Targeting LIM kinases in taxane resistant tumors

International audienc

PD-1/PD-L1 Checkpoint Inhibitors Are Active in the Chicken Embryo Model and Show Antitumor Efficacy <i>In Ovo</i>

(1) Purpose: To assess the use of the chicken embryo (in ovo) model as an alternative in vivo model for immuno-oncology (IO) drug development, focusing on programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors. (2) Methods: First, the presence of immune cells in the model was detected through the immunophenotyping of chicken peripheral blood mononuclear cells (PBMCs) based on fluorescence activated cell sorting (FACS) analysis and the immunohistochemistry (IHC) analysis of in ovo tumor-infiltrating lymphocytes. Second, the cross-reactivity between one anti-human PD-1 Ab, pembrolizumab (KEYTRUDA®), and chicken PD-1 was verified through the labelling of chicken splenocytes with pembrolizumab by FACS analysis. Third, the blockade effect of pembrolizumab on chicken PBMCs was assessed in vitro through cytotoxicity assay based on MTT. Fourth, the CAM assay was used to estimate the anti-tumor performance of pembrolizumab through the analyses of tumor growth and chicken immune cell infiltration in tumors. Finally, the efficacy of several PD-1 or PD-L1 inhibitors (nivolumab, atezolizumab and avelumab) on tumor growth was further assessed using the CAM assay. (3) Results: The presence of CD3+, CD4+, CD8+ T lymphocytes and monocytes was confirmed by FACS and IHC analyses. During in vitro assays, pembrolizumab cross-reacted with chicken lymphocytes and induced PD-1/PD-L1 blockade, which permitted the restoration of chicken T-cell’s cytotoxicity against human lung cancer H460 tumor cells. All these in vitro results were correlated with in ovo findings based on the CAM assay: pembrolizumab inhibited H460 tumor growth and induced evident chicken immune cell infiltration (with significant chicken CD45, CD3, CD4, CD8 and CD56 markers) in tumors. Furthermore, the potency of the CAM assay was not limited to the application of pembrolizumab. Nivolumab, atezolizumab and avelumab also led to tumor growth inhibition in ovo, on different tumor models. (4) Conclusions: The chicken embryo affords a physiological, immune reactive, in vivo environment for IO research, which allows observation of how the immune system defense against tumor cells, as well as the different immune tolerance mechanisms leading to tumor immune escape. The encouraging results obtained with PD-1/PD-L1 inhibitors in this study reveal the potential use of the chicken embryo model as an alternative, fast, and reliable in vivo model in the different fields of IO drug discovery

Breast cancer dormancy is associated with a 4NG1 state and not senescence

Reactivation of dormant cancer cells can lead to cancer relapse, metastasis, and patient death. Dormancy is a nonproliferative state and is linked to late relapse and death. No targeted therapy is currently available to eliminate dormant cells, highlighting the need for a deeper understanding and reliable models. Here, we thoroughly characterize the dormant D2.OR and ZR-75-1, and proliferative D2A1 breast cancer cell line models in vivo and/or in vitro, and assess if there is overlap between a dormant and a senescent phenotype. We show that D2.OR but not D2A1 cells become dormant in the liver of an immunocompetent model. In vitro, we show that D2.OR and ZR-75-1 cells in response to a 3D environment or serum-free conditions are growth-arrested in G1, of which a subpopulation resides in a 4NG1 state. The dormancy state is reversible and not associated with a senescence phenotype. This will aid future research on breast cancer dormancy

Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

International audienceDespite the development of new therapeutic strategies, cancer remains one of the leading causes of mortality worldwide. One of the current major challenges is the resistance of cancers to chemotherapy treatments inducing metastases and relapse of the tumor. The Hedgehog receptor Patched (Ptch1) is overexpressed in many types of cancers. We showed that Ptch1 contributes to the efflux of doxorubicin and plays an important role in the resistance to chemotherapy in adrenocortical carcinoma (ACC), a rare cancer which presents strong resistance to the standard of care chemotherapy treatment. In the present study, we isolated and characterized a subpopulation of the ACC cell line H295R in which Ptch1 is overexpressed and more present at the cell surface. This cell subpopulation is more resistant to doxorubicin, grows as spheroids, and has a greater capability of clonogenicity, migration, and invasion than the parental cells. Xenograft experiments performed in mice and in ovo showed that this cell subpopulation is more tumorigenic and metastatic than the parental cells. These results suggest that this cell subpopulation has cancer stem-like or persistent cell properties which were strengthened by RNA-seq. If present in tumors from ACC patients, these cells could be responsible for therapy resistance, relapse, and metastases

Fibroblast growth factor-2 bound to specific dermal fibroblast-derived extracellular vesicles is protected from degradation

Fibroblast growth factor-2 (FGF2) has multiple roles in cutaneous wound healing but its natural low stability prevents the development of its use in skin repair therapies. Here we show that FGF2 binds the outer surface of dermal fibroblast (DF)-derived extracellular vesicles (EVs) and this association protects FGF2 from fast degradation. EVs isolated from DF cultured in the presence of FGF2 harbor FGF2 on their surface and FGF2 can bind purified EVs in absence of cells. Remarkably, FGF2 binding to EVs is restricted to a specific subpopulation of EVs, which do not express CD63 and CD81 markers. Treatment of DF with FGF2-EVs activated ERK and STAT signaling pathways and increased cell proliferation and migration. Local injection of FGF2-EVs improved wound healing in mice. We further demonstrated that binding to EVs protects FGF2 from both thermal and proteolytic degradation, thus maintaining FGF2 function. This suggests that EVs protect soluble factors from degradation and increase their stability and half-life. These results reveal a novel aspect of EV function and suggest EVs as a potential tool for delivering FGF2 in skin healing therapies

Data from: Genetic control of contagious asexuality in the pea aphid

Although evolutionary transitions from sexual to asexual reproduction are frequent in eukaryotes, the genetic bases of such shifts toward asexuality remain largely unknown. We addressed this issue in an aphid species where both sexual and obligate asexual lineages coexist in natural populations. These sexual and asexual lineages may occasionally interbreed because some asexual lineages maintain a residual production of males potentially able to mate with the females produced by sexual lineages. Hence, this species is an ideal model to study the genetic basis of the loss of sexual reproduction with quantitative genetic and population genomic approaches. Our analysis of the co-segregation of ~300 molecular markers and reproductive phenotype in experimental crosses pinpointed an X-linked region controlling obligate asexuality, this state of character being recessive. A population genetic analysis (>400-marker genome scan) on wild sexual and asexual genotypes from geographically distant populations under divergent selection for reproductive strategies detected a strong signature of divergent selection in the genomic region identified by the experimental crosses. These population genetic data confirm the implication of the candidate region in the control of reproductive mode in wild populations originating from 700 km apart. Patterns of genetic differentiation along chromosomes suggest bidirectional gene flow between populations with distinct reproductive modes, supporting contagious asexuality as a prevailing route to permanent parthenogenesis in pea aphids. This genetic system provides new insights into the mechanisms of coexistence of sexual and asexual aphid lineages

Genetic control of contagious asexuality in the pea aphid.

Although evolutionary transitions from sexual to asexual reproduction are frequent in eukaryotes, the genetic bases of such shifts toward asexuality remain largely unknown. We addressed this issue in an aphid species where both sexual and obligate asexual lineages coexist in natural populations. These sexual and asexual lineages may occasionally interbreed because some asexual lineages maintain a residual production of males potentially able to mate with the females produced by sexual lineages. Hence, this species is an ideal model to study the genetic basis of the loss of sexual reproduction with quantitative genetic and population genomic approaches. Our analysis of the co-segregation of ∼ 300 molecular markers and reproductive phenotype in experimental crosses pinpointed an X-linked region controlling obligate asexuality, this state of character being recessive. A population genetic analysis (>400-marker genome scan) on wild sexual and asexual genotypes from geographically distant populations under divergent selection for reproductive strategies detected a strong signature of divergent selection in the genomic region identified by the experimental crosses. These population genetic data confirm the implication of the candidate region in the control of reproductive mode in wild populations originating from 700 km apart. Patterns of genetic differentiation along chromosomes suggest bidirectional gene flow between populations with distinct reproductive modes, supporting contagious asexuality as a prevailing route to permanent parthenogenesis in pea aphids. This genetic system provides new insights into the mechanisms of coexistence of sexual and asexual aphid lineages

LIM Kinase inhibitor Pyr1 reduces the growth and metastatic load of breast cancers

International audienceLIM kinases (LIMK) are emerging targets for cancer therapy, and they function as network hubs to coordinate actin and microtubule dynamics. When LIMKs are inhibited, actin microfilaments are disorganized and microtubules are stabilized. Owing to their stabilizing effect on microtubules, LIMK inhibitors may provide a therapeutic strategy to treat taxane-resistant cancers. In this study, we investigated the effect of LIMK inhibition on breast tumor development and on paclitaxel-resistant tumors, using a novel selective LIMK inhibitor termed Pyr1. Treatment of breast cancer cells, including paclitaxel-resistant cells, blocked their invasion and proliferation in vitro and their growth in vivo in tumor xenograft assays. The tumor-invasive properties of Pyr1 were investigated in vivo by intravital microscopy of tumor xenografts. A striking change of cell morphology was observed with a rounded phenotype arising in a subpopulation of cells, while other cells remained elongated. Notably, although Pyr1 decreased the motility of elongated cells, it increased the motility of rounded cells in the tumor. Pyr1 administration prevented the growth of metastasis but not their spread. Overall, our results provided a preclinical proof of concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat taxane-resistant breast tumors and metastases. Cancer Res; 76(12); 3541-52. (c)2016 AAC

Genotype data used for genome scans and construction of genetic maps

The Excel sheet entitled "Genotype_genome_scan" contains the genotypes of 109 individuals at 436 microsatelitte markers used for genome scan analyses. The Excel sheet entitled "Genotype_genetic_map" contains genotype data at 343 polymorphic microsatelitte markers on a 3-generation pedigree that have been used to construct genetic maps