Horacio Astudillo-de la Vega

Treatment of malignant, non-resectable, epithelial origin esophageal tumours with the humanized anti-epidermal growth factor antibody nimotuzumab combined with radiation therapy and chemotherapy.

Background: Over-expression of epidermal growth factor receptor in esophageal cancer is associated with poor prognosis. The present study was conducted to evaluate safety and preliminary efficacy of nimotuzumab, a humanized anti-EGFR antibody in combination with radiation and chemotherapy in advanced esophageal tumours. Patients and Methods: A Phase II clinical trial was conducted, where patients received cisplatin, 5-fluorouracil, and radiotherapy, either alone or combined with six weekly infusions of nimotuzumab at the dose of 200 mg. Safety was the primary endpoint. The antitumoral objective response rate was the secondary endpoint. Epidermal growth factor receptor expression, KRAS mutation status and anti-idiotypic response were also evaluated. Results: Sixty-three patients were included in the study. Thirty patients were entered into the control group, and thirty-three patients received the treatment with nimotuzumab. The antibody was very well tolerated. Objective response rate was 47.8 % (nimotuzumab group) and 15.4 % (control group). Disease control rate was 60.9 % (nimotuzumab group) and 26.9 % (control group). Response and disease control rate were higher in patients with EGFR overexpressing tumors. Conclusion: Nimotuzumab plus chemoradiotherapy was safe and provided statistically significant objective response. A Phase III in patients with similar characteristics will be launched.

A molecular analysis by gene expression profiling reveals Bik/NBK overexpression in sporadic breast tumor samples of Mexican females

BackgroundBreast cancer is one of the most frequent causes of death in Mexican women over 35 years of age. At molecular level, changes in many genetic networks have been reported as associated with this neoplasia. To analyze these changes, we determined gene expression profiles of tumors from Mexican women with breast cancer at different stages and compared these with those of normal breast tissue samples.Methods32P-radiolabeled cDNA was synthesized by reverse transcription of mRNA from fresh sporadic breast tumor biopsies, as well as normal breast tissue. cDNA probes were hybridized to microarrays and expression levels registered using a phosphorimager. Expression levels of some genes were validated by real time RT-PCR and immunohistochemical assays.ResultsWe identified two subgroups of tumors according to their expression profiles, probably related with cancer progression. Ten genes, unexpressed in normal tissue, were turned on in some tumors. We found consistent high expression of Bik gene in 14/15 tumors with predominant cytoplasmic distribution.ConclusionRecently, the product of the Bik gene has been associated with tumoral reversion in different neoplasic cell lines, and was proposed as therapy to induce apoptosis in cancers, including breast tumors. Even though a relationship among genes, for example those from a particular pathway, can be observed through microarrays, this relationship might not be sufficient to assign a definitive role to Bik in development and progression of the neoplasia. The findings herein reported deserve further investigation.

Comparative proteomic profiling of triple-negative breast cancer reveals that up-regulation of RhoGDI-2 is associated to the inhibition of caspase 3 and caspase 9☆

UNLABELLED There are no targeted therapeutic modalities for triple-negative breast cancer (TNBC), thus it is associated with poor prognosis and worst clinical outcome. Here, our aim was to identify deregulated proteins in TNBC with potential therapeutic applications. Proteomics profiling of TNBC and normal breast tissues through two-dimensional electrophoresis and ESI-MS/MS mass spectrometry revealed the existence of 16 proteins (RhoGDI-2, HSP27, SOD1, DJ1, UBE2N, PSME1, FTL, SH3BGRL, and eIF5A-1) with increased abundance in carcinomas. We also evidenced for the first time the deregulation of COX5, MTPN and DB1 proteins in TNBC that may represent novel tumor markers. Particularly, we confirmed the overexpression of the Rho-GDP dissociation inhibitor 2 (RhoGDI-2) in distinct breast cancer subtypes, as well as in metastatic cell lines derived from lung, prostate, and breast cancer. Remarkably, targeted disruption of RhoGDI-2 by RNA interference induced mitochondrial dysfunction, and facilitated caspase-3 and -9 activation in two breast cancer cell lines. Moreover, suppression of RhoGDI-2 resulted in a robust sensitization of breast cancer cells to cisplatin therapy. In conclusion, we identified novel proteins deregulated in TNBC, and confirmed the overexpression of RhoGDI-2. We propose that RhoGDI-2 inhibition may be exploited as a potential therapeutic strategy along cisplatin-based chemotherapy in breast cancer. BIOLOGICAL SIGNIFICANCE There are no useful biomarkers neither targeted therapeutic modalities for triple-negative breast cancer, which highly contributes to the poor prognosis of this breast cancer subtype. In this work, we used two-dimensional electrophoresis and ESI-MS/MS spectrometry to identify novel deregulated proteins in breast cancer tissues. Particularly, our results showed that RhoGDI-2, a protein that has been associated to metastasis and poor survival in human cancers, is overexpressed in different subtypes of breast tumors, as well as in metastatic cell lines derived from lung, prostate, and breast cancer. Our data also provided novel insights about the role of RhoGDI-2 in apoptosis through intrinsic pathway inhibition. Importantly, they suggested that targeted modulation of RhoGDI-2 levels might be a useful strategy for breast cancer therapy.

Proteomic profiling reveals that EhPC4 transcription factor induces cell migration through up-regulation of the 16-kDa actin-binding protein EhABP16 in Entamoeba histolytica☆

UNLABELLED Actin cytoskeleton is an essential structure involved in cell migration and invasion in parasites. In Entamoeba histolytica, the protozoan parasite causing human amoebiasis, the mechanisms underlying the expression of migration-related genes are poorly understood. Here, we investigated the biological effects of ectopic overexpression of EhPC4 (positive coactivator 4) in cell migration of E. histolytica trophozoites. Using differential in gel two-dimensional electrophoresis, 33 modulated proteins were detected in EhPC4-overexpressing cells. By electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis, 16 of these proteins were identified. Interestingly, four up-regulated proteins involved in cytoskeleton organization and cell migration were identified. Particularly, we found the up-regulation of a 16-kDa actin-binding protein (EhABP16) which is a putative member of the cofilin/tropomyosin family involved in actin polymerization. EhPC4 overexpression induced a significant increase in migration of trophozoites and in the destruction of human SW480 colon cells. Consistently, silencing of gene expression by RNA interference of EhABP16 significantly impairs cell migration. These changes were associated to alterations in the organization of actin cytoskeleton, and suppression of uropod-like structure formation in EhABP16-deficient cells. In summary, we have uncovered novel proteins modulated by EhPC4, including EhABP16, with a potential role in cell migration, cytopathogenicity and virulence in E. histolytica. BIOLOGICAL SIGNIFICANCE The human pathogen Entamoeba histolytica infects around 50million people worldwide resulting in 40,000-100,000 deaths annually. Cell motility is a complex trait that is critical for parasites adaptation, spread and invasion processes into host tissues; it has been associated with virulence. In this study, we used a differential proteomic approach to demonstrate that E. histolytica EhPC4 induces changes in the expression of actin cytoskeleton proteins, including EhABP16, promoting a significant increase in cell motility and destruction of intestinal human cells. Particularly, we demonstrated for the first time that abrogation of EhABP16 impairs cell migration by altering the actin cytoskeleton dynamics and uropod-like structure formation in trophozoites. These data contribute to the understanding of molecular mechanisms that regulate virulence properties in this neglected protozoan parasite.

Telomerase activity, estrogen receptors (α, β), Bcl-2 expression in human breast cancer and treatment response

BackgroundThe mechanism for maintaining telomere integrity is controlled by telomerase, a ribonucleoprotein enzyme that specifically restores telomere sequences, lost during replication by means of an intrinsic RNA component as a template for polymerization. Among the telomerase subunits, hTERT (human telomerase reverse transcriptase) is expressed concomitantly with the activation of telomerase. The role of estrogens and their receptors in the transcriptional regulation of hTERT has been demonstrated. The current study determines the possible association between telomerase activity, the expression of both molecular forms of estrogen receptor (ERα and ERβ) and the protein bcl-2, and their relative associations with clinical parameters.MethodsTissue samples from 44 patients with breast cancer were used to assess telomerase activity using the TRAP method and the expression of ERα, ERβ and bcl-2 by means of immunocytochemical techniques.ResultsTelomerase activity was detected in 59% of the 44 breast tumors examined. Telomerase activity ranged from 0 to 49.93 units of total product generated (TPG). A correlation was found between telomerase activity and differentiation grade (p = 0.03). The only significant independent marker of response to treatment was clinical stage. We found differences between the frequency of expression of ERα (88%) and ERβ (36%) (p = 0.007); bcl-2 was expressed in 79.5% of invasive breast carcinomas. We also found a significant correlation between low levels of telomerase activity and a lack of ERβ expression (p = 0.03).ConclusionLower telomerase activity was found among tumors that did not express estrogen receptor beta. This is the first published study demonstrating that the absence of expression of ERβ is associated with low levels of telomerase activity.

Methylation Landscape of Human Breast Cancer Cells in Response to Dietary Compound Resveratrol

Aberrant DNA methylation is a frequent epigenetic alteration in cancer cells that has emerged as a pivotal mechanism for tumorigenesis. Accordingly, novel therapies targeting the epigenome are being explored with the aim to restore normal DNA methylation patterns on oncogenes and tumor suppressor genes. A limited number of studies indicate that dietary compound resveratrol modulates DNA methylation of several cancer-related genes; however a complete view of changes in methylome by resveratrol has not been reported yet. In this study we performed a genome-wide survey of DNA methylation signatures in triple negative breast cancer cells exposed to resveratrol. Our data showed that resveratrol treatment for 24 h and 48 h decreased gene promoter hypermethylation and increased DNA hypomethylation. Of 2476 hypermethylated genes in control cells, 1,459 and 1,547 were differentially hypomethylated after 24 h and 48 h, respectively. Remarkably, resveratrol did not induce widespread non-specific DNA hyper- or hypomethylation as changes in methylation were found in only 12.5% of 27,728 CpG loci. Moreover, resveratrol restores the hypomethylated and hypermethylated status of key tumor suppressor genes and oncogenes, respectively. Importantly, the integrative analysis of methylome and transcriptome profiles in response to resveratrol showed that methylation alterations were concordant with changes in mRNA expression. Our findings reveal for the first time the impact of resveratrol on the methylome of breast cancer cells and identify novel potential targets for epigenetic therapy. We propose that resveratrol may be considered as a dietary epidrug as it may exert its anti-tumor activities by modifying the methylation status of cancer -related genes which deserves further in vivo characterization.

Phosphatase and tensin homologue and pituitary tumor-transforming gene in pituitary adenomas. Clinical-pathologic and immunohistochemical analysis

Pituitary tumor-transforming gene (PTTG) is also known to induce angiogenesis during pituitary tumorigenesis. It has not been clarified whether PTTG functions as a cytoplasmic or a nuclear protein. Pituitary tumor-transforming gene-1 is usually expressed in most pituitary tumors, and little is known about phosphatase and tensin homologue (PTEN). In our knowledge, it has not been studied in pituitary tumors. The aim of this study was to determine the correlation between proliferating cell nuclear antigen (PCNA) labeling index (LI), PTEN, and PTTG-1 immunoexpression in pituitary adenomas. Forty-five pituitary adenomas were included-46.7% were males and 53.7% were females. The mean age was 43.18 +/- 9.42 years (27-70 years). For functional pituitary adenoma (PA), it was 41.92 +/- 6.63, and for nonfunctional pituitary adenomas, it was 44.62 +/- 11.85 (P = .003). Proliferating cell nuclear antigen LI range was 19.42 +/- 5.49; in functional pituitary adenomas, it was 41.92 +/- 6.63, and in nonfunctional adenomas, it was 44.62 +/- 11.85 (P = .081). The PTEN immunoreaction was positive-weak in 21 (47%), moderate in 19 (42%), and strong in 5 (11%; P = .000). The PTTG-1 gene was positive-weak in 18 (41%), moderate in 19 (41%), and strong in 6 (13%; P = .000). When we correlated PTEN + PCNA, it was P =.004, and PTEN + PTTG-1, it was P = .019. And when we correlated PCNA + PTGG-1, it was P = .262. In our results, we observed higher expression of PCNA-LI and PTTG-1 and loss of expression of PTEN. Nonfunctional hypophysis adenomas presented a higher PCNA, PTTG-1, and PTEN expression than functional ones. There was no difference between single-hormone-producing hypophysis adenomas or multiple-hormone-producing ones. Necrosis and hemorrhage were associated with PTEN expression, whereas atypias and mitosis figures were associated to PTTG-1 expression.

Dual targeting of ANGPT1 and TGFBR2 genes by miR-204 controls angiogenesis in breast cancer

Deregulated expression of microRNAs has been associated with angiogenesis. Studying the miRNome of locally advanced breast tumors we unsuspectedly found a dramatically repression of miR-204, a small non-coding RNA with no previous involvement in tumor angiogenesis. Downregulation of miR-204 was confirmed in an independent cohort of patients and breast cancer cell lines. Gain-of-function analysis indicates that ectopic expression of miR-204 impairs cell proliferation, anchorage-independent growth, migration, invasion, and the formation of 3D capillary networks in vitro. Likewise, in vivo vascularization and angiogenesis were suppressed by miR-204 in a nu/nu mice model. Genome-wide profiling of MDA-MB-231 cells expressing miR-204 revealed changes in the expression of hundred cancer-related genes. Of these, we focused on the study of pro-angiogenic ANGPT1 and TGFβR2. Functional analysis using luciferase reporter and rescue assays confirmed that ANGPT1 and TGFβR2 are novel effectors downstream of miR-204. Accordingly, an inverse correlation between miR-204 and ANGPT1/TGFβR2 expression was found in breast tumors. Knockdown of TGFβR2, but not ANGPT1, impairs cell proliferation and migration whereas inhibition of both genes inhibits angiogenesis. Taken altogether, our findings reveal a novel role for miR-204/ANGPT1/TGFβR2 axis in tumor angiogenesis. We propose that therapeutic manipulation of miR-204 levels may represent a promising approach in breast cancer.

Differential proteomic analysis reveals that EGCG inhibits HDGF and activates apoptosis to increase the sensitivity of non-small cells lung cancer to chemotherapy.

To search for regulated proteins in response to green tea (–)‐epigallocatechin‐3‐gallate (EGCG) in A549 lung cancer cells.

Proteomic analysis identifies endoribouclease EhL-PSP and EhRRP41 exosome protein as novel interactors of EhCAF1 deadenylase

UNLABELLED In higher eukaryotic cells mRNA degradation initiates by poly(A) tail shortening catalyzed by deadenylases CAF1 and CCR4. In spite of the key role of mRNA turnover in gene expression regulation, the underlying mechanisms remain poorly understood in parasites. Here, we aimed to study the function of EhCAF1 and identify associated proteins in Entamoeba histolytica. By biochemical assays, we evidenced that EhCAF1 has both RNA binding and deadenylase activities in vitro. EhCAF1 was located in cytoplasmic P-bodies that increased in number and size after cellular stress induced by DNA damage, heat shock, and nitric oxide. Using pull-down assays and ESI-MS/MS mass spectrometry, we identified 15 potential EhCAF1-interacting proteins, including the endoribonuclease EhL-PSP. Remarkably, EhCAF1 colocalized with EhL-PSP in cytoplasmic P-bodies in trophozoites. Bioinformatic analysis of EhL-PSP network proteins predicts a potential interaction with EhRRP41 exosome protein. Consistently, we evidenced that EhL-PSP colocalizes and physically interacts with EhRRP41. Strikingly, EhRRP41 did not coimmunoprecipitate EhCAF1, suggesting the existence of two EhL-PSP-containing complexes. In conclusion, our results showed novel interactions between mRNA degradation proteins and evidenced for the first time that EhCAF1 is a functional deadenylase that interacts with EhL-PSP endoribonuclease in P-bodies, while EhL-PSP interacts with EhRRP41 exosome protein in this early-branched eukaryote. BIOLOGICAL SIGNIFICANCE This study provides evidences for the functional deadenylase activity of EhCAF1 and shows a link between different mRNA degradation proteins in E. histolytica. By proteomic tools and pull down assays, we evidenced that EhCAF1 interacts with the putative endoribonuclease EhL-PSP, which in turn interacts with exosome EhRRP41 protein. Our data suggest for the first time the presence of two complexes, one containing the endoribonuclease EhL-PSP and the deadenylase EhCAF1 in P-bodies; and another containing the endoribonuclease EhL-PSP and the exosome EhRRP41 exoribonuclease. Overall, these results provide novel data that may help to understand mRNA decay mechanisms in this parasite.