Cristina Chamizo

Poly (ADP-ribose) polymerase inhibition enhances trastuzumab antitumour activity in HER2 overexpressing breast cancer

AIM Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies. METHODS We tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells. RESULTS In a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis. CONCLUSION Taken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development.

Defective Cyclin B1 Induction in Trastuzumab-emtansine (T-DM1) Acquired Resistance in HER2-positive Breast Cancer.

Purpose: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance. Experimental Design: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2-positive breast cancer explants. Results: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation. Conclusions: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer. Clin Cancer Res; 23(22); 7006–19. ©2017 AACR.

Gene Expression Profiling in True Interval Breast Cancer Reveals Overactivation of the mTOR Signaling Pathway

Background: The development and progression of true interval breast cancers (tumors that truly appear after a negative screening mammogram) is known to be different from screen-detected cancers. However, the worse clinical behavior of true interval cancers is not fully understood from a biologic basis. We described the differential patterns of gene expression through microarray analysis in true interval and screen-detected cancers. Methods: An unsupervised exploratory gene expression profile analysis was performed on 10 samples (true interval cancers = 5; screen-detected cancers = 5) using Affymetrix Human Gene 1.0ST arrays and interpreted by Ingenuity Pathway Analysis. Differential expression of selected genes was confirmed in a validation series of 91 tumors (n = 12; n = 79) by immunohistochemistry and in 24 tumors (n = 8; n = 16) by reverse transcription quantitative PCR (RT-qPCR), in true interval and screen-detected cancers, respectively. Results: Exploratory gene expression analysis identified 1,060 differentially expressed genes (unadjusted P < 0.05) between study groups. On the basis of biologic implications, four genes were further validated: ceruloplasmin (CP) and ribosomal protein S6 kinase, 70 kDa, polypeptide 2 (RPS6KB2), both upregulated in true interval cancers; and phosphatase and tensin homolog (PTEN) and transforming growth factor beta receptor III (TGFBR3), downregulated in true interval cancers. Their differential expression was confirmed by RT-qPCR and immunohistochemistry, consistent with mTOR pathway overexpression in true interval cancers. Conclusions: True interval and screen-detected cancers show differential expression profile both at gene and protein levels. The mTOR signaling is significantly upregulated in true interval cancers, suggesting this pathway may mediate their aggressiveness. Impact: Linking epidemiologic factors and mTOR activation may be the basis for future personalized screening strategies in women at risk of true interval cancers. Cancer Epidemiol Biomarkers Prev; 23(2); 288–99. ©2013 AACR.

c-Jun N-Terminal Kinase Inactivation by Mitogen-Activated Protein Kinase Phosphatase 1 Determines Resistance to Taxanes and Anthracyclines in Breast Cancer

MAPK phosphatase-1 (MKP-1) is overexpressed during malignant transformation of the breast in many patients, and it is usually associated with chemoresistance through interference with JNK-driven apoptotic pathways. Although the molecular settings of the mechanism have been documented, details about the contribution of MKP-1 to the failure of chemotherapeutic interventions are unclear. Transient overexpression of MKP-1 and treatment with JNK-modulating agents in breast carcinoma cells confirmed the mediation of MKP-1 in the resistance to taxanes and anthracyclines in breast cancer, through the inactivation of JNK1/2. We next assessed MKP-1 expression and JNK1/2 phosphorylation status in a large cohort of samples from 350 early breast cancer patients treated with adjuvant anthracycline–based chemotherapy. We detected that MKP-1 overexpression is a recurrent event predominantly linked to dephosphorylation of JNK1/2 with an adverse impact on relapse of the tumor and overall and disease-free survival. Moreover, MKP-1 and p-JNK1/2 determinations in 64 locally advanced breast cancer patients treated with neoadjuvant taxane–based chemotherapy showed an inverse correlation between MKP-1 overexpression (together with JNK1/2 inhibition) and the pathologic response of the tumors. Our results emphasize the importance of MKP-1 as a potential predictive biomarker for a subset of breast cancer patients with worse outcome and less susceptibility to treatment. Mol Cancer Ther; 15(11); 2780–90. ©2016 AACR.

Determination of True erbb2 Gene Amplification in Breast Cancer by Quantitative Pcr Using a Reference and a Novel Control Gene

Human epidermal growth factor receptor 2 (ERBB2/HER2) is amplified and overexpressed in 20% to 25% of breast carcinomas, correlates with poor outcome, and is an indication for treatment with trastuzumab. Accurate assessment of ERBB2 status is crucial for proper prognosis and to offer appropriate treatment for patients. ERBB2 status is generally determined by immunohistochemistry or fluorescence in situ hybridization (FISH), and sporadically by quantitative real-time polymerase chain reaction (PCR). We developed a new algorithm, termed quantitative PCR algorithm (QPA) score, and compared its performance with the gold standard FISH assay. The QPA is a computation of the relative number of copies of the ERBB2 gene with respect to a nonstandard, short-arm centromeric sequence on chromosome 17, and referenced to a single-copy gene, RPP30. This provides a more reliable determination of ERBB2 amplification, reducing the false polysomy 17 error. A total of 69 breast carcinoma samples were tested for quantitative real-time PCR and FISH, and the degree of concordance was analyzed. Sixty-two cases were in agreement between the 2 methods, and the contingency study assigned a &kgr; value of 0.729 for their correlation. A receiver operating characteristic analysis was used to determine the optimal cut-off point for ERBB2 amplification, which was estimated at a QPA=1.53 (sensitivity=0.863; specificity=0.944). Our data conclude that the QPA is able to determine ERBB2 gene status with high accuracy, while also overcoming the limitations of conventional techniques and providing better cost-effectiveness.

Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma

Mutations in Human Epidermal Growth Factor Receptors (HER) are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS), alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE) samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC), ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

Analysis of Potential Alterations Affecting SETBP1 as a Novel Contributing Mechanism to Inhibit PP2A in Colorectal Cancer Patients

BackgroundThe functional loss of the tumor suppressor protein phosphatase 2A (PP2A) occurs in a wide variety of human cancers including colorectal cancer (CRC), and SET overexpression has been reported as a key contributing mechanism to inhibit PP2A. Although SET binding protein 1 (SETBP1) overexpression and gain of function mutations have been described in several hematological malignancies as common events that increase the expression levels of the PP2A inhibitor SET, thereby leading to PP2A inactivation, the potential existence of SETBP1 alterations in CRC still remains unexplored.MethodsWe studied the expression profile of SETBP1 by Western blot in a set of CRC cell lines and patient samples. Moreover, we performed co-immunoprecipitation assays to analyze the formation of the previously reported SETBP1–SET–PP2A inhibitory complex. Furthermore, we evaluated the mutational status of SETBP1 by pyrosequencing assays in a cohort of 55 CRC patients with metastatic disease after the immunohistochemical characterization of SET and p-PP2A expression in this cohort.ResultsWe found high SETBP1 expression in several CRC lines but only in two of the patients analyzed. In addition, we demonstrated the formation of the SETBP1–SET–PP2A heterotrimeric complex in CRC cells. However, we failed to detect SETBP1 mutations in any of the CRC patient samples included in the study.ConclusionsOur results suggest that SETBP1 expression is mainly similar o lower in colorectal cancer tissue compared to normal colonic mucosa. However, its overexpression is a low prevalent alteration which could contribute to inhibit PP2A in CRC through the formation of a SETBP1–SET–PP2A complex in some CRC patients. Moreover, SETBP1 mutations are, if exist, rare events in CRC patients.

Recent Insights into the Development of Preclinical Trastuzumab- Resistant HER2+ Breast Cancer Models

BACKGROUND Overexpression and amplification of the human epidermal growth factor receptor 2 (HER2) occur in 20% of total breast carcinomas. HER2-overexpression is implicated in disease initiation and progression and associated with poor prognosis. Trastuzumab, a humanized monoclonal antibody, is the standard HER2-targeted therapy for early and metastatic HER2-amplified breast cancer patients. Trastuzumab has significantly increased clinical benefit in HER2+ metastatic and adjuvant settings; however, it is not effective for many patients due to primary or acquired resistance to the drug. During the last decade, many studies have revealed a number of novel molecular traits of HER2+ breast cancer, allowing us to uncover the molecular mechanisms involved in trastuzumab resistance and develop strategies to overcome resistance to therapy. OBJECTIVE In this review, we comprehensively addressed the current achievements in preclinical studies; we discussed molecular mechanisms of acquired trastuzumab resistance in HER2+ breast cancer models and potential therapeutic approaches based on the molecular features for HER2+ breast cancer. CONCLUSION Enhanced understanding of the molecular profiles in HER2+ breast cancer may lead to the identification of novel biomarkers for the development of diagnostic approaches and improvement of therapeutic targets for the prevention and treatment of trastuzumab resistant HER2+ breast cancer.

Mutual regulation between BCL6 and a specific set of miRNAs controls TFH phenotype in peripheral T-cell lymphoma

The BCL6 transcriptional repressor protein is upregulated in follicular helper T-cells (TFH) and is considered a master regulator of the TFH cell lineage (Nurieva et al, 2009; Yu et al, 2009). Follicular helper T-cells (TFH) enter the follicles during T-cell-dependent immune responses and help B-cells to differentiate into long-lived antibody-secreting plasma cells or memory B-cells. TFH cells are characterized by the expression of CXCR5 (CXC-chemokine receptor type 5), PDCD1 (programmed cell death protein 1, also termed PD-1), ICOS (inducible T-cell costimulator), CXCL13 (CXC-chemokine ligand 13), and secrete high levels of IL21. The differentiation of T-cells into specialized effector lineages depends on lineage-specific transcription factors, which leads each of them to express and produce specific subsets of cytokines. Nevertheless, the well-known flexibility and plasticity of these phenotypes is highly dependent on the microenvironment. Studies have shown that BCL6 expression can be regulated by microRNAs (miRNAs) and extracellular cytokine signals (Wei et al, 2015). BCL6 is also involved in regulating the expression of many non-coding RNAs and/or transcription factors (Yu et al, 2009). Furthermore, many of the miRNAs downregulated by BCL6 could join multiple target sites within the 30untranslated regions (UTRs) of TFH-related genes, such as CXCR5, CXCR4 and PDCD1 (previously termed PD-1) (Yu et al, 2009). Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of peripheral T-cell lymphomas (n-PTCLs). It shows peculiar morphological features and a similar gene expression profile to TFH cells (de Leval et al, 2007), with expression of BCL6, ICOS, CXCL13, CXCR4, PDCD1, CXCR5 and other TFH markers, suggesting that these cells are the normal counterpart of this lymphoma subtype. Recent findings have associated AITL morphology and a TFH phenotype with the presence of specific subsets of mutations in genes such as RHO, IDH2 and TET2 (Odejide et al, 2014). The aim of this study was to elucidate whether the TFH phenotype in nPTCLs could also be regulated at the transcriptional level by a set of miRNAs. MicroRNA and mRNA from 26 frozen n-PTCLs samples (14 AITL and 12 PTCL-not otherwise specified [NOS]) and 4 reactive lymph nodes were extracted. All cases were hybridized on an 8 9 15 K Human miRNA microarray and a 4 9 44 K Whole Human Genome Oligo microarray (both from Agilent Technologies Inc., Santa Clara, CA, USA), respectively. The gene sets that co-regulated with BCL6 expression were identified by Pearson correlation using gene set enrichment analysis (GSEA) software (http://software.b roadinstitute.org/gsea/index.jsp). A variety of web resources and algorithms for investigating potential miRNA targets were used. The previously published miRNAs 101 and 103 that regulate the TFH phenotype were also included in the analysis(Yu et al, 2009). A t-test was used to evaluate the significance of group differences between upregulated miRNAs with downregulated targets and downregulated miRNAs with upregulated targets (http://babelomics.bioinfo.cipf.es/) (See Data S1 in the Supplementary Information; Tables SI and SII). TFH markers were closely coregulated in this series, with a positive relationship between the expression of BCL6 and of PDCD1, CXCR4, CXCR5, CXCL13 and ICOS (P < 0 001, 0 034, 0 001, 0 005 and <0 001, respectively), and an inverse correlation between the presence of BCL6 and GATA3 (P = 0 008) (Table I). Clustering analysis revealed similar associations between these genes (Figure S1). These patterns were confirmed in the GSEA study, which demonstrated that GC T-helper cells, BCL6 targets, IL21, STAT3 and STAT6 pathways were significantly upregulated in the BCL6-positive subgroup of n-PTCL patients (Table SIII). Interestingly, the levels of expression of a set of miRNAs (miR-212, mir-17 5p, miR-93, miR-302b, miR-520 h, miR-494, miR-124a, miR10a, miR-200b and miR-181a*) were inversely correlated with that of BCL6 (Table I and Figure S1). No significant correlation was found between BCL6 and miR-101 or miR103. An independent group of 135 formalin-fixed paraffinembedded (FFPE) n-PTCLs (80 AITL and 55 PTCL-NOS) was used to confirm these findings. We were able to confirm a direct correlation of BCL6 with CXCR4, CXCR5 and CXCL13 (P < 0 001 in each case). BCL6 and PDCD1 expression was analysed by immunohistochemistry (IHC) on TMA sections and was considered positive when, respectively, more than 10% or 25% of tumoral cells were stained. BCL6 and PDCD1 were found in 62 2% (84/135) and 16 3% (22/135) of the cases, respectively. In addition, there were significant negative correlations between the expression of BCL6 and both miR-494 (P = 0 016) and miR-200b (P = 0 039) (Table II). However, the negative relationships of BCL6 with miR-10a, miR-212 and miR-17 5p narrowly failed to attain statistical significance (P = 0 074, 0 076 and 0 089, respectively).