Jean-Luc Servely

Inhibiting Aurora kinases reduces tumor growth and suppresses tumor recurrence after chemotherapy in patient-derived triple-negative breast cancer xenografts

Triple-negative breast cancers (TNBC) have an aggressive phenotype with a relatively high rate of recurrence and poor overall survival. To date, there is no approved targeted therapy for TNBCs. Aurora kinases act as regulators of mammalian cell division. They are important for cell-cycle progression and are frequently overexpressed or mutated in human tumors, including breast cancer. In this study, we investigated the therapeutic potential of targeting Aurora kinases in preclinical models of human breast cancers using a pan-inhibitor of Aurora kinases, AS703569. In vitro, AS703569 was tested in 15 human breast cancer cell lines. TNBC cell lines were more sensitive to AS703569 than were other types of breast cancer cells. Inhibition of proliferation was associated with cell-cycle arrest, aneuploidy, and apoptosis. In vivo, AS703569 administered alone significantly inhibited tumor growth in seven of 11 patient-derived breast cancer xenografts. Treatment with AS703569 was associated with a decrease of phospho-histone H3 expression. Finally, AS703569 combined to doxorubicin–cyclophosphamide significantly inhibited in vivo tumor recurrence, suggesting that Aurora kinase inhibitors could be used both in monotherapy and in combination settings. In conclusion, these data indicate that targeting Aurora kinases could represent a new effective approach for TNBC treatment. Mol Cancer Ther; 11(12); 2693–703. ©2012 AACR.

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.

Comparative measurement of the lactogenic activity of ovine placental lactogen in rabbit and ewe mammary gland

Ovine placental lactogen is known to bind to prolactin receptors and to initiate milk synthesis in the rabbit mammary gland. However, this hormone exhibited a very low capacity of competing with 125I-labeled human growth hormone for the binding to membranes extracted from ewe mammary gland. Ovine placental lactogen was very efficient in provoking the accumulation of beta-casein mRNA in rabbit mammary explants but was much less active on ewe mammary explants. These data indicate that the placental hormone is not a potent lactogen in the homologous species and that its role in the control of mammary gland development and activity may have been previously overestimated.

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.

Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters

Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells’ metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development.

Induction of β-casein mRNA accumulation by the putative prolactin second messenger added to the culture medium of cultured mammary epithelial cells

Milk protein synthesis is under a hormonal control in which a protein hormone, prolactin, plays an essential role. Experiments in several species have shown that the induction of casein synthesis is coincident with an accumulation of the corresponding mRNAs [l-3]. These phenomena can be reproduced using mammary fragments cultured in synthetic media (4-61. Isolated mammary epithelial cells in primary culture have also been shown to retain their capacity to respond to the prolactin stimulus by the accumulation of casein mRNAs [7,8]. Experiments carried out in the rabbit demonstrated that the accumulation of casein mRNAs results from an acceleration of the transcription rate of the casein genes and from a stabilization of the mRNAs [9]. These effects are amplilied by glucocorticoids which per se are not inducers and they are inhibited by progesterone [lo]. Independently, the translation of casein mRNA is activated by prolactin and inhibited by progesterone [I 11. The evaluation of /?-casein gene transcription also proved possible using isolated nuclei [9,10,12]. This technique has been used for the search of the intracellular relay carrying the hormonal information from the receptors located in the plasma membrane to the target genes. It has been observed that the incubation of mammary membranes with prolactin provokes the release of a factor which is a potent stimulator of /?-casein gene transcription when added to isolated nuclei [ 13151. This factor is specifically generated by lactogenic hormones and it stimulates at least one of the prolactin-sensitive genes. The active fraction liberated from mammary membranes by prolactin has a small M,-value [14]. Here, the supernatant from membranes which contain the putative prolactin second messenger were added to the culture medium of isolated mammary epithelial cells, in an attempt to provoke the accumulation of casein mRNA and mimic prolactin action and in order to overcome possible problems encountered with the use of isolated nuclei.

Control of casein gene expression in isolated cultured rabbit epithelial mammary cells

Abstract Epithelial cells were isolated from mammary glands of pseudo-pregnant rabbits after a digestion of the tissue by collagenase and cell fractionation on a percoll gradient. These cells were cultured for various times in the presence of serum until growth was sufficient. The serum was then withdrawn for two days and hormones were added for one more day in the absence of serum. Cells were bound either directly to the plastic of the flask or to collagen spread in one or two layers. The concentration of β-casein mRNA was evaluated at the end of the culture by using a 3 H-DNA complementary to β-casein mRNA. The concentration of β3-casein mRNA was increased when prolactin was present in the culture medium. Cortisol amplified this effect while being totally inefficient alone. This induction was obtained as well when cells were cultured on plastic or on monolayer, bilayer or floating collagen, and also when they were kept in suspension. However, they apparently responded to prolactin only when fibroblasts were eliminated by adding hydroxy-L-proline (allo) or cholera toxin to the culture medium or by using collagen. The lactose synthetase activity and casein synthesis always remained extremely low regardless of the presence of hormones in the culture medium. Cells cultured on collagen tended to become reorganized in a way somewhat similar to that in mammary tissue, whereas cells spread on plastic remained uniformly distributed. These results suggest that isolated rabbit mammary epithelial cells have kept the essence of their capacity to respond to prolactin regardless of the culture technique. Isolated cultured cells thus appear to be a viable tool for further studies on the mechanism of prolactin action.

Vandetanib as a potential new treatment for estrogen receptor-negative breast cancers.

The receptor tyrosine kinase RET is implicated in the progression of luminal breast cancers (BC) but its role in estrogen receptor (ER) negative tumors is unknown. Here we investigated the expression of RET in breast cancer patients tumors and patient‐derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib, a tyrosin kinase inhibitor with strong activity against RET, EGFR and VEGFR2, in ER negative breast cancer PDX. The RT‐PCR analysis of RET expression in breast tumors of 446 patients and 57 PDX, showed elevated levels of RET in ER+ and HER2+ subtypes and in a small subgroup of triple‐negative breast cancers (TNBC). The activity of Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in PDX models with high expression of RET or EGFR. The effect was associated with inhibition of RET/EGFR phosphorylation and MAP kinase pathway and increased necrosis. In a PDX model with no expression of RET nor EGFR, Vandetanib slowed tumor growth without inducing tumor regression. In addition, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the four PDX models tested, suggesting inhibition of tumor vascularization. In summary, these preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers overexpressing Vandetanibs main targets.

Targeting mTOR pathway inhibits tumor growth in different molecular subtypes of triple-negative breast cancers

Triple-negative breast cancers (TNBC) are characterized by frequent alterations in the PI3K/AKT/mTOR signaling pathway. In this study, we analyzed PI3K pathway activation in 67 patient-derived xenografts (PDX) of breast cancer and investigated the anti-tumor activity of the mTOR inhibitor everolimus in 15 TNBC PDX with different expression and mutational status of PI3K pathway markers. Expression of the tumor suppressors PTEN and INPP4B was lost in 55% and 76% of TNBC PDX, respectively, while mutations in PIK3CA and AKT1 genes were rare. In 7 PDX treatment with everolimus resulted in a tumor growth inhibition higher than 50%, while 8 models were classified as low responder or resistant. Basal-like, LAR (Luminal AR), mesenchymal and HER2-enriched tumors were present in both responder and resistant groups, suggesting that tumor response to everolimus is not restricted to a specific TNBC subtype. Analysis of treated tumors showed a correlation between tumor response and post-treatment phosphorylation of AKT, increased in responder PDX, while PI3K pathway markers at baseline were not sufficient to predict everolimus response. In conclusion, targeting mTOR decreased tumor growth in 7 out of 15 TNBC PDX tested. Response to everolimus occurred in different TNBC subtypes and was associated with post-treatment increase of P-AKT.

Obtaiment of Pudu (Pudu pudu) deer Embryos by the Somatic Nuclear Transfer Technique

Los objetivos de este trabajo fueron los de producir embriones de pudu, obtenidos por la transferencia de nucleos de fibroblastos de la oreja de pudu en ovocitos de un rumiante domesticos que es el bovino. Para posteriormente en un trabajo futuro proceder a la transferencia de embriones de pudu, al utero de hembras receptoras sincronizadas de otra especie. Se obtuvieron biopsias de 1 mm aproximadamente del borde externo de la orejas de dos ciervos pudu machos del jardin zoologico Buin-Zoo, Santiago de Chile. Las lineas celulares han sido establecidas y conservadas segun los protocolos utilizados para las bovinos. Los ovocitos son obtenidos por puncion del complejo cumulos-ovocito (COC).desde ovarios de vacas recuperados del matadero. Cada ovocito es enucleado y fusionado con un fibroblasto aislado insertado bajo la zona pelucida. La fusion de membranas celulares es obtenida por choques electricos. En cuanto a la cronologia, observamos que al segundo dia se forma una etapa de dos blastomeras, al tercer dia morulas de 8 a 16 celulas, y desde el cuarto dia se ha diferenciado como blastocisto, el cual al septimo dia termina por eclosionar de la zona pelucida La obtencion de blastocistos embrionarios indica que es posible obtener embriones de pudu mediante clonaje heteroespecifico, aunque, el porcentaje de exito obtenido es relativamente bajo. Queda aun por verificar la viabilidad de los embriones asi obtenidos despues de la transferencia in utero