Elisabetta Marangoni

A New Model of Patient Tumor-Derived Breast Cancer Xenografts for Preclinical Assays

Purpose: To establish a panel of human breast cancer (HBC) xenografts in immunodeficient mice suitable for pharmacologic preclinical assays. Experimental Design: 200 samples of HBCs were grafted into Swiss nude mice. Twenty-five transplantable xenografts were established (12.5%). Their characterization included histology, p53 status, genetic analysis by array comparative genomic hybridization, gene expression by Western blotting, and quantitative reverse transcription-PCR. Biological profiles of nine xenografts were compared with those of the corresponding patients tumor. Chemosensitivities of 17 xenografts to a combination of Adriamycin and cyclophosphamide (AC), docetaxel, trastuzumab, and Degarelix were evaluated. Results: Almost all patient tumors established as xenografts displayed an aggressive phenotype, i.e., high-grade, triple-negative status. The histology of the xenografts recapitulated the features of the original tumors. Mutation of p53 and inactivation of Rb and PTEN proteins were found in 83%, 30%, and 42% of HBC xenografts, respectively. Two HBCx had an ERBB2 (HER2) amplification. Large variations were observed in the expression of HER family receptors and in genomic profiles. Genomic alterations were close to those of original samples in paired tumors. Three xenografts formed lung metastases. A total of 15 of the 17 HBCx (88%) responded to AC, and 8 (47%) responded to docetaxel. One ERBB2-amplified xenograft responded to trastuzumab, whereas the other did not. The drug response of HBC xenografts was concordant with that of the patients tumor in five of seven analyzable cases. Conclusions: This panel of breast cancer xenografts includes 15 triple-negative, one ER positive and 2 ERBB2 positive. This panel represents a useful preclinical tool for testing new agents and protocols and for further exploration of the biological basis of drug responses.

Tyrphostin AG 1024 modulates radiosensitivity in human breast cancer cells

Insulin-like growth factor-1 (IGF-1) plays an important growth-promoting effect by activating the PI3K/Akt signalling pathway, inhibiting apoptotic pathways and mediating mitogenic actions. Tyrphostin AG 1024, one selective inhibitor of IGF-1R, was used to evaluate effects on proliferation, radiosensitivity, and radiation-induced cell apoptosis in a human breast cancer cell line MCF-7. Exposure to Tyrphostin AG 1024 inhibited proliferation and induced apoptosis in a time-dependent manner, and the degree of growth inhibition for IC20 plus irradiation (4 Gy) was up to 50% compared to the control. Examination of Tyrphostin AG 1024 effects on radiation response demonstrated a marked enhancement in radiosensitivity and amplification of radiation-induced apoptosis. Western blot analysis indicated that Tyrphostin AG 1024-induced apoptosis was associated with a downregulation of expression of phospho-Akt1, increased expression of Bax, p53 and p21, and a decreased expression of bcl-2 expression, especially when combined with irradiation. To our knowledge, this is the first report showing that an IGF-1 inhibitor was able to markedly increase the response of tumour cells to ionizing radiation. These results suggest that Tyrphostin AG 1024 could be used as a potential therapeutic agent in combination with irradiation.

CD44 targeting reduces tumour growth and prevents post-chemotherapy relapse of human breast cancers xenografts

CD44 is a marker of tumour-initiating cells and is upregulated in invasive breast carcinoma; however, its role in the cancer progression is unknown. Here, we show that antibody-mediated CD44-targeting in human breast cancer xenografts (HBCx) significantly reduces tumour growth and that this effect is associated to induction of growth-inhibiting factors. Moreover, treatment with this antibody prevents tumour relapse after chemotherapy-induced remission in a basal-like HBCx.

Decreased DNA-PK activity in human cancer cells exhibiting hypersensitivity to low-dose irradiation.

Low-dose hyper-radiosensitivity (HRS) (below 0.5 Gy) has been extensively documented in the past few years. The molecular basis of this phenomenon remains largely unknown and the purpose of this study was to investigate the possible implication of the DNA repair DNA-PK complex. The activity of the DNA-PK complex, i.e. Ku DNA-end binding activity and kinase activity of the whole complex, was studied in 10 human cancer cell lines, 2 h after 0.2, 0.5 and 1 Gy irradiation. After low-dose irradiation (0.2 Gy), a marked decrease in DNA-PK activity was found in all six cell lines exhibiting HRS, whereas the DNA-PK activity was increased in the four cell lines which did not exhibit HRS. This modulation of DNA-PK activity was a rapid phenomenon occurring within the 2 h following low-dose radiation exposure. These data strongly suggest the implication of the DNA-PK repair complex in the HRS phenomenon.

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts.

Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10–20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy.

Radiation-induced expression of functional Fas ligand in EBV-positive human nasopharyngeal carcinoma cells

Ionizing radiation remains a major therapeutic tool against human cancers, especially epithelial tumors, which account for the majority of human malignancies. Although Fas and Fas‐L are essential determinants of apoptosis, few data support their role in the cytotoxic effect of ionizing radiation. Epstein‐Barr‐virus (EBV)‐positive nasopharyngeal carcinoma (NPC) were chosen to address this question owing to their known sensitivity to ionizing radiation and their constitutive expression of the Fas‐receptor. We here report that, in xenografted NPC cells, Fas‐L expression, which was very low in basal conditions, was dramatically increased by tumor irradiation. Both the Fas receptor and the Fas ligand were found to be functional in this model, and a high proportion of irradiated NPC cells underwent apoptosis following tumor irradiation. Induction of Fas‐L expression and apoptosis were observed for doses as low as 2 Gy. These data show an increase in Fas‐L expression upon irradiation exposure, and strongly suggest that, in some epithelial malignancies, Fas‐mediated apoptosis can play a major role in the anti‐tumor effect of ionizing radiation, in the range of doses used for therapeutic applications. Int. J. Cancer 86:229–237, 2000.

Acquired Resistance to Endocrine Treatments Is Associated with Tumor-Specific Molecular Changes in Patient-Derived Luminal Breast Cancer Xenografts

Purpose: Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC. Experimental Design: Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays. Results: HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival. Conclusions: PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314–25. ©2014 AACR.

Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

Background:Oestrogen receptor-negative (ER−) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer.Methods:Gene and protein expression profiles were analysed in a panel of ER− breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively.Results:The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells.Conclusions:Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER− breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.

Patient-derived tumour xenografts as models for breast cancer drug development.

Purpose of review Patient-derived xenografts (PDXs) are becoming increasing popular as a preclinical tool for evaluating novel therapeutic strategies in cancer. These models maintain the biological characteristics of the donor tumours and have a predictive power in the translation of cancer therapeutics into clinical settings. This review focuses on the rapidly growing body of literature on PDX models of breast cancer and their applications and challenges in cancer drug development. Recent findings Several articles in the last 2 years have reported that breast cancer PDXs can reproduce the phenotype and diversity of patients’ tumours. This preservation of breast cancer biology involves a number of different aspects, including gene expression patterns, mutational status, drug response and tumour architecture. These models have been shown to be a valuable tool for the identification of new treatment targets, rational drug combinations, biomarkers and mechanisms of drug resistance. Summary The development of relevant, predictive models is key to increase the success rate for new drugs. PDX models of breast cancer hold the promise for the development of new and more efficient therapeutic strategies.

Vasculature analysis of patient derived tumor xenografts using species-specific PCR assays: evidence of tumor endothelial cells and atypical VEGFA-VEGFR1/2 signalings.

BackgroundTumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types.MethodsTo characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts.ResultsAs expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated.ConclusionsTaken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies.