Elena Arechaga-Ocampo

Randomized Phase II Trial of All-Trans-Retinoic Acid With Chemotherapy Based on Paclitaxel and Cisplatin As First-Line Treatment in Patients With Advanced Non–Small-Cell Lung Cancer

PURPOSE This randomized phase II trial evaluated whether the combination of cisplatin and paclitaxel (PC) plus all-trans retinoic acid (ATRA) increases response rate (RR) and progression-free survival (PFS) in patients with advanced non-small-cell lung cancer (NSCLC) with an acceptable toxicity profile and its association with the expression of retinoic acid receptor beta 2 (RAR-beta2) as a response biomarker. PATIENTS AND METHODS Patients with stages IIIB with pleural effusion and IV NSCLC were included to receive PC, and randomly assigned to receive ATRA 20 mg/m(2)/d (RA/PC) or placebo (P/PC) 1 week before treatment until two cycles were completed. RAR-beta2 expression was analyzed in tumor and adjacent lung tissue. RESULTS One hundred seven patients were included, 55 in the P/PC group and 52 in the RA/PC group. RR for RA/PC was 55.8% (95% CI, 46.6% to 64.9%) and for P/PC, 25.4% (95% CI, 21.3 to 29.5%; P = .001). The RA/PC group had a longer median PFS (8.9 v 6.0 months; P = .008). Multivariate analysis of PFS showed significant differences for the RA/PC group (hazard ratio, 0.62; 95% CI, 0.4 to 0.95). No significant differences in toxicity grade 3/4 were found between groups, except for hypertriglyceridemia (10% v 0%) in RA/PC (P = .05). Immunohistochemistry and reverse-transcriptase polymerase chain reaction assays showed expression of RAR-beta2 in normal tissues of all tumor samples, but only 10% of samples in the tumor tissue. CONCLUSION Adding ATRA to chemotherapy could increase RR and PFS in patients with advanced NSCLC with an acceptable toxicity profile. A phase III clinical trial is warranted to confirm these findings.

Proteomic Profiling Reveals That Resveratrol Inhibits HSP27 Expression and Sensitizes Breast Cancer Cells to Doxorubicin Therapy

The use of chemopreventive natural compounds represents a promising strategy in the search for novel therapeutic agents in cancer. Resveratrol (3,4′,5-trans-trihydroxystilbilene) is a dietary polyphenol found in fruits, vegetables and medicinal plants that exhibits chemopreventive and antitumor effects. In this study, we searched for modulated proteins with preventive or therapeutic potential in MCF-7 breast cancer cells exposed to resveratrol. Using two-dimensional electrophoresis we found significant changes (FC >2.0; p≤0.05) in the expression of 16 proteins in resveratrol-treated MCF-7 cells. Six down-regulated proteins were identified by tandem mass spectrometry (ESI-MS/MS) as heat shock protein 27 (HSP27), translationally-controlled tumor protein, peroxiredoxin-6, stress-induced-phosphoprotein-1, pyridoxine-5′-phosphate oxidase-1 and hypoxanthine-guanine phosphoribosyl transferase; whereas one up-regulated protein was identified as triosephosphate isomerase. Particularly, HSP27 overexpression has been associated to apoptosis inhibition and resistance of human cancer cells to therapy. Consistently, we demonstrated that resveratrol induces apoptosis in MCF-7 cells. Apoptosis was associated with a significant increase in mitochondrial permeability transition, cytochrome c release in cytoplasm, and caspases -3 and -9 independent cell death. Then, we evaluated the chemosensitization effect of increasing concentrations of resveratrol in combination with doxorubicin anti-neoplastic agent in vitro. We found that resveratrol effectively sensitize MCF-7 cells to cytotoxic therapy. Next, we evaluated the relevance of HSP27 targeted inhibition in therapy effectiveness. Results evidenced that HSP27 inhibition using RNA interference enhances the cytotoxicity of doxorubicin. In conclusion, our data indicate that resveratrol may improve the therapeutic effects of doxorubicin in part by cell death induction. We propose that potential modulation of HSP27 levels using natural alternative agents, as resveratrol, may be an effective adjuvant in breast cancer therapy.

MetastamiRs: Non-Coding MicroRNAs Driving Cancer Invasion and Metastasis

MicroRNAs (miRNAs) are small non-coding RNAs of ~22 nucleotides that function as negative regulators of gene expression by either inhibiting translation or inducing deadenylation-dependent degradation of target transcripts. Notably, deregulation of miRNAs expression is associated with the initiation and progression of human cancers where they act as oncogenes or tumor suppressors contributing to tumorigenesis. Abnormal miRNA expression may provide potential diagnostic and prognostic tumor biomarkers and new therapeutic targets in cancer. Recently, several miRNAs have been shown to initiate invasion and metastasis by targeting multiple proteins that are major players in these cellular events, thus they have been denominated as metastamiRs. Here, we present a review of the current knowledge of miRNAs in cancer with a special focus on metastamiRs. In addition we discuss their potential use as novel specific markers for cancer progression.

Activation of Akt pathway by transcription-independent mechanisms of retinoic acid promotes survival and invasion in lung cancer cells.

BackgroundAll-trans retinoic acid (ATRA) is currently being used in clinical trials for cancer treatment. The use of ATRA is limited because some cancers, such as lung cancer, show resistance to treatment. However, little is known about the molecular mechanisms that regulate resistance to ATRA treatment. Akt is a kinase that plays a key role in cell survival and cell invasion. Akt is often activated in lung cancer, suggesting its participation in resistance to chemotherapy. In this study, we explored the hypothesis that activation of the Akt pathway promotes resistance to ATRA treatment at the inhibition of cell survival and invasion in lung cancer. We aimed to provide guidelines for the proper use of ATRA in clinical trials and to elucidate basic biological mechanisms of resistance.ResultsWe performed experiments using the A549 human lung adenocarcinoma cell line. We found that ATRA treatment promotes PI3k-Akt pathway activation through transcription-independent mechanisms. Interestingly, ATRA treatment induces the translocation of RARα to the plasma membrane, where it colocalizes with Akt. Immunoprecipitation assays showed that ATRA promotes Akt activation mediated by RARα-Akt interaction. Activation of the PI3k-Akt pathway by ATRA promotes invasion through Rac-GTPase, whereas pretreatment with 15e (PI3k inhibitor) or over-expression of the inactive form of Akt blocks ATRA-induced invasion. We also found that treatment with ATRA induces cell survival, which is inhibited by 15e or over-expression of an inactive form of Akt, through a subsequent increase in the levels of the active form of caspase-3. Finally, we showed that over-expression of the active form of Akt significantly decreases expression levels of the tumor suppressors RARβ2 and p53. In contrast, over-expression of the inactive form of Akt restores RARβ2 expression in cells treated with ATRA, indicating that activation of the PI3k-Akt pathway inhibits the expression of ATRA target genes.ConclusionOur results demonstrate that rapid activation of Akt blocks transcription-dependent mechanism of ATRA, promotes invasion and cell survival and confers resistance to retinoic acid treatment in lung cancer cells. These findings provide an incentive for the design and clinical testing of treatment regimens that combine ATRA and PI3k inhibitors for lung cancer treatment.

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.

RAD50 targeting impairs DNA damage response and sensitizes human breast cancer cells to cisplatin therapy

In tumor cells the effectiveness of anti-neoplastic agents that cause cell death by induction of DNA damage is influenced by DNA repair activity. RAD50 protein plays key roles in DNA double strand breaks repair (DSBs), which is crucial to safeguard genome integrity and sustain tumor suppression. However, its role as a potential therapeutic target has not been addressed in breast cancer. Our aim in the present study was to analyze the expression of RAD50 protein in breast tumors, and evaluate the effects of RAD50-targeted inhibition on the cytotoxicity exerted by cisplatin and anthracycline and taxane-based therapies in breast cancer cells. Immunohistochemistry assays on tissue microarrays indicate that the strong staining intensity of RAD50 was reduced in 14% of breast carcinomas in comparison with normal tissues. Remarkably, RAD50 silencing by RNA interference significantly enhanced the cytotoxicity of cisplatin. Combinations of cisplatin with doxorubicin and paclitaxel drugs induced synergistic effects in early cell death of RAD50-deficient MCF-7, SKBR3, and T47D breast cancer cells. Furthermore, we found an increase in the number of DSBs, and delayed phosphorylation of histone H2AX after cisplatin treatment in RAD50-silenced cells. These cellular events were associated to a dramatical increase in the frequency of chromosomal aberrations and a decrease of cell number in metaphase. In conclusion, our data showed that RAD50 abrogation impairs DNA damage response and sensitizes breast cancer cells to cisplatin-combined therapies. We propose that the development and use of inhibitors to manipulate RAD50 levels might represent a promising strategy to sensitize breast cancer cells to DNA damaging agents.

Tumor suppressor miR-29c regulates radioresistance in lung cancer cells

Radiotherapy is an important treatment option for non-small cell lung carcinoma patients. Despite the appropriate use of radiotherapy, radioresistance is a biological behavior of cancer cells that limits the efficacy of this treatment. Deregulation of microRNAs contributes to the molecular mechanism underlying resistance to radiotherapy in cancer cells. Although the functional roles of microRNAs have been well described in lung cancer, their functional roles in radioresistance are largely unclear. In this study, we established a non-small cell lung carcinoma Calu-1 radioresistant cell line by continuous exposure to therapeutic doses of ionizing radiation as a model to investigate radioresistance-associated microRNAs. Our data show that 50 microRNAs were differentially expressed in Calu-1 radioresistant cells (16 upregulated and 34 downregulated); furthermore, well-known and novel microRNAs associated with resistance to radiotherapy were identified. Gene ontology and enrichment analysis indicated that modulated microRNAs might regulate signal transduction, cell survival, and apoptosis. Accordingly, Calu-1 radioresistant cells were refractory to radiation by increasing cell survival and reducing the apoptotic response. Among deregulated microRNAs, miR-29c was significantly suppressed. Reestablishment of miR-29c expression in Calu-1 radioresistant cells overcomes the radioresistance through the activation of apoptosis and downregulation of Bcl-2 and Mcl-1 target genes. Analysis of The Cancer Genome Atlas revealed that miR-29c is also suppressed in tumor samples of non-small cell lung carcinoma patients. Notably, we found that low miR-29c levels correlated with shorter relapse-free survival of non-small cell lung carcinoma patients treated with radiotherapy. Together, these results indicate a new role of miR-29c in radioresistance, highlighting their potential as a novel biomarker for outcomes of radiotherapy in lung cancer.

All-Trans Retinoic Acid Induces Proliferation, Survival, and Migration in A549 Lung Cancer Cells by Activating the ERK Signaling Pathway through a Transcription-Independent Mechanism.

All-trans retinoic acid (ATRA) has been used as an antineoplastic because of its ability to promote proliferation, inhibition, and differentiation, primarily in leukemia; however, in other types of cancer, such as lung cancer, treatment with ATRA is restricted because not all the patients experience the same results. The ERK signaling pathway is dysregulated in cancer cells, including lung cancer, and this dysregulation promotes proliferation and cell invasion. In this study, we demonstrate that treatment with ATRA can activate the ERK signaling pathway by a transcription-independent mechanism through a signaling cascade that involves RARα and PI3K, promoting growth, survival, and migration in lung cancer cells. Until now, this mechanism was unknown in lung cancer cells. The inhibition of the ERK signaling pathway restores the beneficial effects of ATRA, reduces proliferation, increases apoptosis, and blocks the cell migration process in lung cancer cells. In conclusion, our results suggest that the combination of ATRA with ERK inhibitor in clinical trials for lung cancer is warranted.

Functional Roles of microRNAs in Cancer: microRNomes and oncomiRs Connection

Cancer is a complex group of diseases characterized by the presence of cells with uncontrolled growth, and high proliferation capacity. The complexity of cancer properties was outlined as the “hallmarks of cancer” a decade ago by Hanahan and Weinberg [1], and it comprises six alterations in cell physiology that dictate malignant growth including: (i) self-sufficiency in growth signals and uncontrolled growth of cells; (ii) insensitivity to antigrowth signals; (iii) evasion of apoptosis; 4 (iv) limitless replicative potential; (v) sustained angiogenesis; and (vi) acquisition of invasive properties to adjacent tissues and organs [1, 2]. These processes are regulated by protein-encoding genes whose expression switches-on or off during development and in response to cellular environment. Altered versions of the genes (tumor-suppressor genes and proto-oncogenes) which control the normal cellular processes arise from mutations, or expression deregulation in a multistep process resulting in cancer [3]. At the end of the transformation process, the malignant cells acquire growth independence, invasiveness and resistance to senescence and apoptosis. The acquired capabilities of cells to metastasize to other tissues and organs represent the most deadly hallmark of cancer [4-6].

C33-A cells transfected with E6*I or E6*II the short forms of HPV-16 E6, displayed opposite effects on cisplatin-induced apoptosis

The HPV-16 E6/E7 bicistronic immature transcript produces 4 mature RNAs: the unspliced HPV-16 E6/E7pre-mRNA product and 3 alternatively spliced mRNAs. The 3 spliced mRNAs encode short forms of the E6 oncoprotein, namely E6*I, E6*II and E6^E7. In this study we showed that transfection of C-33A cells with monocistronic constructs of these cDNAs fused to GFP, produced different effects on apoptosis, after the treatment with cisplatin. Transfection of C-33A cells with the full-length E6-GFP oncoprotein resulted in a 50% decrease in cell death, while the transfection with the E6*I-GFP construct showed only a 25% of diminution of cell death, compared to the control cells. Transfection with the E6^E7-GFP or E7-GFP construct had no effect on the number of the apoptotic cells, compared with control cells. Conversely, transfection with the E6*II construct resulted in higher cell death than the control cells. Taken together, these results suggested that E6*I or E6*II, the short forms of HPV-16 E6, displayed opposite effects on cisplatin-induced apoptosis, when transfected in C-33A cells.