Pak1 pathway hyper-activation mediates resistance to endocrine therapy and CDK4/6 inhibitors in ER+ breast cancer.

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) have been approved in combination with endocrine therapy (ET) to treat estrogen receptor-positive (ER+) metastatic breast cancer (BC). However, drug resistance represents the leading cause of breast cancer patients mortality. This study aimed to identify novel resistance mechanisms to ER antagonists in combination with CDK4/6 inhibitors. We generated two ER+ BC cell lines, T47D and MCF7, resistant to the combination of the ER antagonist fulvestrant and CDK4/6i abemaciclib, named T47D-FAR and MCF7-FAR. Transcriptomic analysis revealed common up-regulation of genes involved in MAPK and epithelial to mesenchymal transition (EMT) pathways in FAR cells, sustaining their hyper-invasive phenotype and increased anchorage-independent growth, compared to sensitive cells. FAR cells showed higher p21-activated kinase 1 (Pak1) expression and phosphorylation levels than parental cells. PAK1 knockdown by siRNAs hampered cell proliferation, reduced anchorage-independent growth and invasive properties of T47D-FAR and MCF7-FAR, re-sensitizing them to fulvestrant and abemaciclib. Conversely, over-expression of PAK1 in MCF7 and T47D cells increased tumor spheroids' growth and invasion and reduced sensitivity to fulvestrant and abemaciclib, confirming its role in inducing drug resistance. Finally, treatment with Pak1 inhibitors, PF-3758309 (PF309) and NVS-PAK1-1, restored cell sensitivity to fulvestrant and abemaciclib of MCF7-FAR and T47D-FAR cells, both in vitro and in vivo. In conclusion, our data suggested a pivotal role for Pak1 in resistance to ET and CDK4/6i in ER+ breast cancers. These data might promote the rationale for the development of novel Pak1 inhibitors for treatment of patients with ER+ BC progressing on ET plus CDK4/6i.

A Tempered Particle Filter to Enhance the Assimilation of SAR-Derived Flood Extent Maps Into Flood Forecasting Models.

Data assimilation (DA) is a powerful tool to optimally combine uncertain model simulations and observations. Among DA techniques, the particle filter (PF) has gained attention for its capacity to deal with nonlinear systems and for its relaxation of the Gaussian assumption. However, the PF may suffer from degeneracy and sample impoverishment. In this study, we propose an innovative approach, based on a tempered particle filter (TPF), aiming at mitigating PFs issues, thus extending over time the assimilation benefits. Probabilistic flood maps derived from synthetic aperture radar data are assimilated into a flood forecasting model through an iterative process including a particle mutation in order to keep diversity within the ensemble. Results show an improvement of the model forecasts accuracy, with respect to the Open Loop: on average the root mean square error (RMSE) of water levels decrease by 80% at the assimilation time and by 60% 2 days after the assimilation. A comparison with the Sequential Importance Sampling (SIS) is carried out showing that although SIS performances are generally comparable to the TPF ones at the assimilation time, they tend to decrease more quickly. For instance, on average TPF-based RMSE are 20% lower compared to the SIS-based ones 2 days after the assimilation. The application of the TPF determines higher critical success index values compared to the SIS. On average the increase in performances lasts for almost 3 days after the assimilation. Our study provides evidence that the application of the variant of the TPF enables more persistent benefits compared to the SIS.

Antitumor efficacy of Kisspeptin in human malignant mesothelioma cells.

PURPOSE: Kisspeptin signaling, via its receptors GPR54, could be an essential players in the inhibition of mesothelioma progression, invasion and metastasis formation. The loss of KiSS1 by tumor cells has been associated with a metastatic phenotype but the mechanistic insights of this process are still unknown. EXPERIMENTAL DESIGN: The blockade of the metastatic process at early stage is a hot topic in cancer research. We studied the role of KiSS1 on proliferation, invasiveness, migration abilities of mesothelioma cell lines focusing on the effect on epithelial-to-mesenchymal transition (EMT). RESULTS: Treatment with the KiSS1 peptide or with a synthesis peptide with longer half-life, the FTM080, significantly inhibited cell proliferation, migration and invasion of mesothelioma cell lines; the same treatment reduced the activity of MMP-2 and MMP-9 determining consequently a marked reduction in the invasiveness of primary tumors and metastases. Thespecificexpression of EMT markers, as E-caderin, Vimentin, Slug and Snail, suggested the inhibition of EMT after treatment with KiSS1 as well as the preservation of epithelial components. CONCLUSION: Our results support anti-proliferative effect of KiSS1 in cancer cells and suggest that targeting the KiSS1/GPR54 system may represent a novel therapeutic approach for mesothelioma.

Hedgehog signalling pathway orchestrates angiogenesis in triple-negative breast cancers.

BACKGROUND: Several evidences suggest a marked angiogenic dependency in triple-negative breast cancer (TNBC) tumorigenesis and a potential sensitivity to anti-angiogenic agents. Herein, the putative role of Hedgehog (Hh) pathway in regulating TNBC-dependent angiogenesis was investigated. METHODS: Expression and regulation of the Hh pathway transcription factor glioma-associated oncogene homolog1 protein (GLI1) were studied on the endothelial compartment and on TNBC-initiated angiogenesis. To evaluate the translational relevance of our findings, the combination of paclitaxel with the Smo inhibitor NVP-LDE225 was tested in TNBC xenografted mice. RESULTS: Tissue microarray analysis on 200 TNBC patients showed GLI1 overexpression paired with vascular endothelial growth factor receptor 2 (VEGFR2) expression. In vitro, Hh pathway promotes TNBC progression in an autocrine manner, regulating the VEGF/VEGFR2 loop on cancer cell surface, and in a paracrine manner, orchestrating tumour vascularisation. These effects were counteracted by Smo pharmacological inhibition. In TNBC xenografted mice, scheduling NVP-LDE225 rather than bevacizumab provided a better sustained inhibition of TNBC cells proliferation and endothelial cells organisation. CONCLUSIONS: This study identifies the Hh pathway as one of the main regulators of tumour angiogenesis in TNBC, thus suggesting Hh inhibition as a potential new anti-angiogenic therapeutic option to be clinically investigated in GLI1 overexpressing TNBC patients.

Everolimus induces Met inactivation by disrupting the FKBP12/Met complex.

Inhibition of the mechanistic target of rapamycin (mTOR) is a promising treatment strategy for several cancer types. Rapamycin derivatives such as everolimus are allosteric mTOR inhibitors acting through interaction with the intracellular immunophilin FKBP12, a prolyl isomerase with different cellular functions. Although mTOR inhibitors have significantly improved survival of different cancer patients, resistance and lack of predictive factors of response remain unsolved issues. To elucidate the mechanisms of resistance to everolimus, we evaluated Met activation in everolimus-sensitive/resistant human cancer cells, in vitro and in vivo. Biochemical and computational analyses were performed. Everolimus-resistant cells were xenografted into mice (10/group) and studied for their response to everolimus and Met inhibitors. The statistical significance of the in vitro results was evaluated by Student's t test.Everolimus reduced Met phosphorylation in everolimus-sensitive cells. This event was mediated by the formation of a Met-FKBP12 complex, which in turn is disrupted by everolimus. Aberrant Met activation in everolimus-resistant cells and overexpression of wild-type/mutant Met caused everolimus resistance. Pharmacological inhibition and RNA silencing of Met are effective in condition of everolimus resistance (P<0.01). In mice xenografted with everolimus-resistant cells, the combination of everolimus with the Met inhibitor PHA665752 reduced tumor growth and induced a statistically significant survival advantage (combination vs control P=0.0005).FKBP12 binding is required for full Met activation and everolimus can inhibit Met. Persistent Met activation might sustain everolimus resistance. These results identify a novel everolimus mechanism of action and suggest the development of clinical strategies based on Met inhibitors in everolimus-resistant cancers.

Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen.

PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG) conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments.

Metformin increases antitumor activity of MEK inhibitors through GLI1 downregulation in LKB1 positive human NSCLC cancer cells.

PURPOSE: Metformin, widely used as antidiabetic drug, showed antitumoral effects expecially in combination with chemotherapy. Our group recently has demonstrated that metformin and gefitinib are synergistic in LKB1-wild-type NSCLC cells. In these models, metformin as single agent induced an activation and phosphorylation of mitogen-activated-protein-kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. EXPERIMENTAL DESIGN: Since single agent metformin enhances proliferating signals through the RAS/RAF/MAPK pathway, and several MEK inhibitors (MEK-I) demonstrated clinical efficacy in combination with other agents in NSCLC, we tested the effects of metformin plus MEK-I (selumetinib or pimasertib) on proliferation, invasiveness, migration abilities in vitro and in vivo in LKB1 positive NSCLC models harboring KRAS wild type and mutated gene. RESULTS: The combination of metformin with MEK-I showed a strong anti-proliferative and proapoptotic effect in Calu-3, H1299, H358 and H1975 human NSCLC cell lines, independently from the KRAS mutational status. The combination reduced the metastatic behaviour of NSCLC cells, via a downregulation of GLI1 trascritional activity, thus affecting the transition from an epithelial to a mesenchymal phenotype. Metformin and MEK-Is combinations also decreased the production and activity of MMP-2 and MMP-9 by reducing the NF-jB (p65) binding to MMP-2 and MMP-9 promoters. CONCLUSIONS: Metformin potentiates the antitumor activity of MEK-Is in human LKB1-wild-type NSCLC cell lines, independently from the KRAS mutational status, through GLI1 downregulation and by reducing the NF-jB (p65)-mediated transcription of MMP-2 and MMP-9.

Src inhibitors act through different mechanisms in Non-Small Cell Lung Cancer models depending on EGFR and RAS mutational status.

Resistance to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, often related to Ras or secondary EGFR mutations, is a relevant clinical issue in Non-Small Cell Lung Cancer (NSCLC). Although Src TK has been involved in such resistance, clinical development of its inhibitors has been so far limited. To better define the molecular targets of the Src TKIs saracatinib, dasatinib and bosutinib, we used a variety of in vitro/in vivo studies. Kinase assays supported by docking analysis demonstrated that all the compounds directly inhibit EGFR TK variants. However, in live cells only saracatinib efficiently reduced EGFR activation, while dasatinib was the most effective agent in inhibiting Src TK. Consistently, a pronounced anti-proliferative effect was achieved with saracatinib, in EGFR mutant cells, or with dasatinib, in wt EGFR/Ras mutant cells, poorly dependent on EGFR and erlotinib-resistant. We then identified the most effective drug combinations to overcome resistance to EGFR inhibitors, both in vitro and in nude mice: in T790M EGFR erlotinib-resistant cells, saracatinib with the anti-EGFR mAb cetuximab; in Ras mutant erlotinib-resistant models, dasatinib with the MEK inhibitor selumetinib. Src inhibitors may act with different mechanisms in NSCLCs, depending on EGFR/Ras mutational profile, and may be integrated with EGFR or MEK inhibitors for different cohorts of NSCLCs.

A new human dyskerin isoform with cytoplasmic localization.

BACKGROUND: The human DKC1 gene is causative of X-linked dyskeratosis congenita (X-DC), a syndrome characterized by mucocutaneous features, bone marrow failure, tumor susceptibility, perturbation of stem cell function, and premature aging. DKC1 is thought to produce a single protein, named dyskerin, which shows strict nucleolar localization and participates in at least two distinct nuclear functional complexes: the H/ACA small nucleolar ribonucleoproteic complex involved in RNA pseudouridylation and the active telomerase complex. METHODS: By bioinformatics and molecular analyses we identified a DKC1 splice variant able to encode a truncated form of dyskerin, confirmed its active expression in diverse human tissues by RT-PCR, and showed by immunoblotting and immunocytochemistry experiments that it actually encodes a novel protein. Stably transfected clones over-expressing the new isoform were analyzed for growth, morphology and adhesion properties. RESULTS: Our results show that DKC1 encodes a new alternatively spliced mRNA able to direct the synthesis of a variant dyskerin with unexpected cytoplasmic localization. Intriguingly, when over-expressed in HeLa cells, the new isoform promotes cell to cell and cell to substratum adhesion, increases the cell proliferation rate and leads to cytokeratin hyper-expression. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our results highlight a novel degree of complexity and regulation of the human DKC1 gene and reveal that it can play a further, unpredicted role in cell adhesion. The identification of a dyskerin cytoplasmic variant reinforces the view that other mechanisms, in addition to telomere instability, can significantly contribute to the pathogenesis of the X-DC, and suggests that DKC1 nucleolar and cytoplasmic functions might cumulatively account for the plethora of manifestations displayed by this syndrome.