Pathway profiling of a novel SRC inhibitor, AZD0424, in combination with MEK inhibitors

A more comprehensive understanding of how cells respond to drug intervention, the likely immediate signalling responses and how resistance may develop within different microenvironments will help inform treatment regimes. The nonreceptor tyrosine kinase SRC regulates many cellular signalling processes, and pharmacological inhibition has long been a target of cancer drug discovery projects. Here, we describe the in vitro and in vivo characterisation of the small‐molecule SRC inhibitor AZD0424. We show that AZD0424 potently inhibits the phosphorylation of tyrosine‐419 of SRC (IC50 ~ 100 nm) in many cancer cell lines; however, inhibition of cell viability, via a G1 cell cycle arrest, was observed only in a subset of cancer cell lines in the low (on target) micromolar range. We profiled the changes in intracellular pathway signalling in cancer cells following exposure to AZD0424 and other targeted therapies using reverse‐phase protein array (RPPA) analysis. We demonstrate that SRC is activated in response to treatment of KRAS‐mutant colorectal cell lines with MEK inhibitors (trametinib or AZD6244) and that AZD0424 abrogates this. Cell lines treated with trametinib or AZD6244 in combination with AZD0424 had reduced EGFR, FAK and SRC compensatory activation, and cell viability was synergistically inhibited. In vivo, trametinib treatment of mice‐bearing HCT116 tumours increased phosphorylation of SRC on Tyr419, and, when combined with AZD0424, inhibition of tumour growth was greater than with trametinib alone. We also demonstrate that drug‐induced resistance to trametinib is not re‐sensitised by AZD0424 treatment in vitro, likely as a result of multiple compensatory signalling mechanisms; however, inhibition of SRC remains an effective way to block invasion of trametinib‐resistant tumour cells. These data imply that SRC inhibition may offer a useful addition to MEK inhibitor combination strategies

A synergistic anti-cancer FAK and HDAC inhibitor combination discovered by a novel chemical-genetic high-content phenotypic screen

We mutated the focal adhesion kinase (FAK) catalytic domain to inhibit binding of the chaperone Cdc37 and ATP, mimicking the actions of a FAK kinase inhibitor. We re-expressed mutant and wild-type FAK in squamous cell carcinoma (SCC) cells from which endogenous FAK had been deleted, genetically fixing one axis of a FAK inhibitor combination high-content phenotypic screen to discover drugs that may synergize with FAK inhibitors. Histone deacetylase (HDAC) inhibitors represented the major class of compounds that potently induced multiparametric phenotypic changes when FAK was rendered kinase-defective or inhibited pharmacologically in SCC cells. Combined FAK and HDAC inhibitors arrest proliferation and induce apoptosis in a sub-set of cancer cell lines in vitro and efficiently inhibit their growth as tumors in vivo. Mechanistically, HDAC inhibitors potentiate inhibitor-induced FAK inactivation and impair FAK-associated nuclear YAP in sensitive cancer cell lines. Here we report the discovery of a new, clinically actionable, synergistic combination between FAK and HDAC inhibitors

WT1 expression in breast cancer disrupts the epithelial/mesenchymal balance of tumour cells and correlates with the metabolic response to docetaxel

WT1 is a transcription factor which regulates the epithelial-mesenchymal balance during embryonic development and, if mutated, can lead to the formation of Wilms' tumour, the most common paediatric kidney cancer. Its expression has also been reported in several adult tumour types, including breast cancer, and usually correlates with poor outcome. However, published data is inconsistent and the role of WT1 in this malignancy remains unclear. Here we provide a complete study of WT1 expression across different breast cancer subtypes as well as isoform specific expression analysis. Using in vitro cell lines, clinical samples and publicly available gene expression datasets, we demonstrate that WT1 plays a role in regulating the epithelial-mesenchymal balance of breast cancer cells and that WT1-expressing tumours are mainly associated with a mesenchymal phenotype. WT1 gene expression also correlates with CYP3A4 levels and is associated with poorer response to taxane treatment. Our work is the first to demonstrate that the known association between WT1 expression in breast cancer and poor prognosis is potentially due to cancer-related epithelial-to-mesenchymal transition (EMT) and poor chemotherapy response

Loss of Integrin-linked kinase sensitizes breast cancer to SRC inhibitors

SRC is a nonreceptor tyrosine kinase with key roles in breast cancer development and progression. Despite this, SRC tyrosine kinase inhibitors have so far failed to live up to their promise in clinical trials, with poor overall response rates. We aimed to identify possible synergistic gene–drug interactions to discover new rational combination therapies for SRC inhibitors. An unbiased genome-wide CRISPR-Cas9 knockout screen in a model of triple-negative breast cancer revealed that loss of integrin-linked kinase (ILK) and its binding partners α-Parvin and PINCH-1 sensitizes cells to bosutinib, a clinically approved SRC/ABL kinase inhibitor. Sensitivity to bosutinib did not correlate with ABL dependency; instead, bosutinib likely induces these effects by acting as a SRC tyrosine kinase inhibitor. Furthermore, in vitro and in vivo models showed that loss of ILK enhanced sensitivity to eCF506, a novel and highly selective inhibitor of SRC with a unique mode of action. Whole-genome RNA sequencing following bosutinib treatment in ILK knockout cells identified broad changes in the expression of genes regulating cell adhesion and cell–extracellular matrix. Increased sensitivity to SRC inhibition in ILK knockout cells was associated with defective adhesion, resulting in reduced cell number as well as increased G(1) arrest and apoptosis. These findings support the potential of ILK loss as an exploitable therapeutic vulnerability in breast cancer, enhancing the effectiveness of clinical SRC inhibitors. SIGNIFICANCE: A CRISPR-Cas9 screen reveals that loss of integrin-linked kinase synergizes with SRC inhibition, providing a new opportunity for enhancing the clinical effectiveness of SRC inhibitors in breast cancer

Cytoplasmic innate immune sensing by the caspase-4 non-canonical inflammasome promotes cellular senescence

Cytoplasmic recognition of microbial lipopolysaccharides (LPS) in human cells is elicited by the caspase-4 and caspase-5 noncanonical inflammasomes, which induce a form of inflammatory cell death termed pyroptosis. Here we show that LPS-mediated activation of caspase-4 also induces a stress response promoting cellular senescence, which is dependent on the caspase-4 substrate gasdermin-D and the tumor suppressor p53. Furthermore, we found that the caspase-4 noncanonical inflammasome is induced and assembled in response to oncogenic RAS signaling during oncogene-induced senescence (OIS). Moreover, targeting caspase-4 expression in OIS showed its critical role in the senescence-associated secretory phenotype and the cell cycle arrest induced in cellular senescence. Finally, we observed that caspase-4 induction occurs in vivo in mouse models of tumor suppression and ageing. Altogether, we are showing that cellular senescence is induced by cytoplasmic LPS recognition by the noncanonical inflammasome and that this pathway is conserved in the cellular response to oncogenic stress.This work was funded by Cancer Research UK (CRUK) (C47559/A16243 Training & Career Development Board - Career Development Fellowship), the University of Edinburgh Chancellor’s Fellowship R42576 MRC, and the Ministry of Science and Innovation of the Government of Spain (Proyecto PID2020-117860GB-I00 financiado por MCIN/ AEI /10.13039/501100011033). J.C.A. was supported by CRUK, the University of Edinburgh and is supported by the Spanish National Research Council (CSIC). P.H., I.F.D and N.T. were funded by the University of Edinburgh. A.Q. was funded by CRUK. J.F.P and A.B.L. are funded by NIH grants: 1R01AG068048-01; P01 AG062413; 1UG3 CA268103-01. J.B. was funded by BBSRC (BB/K017314/1). V.S-B is supported by funding from the University of Edinburgh and Medical Research Council (MC_UU_00009/2). F.R.M is funded by a Wellcome Trust Clinical Research Fellowship through the Edinburgh Clinical Academic Track (ECAT) (203913/Z/16/Z). M.M. was supported by CRUK Edinburgh Centre Award (C157/A25140). V.G.B. is funded by CRUK (C157/A24837) and the University of Edinburgh

ISGylation drives basal breast tumour progression by promoting EGFR recycling and Akt signalling

ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander.</p

Kindlin-1 promotes pulmonary breast cancer metastasis

Abstract In breast cancer, increased expression of the cytoskeletal adaptor protein Kindlin-1 has been linked to increased risks of lung metastasis, but the functional basis is unknown. Here, we show that in a mouse model of polyomavirus middle T antigen–induced mammary tumorigenesis, loss of Kindlin-1 reduced early pulmonary arrest and later development of lung metastasis. This phenotype relied on the ability of Kindlin-1 to bind and activate β integrin heterodimers. Kindlin-1 loss reduced α4 integrin–mediated adhesion of mammary tumor cells to the adhesion molecule VCAM-1 on endothelial cells. Treating mice with an anti–VCAM-1 blocking antibody prevented early pulmonary arrest. Kindlin-1 loss also resulted in reduced secretion of several factors linked to metastatic spread, including the lung metastasis regulator tenascin-C, showing that Kindlin-1 regulated metastatic dissemination by an additional mechanism in the tumor microenvironment. Overall, our results show that Kindlin-1 contributes functionally to early pulmonary metastasis of breast cancer. Significance: These findings provide a mechanistic proof in mice that Kindin-1, an integrin-binding adaptor protein, is a critical mediator of early lung metastasis of breast cancer. Cancer Res; 78(6); 1484–96. ©2018 AACR.</jats:p

Transcript and protein profiling identifies signaling, growth arrest, apoptosis, and NF-κB survival signatures following GNRH receptor activation

GNRH significantly inhibits proliferation of a proportion of cancer cell lines by activating GNRH receptor (GNRHR)-G protein signaling. Therefore, manipulation of GNRHR signaling may have an under-utilized role in treating certain breast and ovarian cancers. However, the precise signaling pathways necessary for the effect and the features of cellular responses remain poorly defined. We used transcriptomic and proteomic profiling approaches to characterize the effects of GNRHR activation in sensitive cells (HEK293-GNRHR, SCL60) in vitro and in vivo, compared to unresponsive HEK293. Analyses of gene expression demonstrated a dynamic response to the GNRH superagonist Triptorelin. Early and mid-phase changes (0.5–1.0 h) comprised mainly transcription factors. Later changes (8–24 h) included a GNRH target gene, CGA, and up- or downregulation of transcripts encoding signaling and cell division machinery. Pathway analysis identified altered MAPK and cell cycle pathways, consistent with occurrence of G(2)/M arrest and apoptosis. Nuclear factor kappa B (NF-κB) pathway gene transcripts were differentially expressed between control and Triptorelin-treated SCL60 cultures. Reverse-phase protein and phospho-proteomic array analyses profiled responses in cultured cells and SCL60 xenografts in vivo during Triptorelin anti-proliferation. Increased phosphorylated NF-κB (p65) occurred in SCL60 in vitro, and p-NF-κB and IκBϵ were higher in treated xenografts than controls after 4 days Triptorelin. NF-κB inhibition enhanced the anti-proliferative effect of Triptorelin in SCL60 cultures. This study reveals details of pathways interacting with intense GNRHR signaling, identifies potential anti-proliferative target genes, and implicates the NF-κB survival pathway as a node for enhancing GNRH agonist-induced anti-proliferation