PI3K activation promotes resistance to eribulin in HER2-negative breast cancer.

BACKGROUND: Eribulin is a microtubule-targeting agent approved for the treatment of advanced or metastatic breast cancer (BC) previously treated with anthracycline- and taxane-based regimens. PIK3CA mutation is associated with worse response to chemotherapy in oestrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) metastatic BC. We aimed to evaluate the role of phosphoinositide 3-kinase (PI3K)/AKT pathway mutations in eribulin resistance. METHODS: Resistance to eribulin was evaluated in HER2- BC cell lines and patient-derived tumour xenografts, and correlated with a mutation in the PI3K/AKT pathway. RESULTS: Eleven out of 23 HER2- BC xenografts treated with eribulin exhibited disease progression. No correlation with ER status was detected. Among the resistant models, 64% carried mutations in PIK3CA, PIK3R1 or AKT1, but only 17% among the sensitive xenografts (P = 0.036). We observed that eribulin treatment induced AKT phosphorylation in vitro and in patient tumours. In agreement, the addition of PI3K inhibitors reversed primary and acquired resistance to eribulin in xenograft models, regardless of the genetic alterations in PI3K/AKT pathway or ER status. Mechanistically, PI3K blockade reduced p21 levels likely enabling apoptosis, thus sensitising to eribulin treatment. CONCLUSIONS: PI3K pathway activation induces primary resistance or early adaptation to eribulin, supporting the combination of PI3K inhibitors and eribulin for the treatment of HER2- BC patients.

PI3K inhibition impairs BRCA1/2 expression and sensitizes BRCA-proficient triple-negative breast cancer to PARP inhibition.

UNLABELLED: PARP inhibitors are active in tumors with defects in DNA homologous recombination (HR) due to BRCA1/2 mutations. The phosphoinositide 3-kinase (PI3K) signaling pathway preserves HR steady state. We hypothesized that in BRCA-proficient triple-negative breast cancer (TNBC), PI3K inhibition would result in HR impairment and subsequent sensitization to PARP inhibitors. We show in TNBC cells that PI3K inhibition leads to DNA damage, downregulation of BRCA1/2, gain in poly-ADP-ribosylation, and subsequent sensitization to PARP inhibition. In TNBC patient-derived primary tumor xenografts, dual PI3K and PARP inhibition with BKM120 and olaparib reduced the growth of tumors displaying BRCA1/2 downregulation following PI3K inhibition. PI3K-mediated BRCA downregulation was accompanied by extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of an active form of MEK1 resulted in ERK activation and downregulation of BRCA1, whereas the MEK inhibitor AZD6244 increased BRCA1/2 expression and reversed the effects of MEK1. We subsequently identified that the ETS1 transcription factor was involved in the ERK-dependent BRCA1/2 downregulation and that knockdown of ETS1 led to increased BRCA1/2 expression, limiting the sensitivity to combined BKM120 and olaparib in 3-dimensional culture. SIGNIFICANCE: Treatment options are limited for patients with TNBCs. PARP inhibitors have clinical activity restricted to a small subgroup of patients with BRCA mutations. Here, we show that PI3K blockade results in HR impairment and sensitization to PARP inhibition in TNBCs without BRCA mutations, providing a rationale to combine PI3K and PARP inhibitors in this indication. Our findings could greatly expand the number of patients with breast cancer that would benefit from therapy with PARP inhibitors. On the basis of our findings, a clinical trial with BKM120 and olaparib is being initiated in patients with TNBCs.

A role for stroma-derived annexin A1 as mediator in the control of genetic susceptibility to T-cell lymphoblastic malignancies through prostaglandin E2 secretion.

Cancer susceptibility is essentially attributable to multiple low-penetrance genes. Using interspecific consomic and congenic mice between the tumor-resistant SEG/Pas and the tumor-sensitive C57BL/6J strains, a region on chromosome 19 involved in the genetic resistance to gamma-irradiation-induced T-cell lymphomas (Tlyr1) has been identified. Through the development of nonoverlapping subcongenic strains, it has been further shown that Anxa1 may be a candidate resistance gene on the basis of its differential expression in thymus stroma cells after gamma-radiation exposure. In addition, thymus stroma cells of thymic lymphomas exhibited a significant reduction in the expression levels of Anxa1. Interestingly, the activity of Anxa1 relies on prostaglandin E(2) (PGE(2)) induction that brings about apoptosis in thymocytes. In fact, in vitro transfection experiments revealed that PGE(2) production was enhanced when HEK 293 cells were transfected with full-length cDNAs of Anxa1, with PGE(2) production in the cells transfected with the allele of the resistant strain (Anxa1(Tyr)) being higher than that in cells transfected with the allele of the susceptible strain (Anxa1(Phe)). Furthermore, the presence of this compound in the medium induced apoptosis of immature CD4(+)CD8(+)CD3(low) cells in a dose-dependent manner. These results improve our knowledge of the molecular mechanisms triggering T-cell lymphoblastic lymphoma development while highlighting the relevance of the stroma in controlling genetic susceptibility and the use of PGE(2) as a new therapeutic approach in T-cell hematologic malignancies.

Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection.

The production of animals with large transgenes is an increasingly valuable tool in biotechnology and for genetic studies, including the characterization and manipulation of large genes and polygenic traits. In the present study, we describe an intracytoplasmic sperm injection (ICSI) method for the stable incorporation and phenotypic expression of large yeast artificial chromosomes (YAC) constructs of submegabase and megabase magnitude. By coinjecting spermatozoa and YACs into metaphase II oocytes, we were able to produce founders exhibiting germline transmission of an intact and functional transgene of 250 kilobases, carrying the mouse tyrosinase locus, used here as a reporter gene to rescue the albinism of recipient mice. More than 35% transgenesis was obtained for this YAC transgene. When compared with the pronuclear microinjection standard method, the efficiency of the ICSI-mediated YAC transfer system was significantly greater. In summary, we describe, for the first time, stable incorporation in the host genome and correct phenotypic expression of large DNA constructs mediated by ICSI.

A transgenic mouse model with inducible Tyrosinase gene expression using the tetracycline (Tet-on) system allows regulated rescue of abnormal chiasmatic projections found in albinism.

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET-ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.

Generation and phenotypic analysis of sigma receptor type I (sigma 1) knockout mice.

Sigma (sigma) sites are a type of nonopiate receptor whose role has been associated with several behaviours, including anxiety, depression, analgesia, learning processes and psychosis. Although there are several known sigma receptor types, only the type I receptor (sigma 1) has been cloned. To uncover the in vivo relevance of sigma-receptors, we have generated knockout mice for sigma 1. Despite the broad expression pattern found for the sigma 1-gene, homozygous mutant mice are viable, fertile and do not display any overt phenotype, compared with their wild-type litter-mates, in mixed genetic backgrounds. However, a significant decrease in the hypermotility response has been measured in knockout mice upon challenge with (+)SKF-10 047, in agreement with the involvement of sigma 1-receptors in the induction of psychostimulant actions. The activity of sigma 2-receptors seems to be unaffected in sigma 1-mutant mice. These knockout mice could contribute to better understand the in vivo role of sigma-receptors.