumors and in 39% of HPV16-negative tumors. This difference was statistically significant (P = 0.01) suggesting that loss of Smad4 expression may be involved in HPV16-induced carcinogenesis of HNSCC.

Assessing needs and assets for building a regional network infrastructure to reduce cancer related health disparities

Significant cancer health disparities exist in the United States and Puerto Rico. While numerous initiatives have been implemented to reduce cancer disparities, regional coordination of these efforts between institutions is often limited. To address cancer health disparities nation-wide, a series of regional transdisciplinary networks through the Geographic Management Program (GMaP) and the Minority Biospecimen/Biobanking Geographic Management Program (BMaP) were established in six regions across the country. This paper describes the development of the Region 3 GMaP/BMaP network composed of over 100 investigators from nine institutions in five Southeastern states and Puerto Rico to develop a state-of-the-art network for cancer health disparities research and training. We describe a series of partnership activities that led to the formation of the infrastructure for this network, recount the participatory processes utilized to develop and implement a needs and assets assessment and implementation plan, and describe our approach to data collection. Completion, by all nine institutions, of the needs and assets assessment resulted in several beneficial outcomes for Region 3 GMaP/BMaP. This network entails ongoing commitment from the institutions and institutional leaders, continuous participatory and engagement activities, and effective coordination and communication centered on team science goals.

Enhancing Oncology Health Care Provider's Sensitivity to Cultural Communication to Reduce Cancer Disparities: A Pilot Study

Under the auspices of a partnership grant to reduce cancer health disparities, Moffitt Cancer Center (MCC) partnered with the Ponce School of Medicine to identify the perceived cultural communication needs of MCC healthcare providers regarding Hispanic patients with limited or no English skills. Oncologists (N = 72) at MCC were surveyed to identify the specific areas of cultural communication techniques for which they desired to receive additional training. The majority of participants (66%) endorsed an interest in obtaining training to communicate difficult issues (terminal illness, controversial diagnosis) in a manner respectful to Hispanic culture. A workshop was conducted with providers (N = 55) to improve cultural communication between Hispanic patients and families focusing on culture, terminal illness, and communication strategies. Findings from a pre–post test indicate an overall positive response to the workshop. Results from this study can help inform future efforts to enhance cultural competency among health providers.