The molecular basis of disease progression from UV-induced precancerous actinic keratosis (AK) to malignant invasive cutaneous squamous cell carcinoma (cSCC) and potentially lethal metastatic disease ...remains unclear. DNA sequencing studies have revealed a massive mutational burden but have yet to illuminate mechanisms of disease progression. Here we perform RNAseq transcriptomic profiling of 110 patient samples representing normal sun-exposed skin, AK, primary and metastatic cSCC and reveal a disease continuum from a differentiated to a progenitor-like state. This is accompanied by the orchestrated suppression of master regulators of epidermal differentiation, dynamic modulation of the epidermal differentiation complex, remodelling of the immune landscape and an increase in the preponderance of tumour specific keratinocytes. Comparative systems analysis of human cSCC coupled with the generation of genetically engineered murine models reveal that combinatorial sequential inactivation of the tumour suppressor genes Tgfbr2, Trp53, and Notch1 coupled with activation of Ras signalling progressively drives cSCC progression along a differentiated to progenitor axis. Taken together we provide a comprehensive map of the cSCC disease continuum and reveal potentially actionable events that promote and accompany disease progression.
The Rab GTPase effector, Rab-coupling protein (RCP) is known to promote invasive behaviour in vitro by controlling integrin and receptor tyrosine kinase (RTK) trafficking, but how RCP influences ...metastasis in vivo is unclear. Here we identify an RTK of the Eph family, EphA2, to be a cargo of an RCP-regulated endocytic pathway which controls cell:cell repulsion and metastasis in vivo. Phosphorylation of RCP at Ser
by Lemur tyrosine kinase-3 (LMTK3) and of EphA2 at Ser
by Akt are both necessary to promote Rab14-dependent (and Rab11-independent) trafficking of EphA2 which generates cell:cell repulsion events that drive tumour cells apart. Genetic disruption of RCP or EphA2 opposes cell:cell repulsion and metastasis in an autochthonous mouse model of pancreatic adenocarcinoma-whereas conditional knockout of another RCP cargo, α5 integrin, does not suppress pancreatic cancer metastasis-indicating a role for RCP-dependent trafficking of an Eph receptor to drive tumour dissemination in vivo.
Pancreatic ductal adenocarcinoma (PDAC) features a near-universal mutation in KRAS. Additionally, the tumor suppressor PTEN is lost in ∼10% of patients, and in mouse models, this dramatically ...accelerates tumor progression. While oncogenic KRAS and phosphatidylinositol 3-kinase (PI3K) cause divergent metabolic phenotypes individually, how they synergize to promote tumor metabolic alterations and dependencies remains unknown. We show that in KRAS-driven murine PDAC cells, loss of Pten strongly enhances both mTOR signaling and macropinocytosis. Protein scavenging alleviates sensitivity to mTOR inhibition by rescuing AKT phosphorylation at serine 473 and consequently cell proliferation. Combined inhibition of mTOR and lysosomal processing of internalized protein eliminates the macropinocytosis-mediated resistance. Our results indicate that mTORC2, rather than mTORC1, is an important regulator of protein scavenging and that protein-mediated resistance could explain the lack of effectiveness of mTOR inhibitors in certain genetic backgrounds. Concurrent inhibition of mTOR and protein scavenging might be a valuable therapeutic approach.
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•PTEN loss increases scavenging, conferring resistance to mTOR inhibition in PDAC•mTORC2 selectively regulates macropinocytosis independently of its kinase activity•Protein scavenging restores pAkt serine 473, leading to recovery of proliferation•Lysosomal blockade eliminates the protein-mediated resistance to mTOR inhibition
Michalopoulou et al. show that in pancreatic cancer cells with oncogenic KRAS and PTEN loss, scavenging of extracellular protein provides resistance against mTOR inhibition. Protein scavenging circumvents mTORC2 signaling by rescuing AKT phosphorylation. Additional treatment with AKT or lysosomal inhibitors strongly potentiates the effect of mTOR inhibitors.
Cancer invasion and metastasis occur in a complex three-dimensional (3D) environment, with reciprocal feedback from the surrounding host tissue and vasculature-governing behavior. In this study, we ...used a novel intravital method that revealed spatiotemporal regulation of Src activity in response to the anti-invasive Src inhibitor dasatinib. A fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) Src biosensor was used to monitor drug-targeting efficacy in a transgenic p53-mutant mouse model of pancreatic cancer. In contrast to conventional techniques, FLIM-FRET analysis allowed for accurate, time-dependent, live monitoring of drug efficacy and clearance in live tumors. In 3D organotypic cultures, we showed that a spatially distinct gradient of Src activity exists within invading tumor cells, governed by the depth of penetration into complex matrices. In parallel, this gradient was also found to exist within live tumors, where Src activity is enhanced at the invasive border relative to the tumor cortex. Upon treatment with dasatinib, we observed a switch in activity at the invasive borders, correlating with impaired metastatic capacity in vivo. Src regulation was governed by the proximity of cells to the host vasculature, as cells distal to the vasculature were regulated differentially in response to drug treatment compared with cells proximal to the vasculature. Overall, our results in live tumors revealed that a threshold of drug penetrance exists in vivo and that this can be used to map areas of poor drug-targeting efficiency within specific tumor microenvironments. We propose that using FLIM-FRET in this capacity could provide a useful preclinical tool in animal models before clinical translation.
Inhibition of the PARP superfamily tankyrase enzymes suppresses Wnt/β-catenin signalling in tumour cells. Here, we describe here a novel, drug-like small molecule inhibitor of tankyrase MSC2504877 ...that inhibits the growth of APC mutant colorectal tumour cells. Parallel siRNA and drug sensitivity screens showed that the clinical CDK4/6 inhibitor palbociclib, causes enhanced sensitivity to MSC2504877. This tankyrase inhibitor-CDK4/6 inhibitor combinatorial effect is not limited to palbociclib and MSC2504877 and is elicited with other CDK4/6 inhibitors and toolbox tankyrase inhibitors. The addition of MSC2504877 to palbociclib enhances G
cell cycle arrest and cellular senescence in tumour cells. MSC2504877 exposure suppresses the upregulation of Cyclin D2 and Cyclin E2 caused by palbociclib and enhances the suppression of phospho-Rb, providing a mechanistic explanation for these effects. The combination of MSC2504877 and palbociclib was also effective in suppressing the cellular hyperproliferative phenotype seen in Apc defective intestinal stem cells in vivo. However, the presence of an oncogenic Kras p.G12D mutation in mice reversed the effects of the MSC2504877/palbociclib combination, suggesting one molecular route that could lead to drug resistance.
Myeloid cells are pivotal within the immunosuppressive tumour microenvironment. The accumulation of tumour-modified myeloid cells derived from monocytes or neutrophils - termed 'myeloid-derived ...suppressor cells' - and tumour-associated macrophages is associated with poor outcome and resistance to treatments such as chemotherapy and immune checkpoint inhibitors. Unfortunately, there has been little success in large-scale clinical trials of myeloid cell modulators, and only a few distinct strategies have been used to target suppressive myeloid cells clinically so far. Preclinical and translational studies have now elucidated specific functions for different myeloid cell subpopulations within the tumour microenvironment, revealing context-specific roles of different myeloid cell populations in disease progression and influencing response to therapy. To improve the success of myeloid cell-targeted therapies, it will be important to target tumour types and patient subsets in which myeloid cells represent the dominant driver of therapy resistance, as well as to determine the most efficacious treatment regimens and combination partners. This Review discusses what we can learn from work with the first generation of myeloid modulators and highlights recent developments in modelling context-specific roles for different myeloid cell subtypes, which can ultimately inform how to drive more successful clinical trials.
RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine and the ...mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation. Knocking down
from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to influencing stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumourigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.
IMPORTANCE: Postpartum depression (PPD) is one of the most common medical complications during and after pregnancy, negatively affecting both mother and child. OBJECTIVE: To demonstrate the efficacy ...and safety of zuranolone, a neuroactive steroid γ-aminobutyric acid receptor–positive allosteric modulator, in PPD. DESIGN, SETTING, AND PARTICIPANTS: This phase 3, double-blind, randomized, outpatient, placebo-controlled clinical trial was conducted between January 2017 and December 2018 in 27 enrolling US sites. Participant were women aged 18 to 45 years, 6 months or fewer post partum, with PPD (major depressive episode beginning third trimester or ≤4 weeks postdelivery), and baseline 17-item Hamilton Rating Scale for Depression (HAMD-17) score of 26 or higher. Analysis was intention to treat and began December 2018 and ended March 2019. INTERVENTIONS: Randomization 1:1 to placebo:zuranolone, 30 mg, administered orally each evening for 2 weeks. MAIN OUTCOMES AND MEASURES: Primary end point was change from baseline in HAMD-17 score for zuranolone vs placebo at day 15. Secondary end points included changes from baseline in HAMD-17 total score at other time points, HAMD-17 response (≥50% score reduction) and remission (score ≤7) rates, Montgomery-Åsberg Depression Rating Scale score, and Hamilton Rating Scale for Anxiety score. Safety was assessed by adverse events and clinical assessments. RESULTS: Of 153 randomized patients, the efficacy set comprised 150 patients (mean SD age, 28.3 5.4 years), and 148 (98.7%) completed treatment. A total of 76 patients were randomized to placebo, and 77 were randomized to zuranolone, 30 mg. Zuranolone demonstrated significant day 15 HAMD-17 score improvements from baseline vs placebo (−17.8 vs −13.6; difference, −4.2; 95% CI, −6.9 to −1.5; P = .003). Sustained differences in HAMD-17 scores favoring zuranolone were observed from day 3 (difference, −2.7; 95% CI, −5.1 to −0.3; P = .03) through day 45 (difference, −4.1; 95% CI, −6.7 to −1.4; P = .003). Sustained differences at day 15 favoring zuranolone were observed in HAMD-17 response (odds ratio, 2.63; 95% CI, 1.34-5.16; P = .005), HAMD-17 score remission (odds ratio, 2.53; 95% CI, 1.24-5.17; P = .01), change from baseline for Montgomery-Åsberg Depression Rating Scale score (difference, −4.6; 95% CI, −8.3 to −0.8; P = .02), and Hamilton Rating Scale for Anxiety score (difference, −3.9; 95% CI, −6.7 to −1.1; P = .006). One patient per group experienced a serious adverse event (confusional state in the zuranolone group and pancreatitis in the placebo group). One patient in the zuranolone group discontinued because of an adverse event vs none for placebo. CONCLUSIONS AND RELEVANCE: In this randomized clinical trial, zuranolone improved the core symptoms of depression as measured by HAMD-17 scores in women with PPD and was generally well tolerated, supporting further development of zuranolone in the treatment of PPD. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02978326
The original version of this Article contained an error in the spelling of the author Miryam Müller, which was incorrectly given as Miryam Müeller. This has now been corrected in both the PDF and ...HTML versions of the Article.
Expression of the Rac-guanine nucleotide exchange factor (RacGEF), P-Rex1 is a key determinant of progression to metastasis in a number of human cancers. In accordance with this proposed role in ...cancer cell invasion and metastasis, we find that ectopic expression of P-Rex1 in an immortalised human fibroblast cell line is sufficient to drive multiple migratory and invasive phenotypes. The invasive phenotype is greatly enhanced by the presence of a gradient of serum or platelet-derived growth factor, and is dependent upon the expression of functional PDGF receptor β. Consistently, the invasiveness of WM852 melanoma cells, which endogenously express P-Rex1 and PDGFRβ, is opposed by siRNA of either of these proteins. Furthermore, the current model of P-Rex1 activation is advanced through demonstration of P-Rex1 and PDGFRβ as components of the same macromolecular complex. These data suggest that P-Rex1 has an influence on physiological migratory processes, such as invasion of cancer cells, both through effects upon classical Rac1-driven motility and a novel association with RTK signalling complexes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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