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The RAS–RAF–MEK–ERK signaling cascade is among the most frequently mutated pathways in human cancer. Approximately 50% of melanoma patients possess a druggable hotspot V600E/K mutation in the BRAF protein kinase. FDA-approved combination therapies of BRAF and MEK inhibitors are available that provide survival benefits to patients with a BRAF V600 mutation. Non-V600 BRAF mutants are found in many cancers, and are more prevalent than V600 mutations in certain tumor types. For example, between 50–80% of BRAF mutations in non-small cell lung cancer and 22–30% in colorectal cancer encode for non-V600 mutants. As next generation sequencing becomes increasingly used in clinical practice, oncologists are frequently identifying non-V600 BRAF mutations in their patient’s tumors, but are uncertain of viable therapeutic options that could be employed for optimal treatment. From recent studies, a new classification system is emerging for BRAF mutations based on biochemical and signaling mechanisms associated with these mutants. Class I BRAF mutations affect amino acid V600 and signal as RAS-independent active monomers, class II mutations function as RAS-independent activated dimers, and class III mutations are kinase impaired but increase signaling through the MAPK pathway due to enhanced RAS binding and subsequent CRAF activation. These distinct classes of BRAF mutations predict response to targeted therapies and have important implications for future drug development. Herein, we discuss pre-clinical and clinical findings that may lead to improved treatments for all classes of BRAF mutant cancers.

Single-cell technologies have revealed the complexity of the tumour immune microenvironment with unparalleled resolution 1 – 9 . Most clinical strategies rely on histopathological stratification of tumour subtypes, yet the spatial context of single-cell phenotypes within these stratified subgroups is poorly understood. Here we apply imaging mass cytometry to characterize the tumour and immunological landscape of samples from 416 patients with lung adenocarcinoma across five histological patterns. We resolve more than 1.6 million cells, enabling spatial analysis of immune lineages and activation states with distinct clinical correlates, including survival. Using deep learning, we can predict with high accuracy those patients who will progress after surgery using a single 1-mm 2 tumour core, which could be informative for clinical management following surgical resection. Our dataset represents a valuable resource for the non-small cell lung cancer research community and exemplifies the utility of spatial resolution within single-cell analyses. This study also highlights how artificial intelligence can improve our understanding of microenvironmental features that underlie cancer progression and may influence future clinical practice. Using imaging mass cytometry, the tumour and immunological spatial landscapes of 416 lung adenocarcinomas are characterized, which, when combined with deep learning, can predict clinical outcomes with high accuracy.

Nada Jabado and colleagues report identification of gain-of-function mutations in ACVR1 , which encodes activin A receptor type I, in midline pediatric high-grade astrocytomas. Pediatric midline high-grade astrocytomas (mHGAs) are incurable with few treatment targets identified. Most tumors harbor mutations encoding p.Lys27Met in histone H3 variants. In 40 treatment-naive mHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with histone H3.1 p.Lys27Met substitution, whereas FGFR1 mutations or fusions occur in thalamic tumors associated with histone H3.3 p.Lys27Met substitution. Hyperactivation of the bone morphogenetic protein (BMP)-ACVR1 developmental pathway in mHGAs harboring ACVR1 mutations led to increased levels of phosphorylated SMAD1, SMAD5 and SMAD8 and upregulation of BMP downstream early-response genes in tumor cells. Global DNA methylation profiles were significantly associated with the p.Lys27Met alteration, regardless of the mutant histone H3 variant and irrespective of tumor location, supporting the role of this substitution in driving the epigenetic phenotype. This work considerably expands the number of potential treatment targets and further justifies pretreatment biopsy in pediatric mHGA as a means to orient therapeutic efforts in this disease.

There is increasing evidence that the expression of CD109, a GPI-anchored cell surface protein is dysregulated in squamous cell carcinoma (SCC). However, the functional role of CD109 in SCC progression is poorly understood. In current study, we demonstrate that CD109 is a critical regulator of epithelial phenotype in SSC cells. CD109 levels inversely correlate with TGF-β signaling, EMT, migration, and invasion in cultured SCC cells. CRISPR/Cas9-mediated knockout CD109 (CD109 KO) in SCC cells represses epithelial traits and promotes the mesenchymal phenotype, as evidenced by elevated expression of mesenchymal proteins and markers of epithelial to mesenchymal transition. Treatment with recombinant CD109 protein causes CD109 KO cells to regain their epithelial traits. CD109 loss results in pronounced alterations of gene expression as detected by microarray analysis and in dysregulation of 15 important signalling pathways as shown by KEGG pathway cluster analysis. Validation using 52 human oral SCC tumor samples show that CD109 levels inversely correlate with tumor grade and the activation state of one such pathway, the TGF-β signaling pathway. Taken together, our findings highlight a novel role for CD109 as a gatekeeper of the epithelial phenotype by regulating TGF-β pathway in SCC cells.

Neutrophils are the first leukocytes recruited to sites of inflammation, where they execute anti-microbial functions to eliminate infectious agents. These functions include phagocytosis, release of reactive oxygen species and the formation of neutrophil extracellular traps via NETosis. Neutrophils are receiving increasing attention in the context of cancer, where these same neutrophil-associated functions are also important for modulating tumor growth and metastatic progression. Neutrophils are phenotypically heterogeneous and, depending on the context, exert anti- or pro-tumorigenic functions. Increasing evidence also suggests an important role of neutrophils and their involvement in promoting multiple steps of the metastatic cascade. The steps include: (1) local invasion and intravasation of cancer cells into circulation, (2) survival of cancer cells in the bloodstream and extravasation at a distant site, (3) early cancer cell seeding/survival, and (4) progressive growth of cancer cells to form macroscopic metastases. Although neutrophil functions designed to eliminate infectious agents can also eliminate tumor cells, their dysregulation can promote tumor growth and enable metastasis at multiple steps along the metastatic cascade. In this review, we will provide an overview of the current advances in neutrophil biology in the context of cancer. We also discuss the emerging field of immunometabolism, in which the rewiring of alternative metabolic pathways within neutrophils can impact their pro-tumorigenic/pro-metastatic functions.

Background Cold-inducible RNA-binding protein (CIRBP) is a stress-induced mRNA-binding protein associated with clinical outcomes in a variety of human disease states. The role of CIRBP as a role as a prognostic biomarker in breast cancer (BC) has yet to be established. Findings We describe a clinically annotated tissue micro-array cohort of 1406 hormone receptor positive (HR +) and 281 triple negative primary breast cancers (TNBC) stained by immunohistochemistry (IHC) for CIRBP. Statistical analyses were performed with the Kaplan–Meier estimator, as well as univariate and multivariate Cox proportional-hazards models. Multivariate models incorporated tumor size, lymph node status, grade and CIRBP expression levels. Co-primary endpoints were overall survival (OS) and progression-free survival (PFS). In N = 281 primary TNBCs, high levels of CIRBP expression by IHC was associated with poor prognosis in multivariate analysis (OS: adjusted hazard ratio (aHR) 2.05, 95% confidence interval (CI) 1.24–3.41, P = 0.005. PFS: aHR 2.46, 95% CI 1.33–4.57, P = 0.004). However, in N = 1406 HR + primary BC, CIRBP expression was correlated with favorable prognosis (OS: aHR 0.927, 95% CI 0.88–0.98, P = 0.005. PFS: aHR 0.904, 95% CI 0.85–0.96, P = 0.002). Conclusions CIRBP expression is associated with poor prognosis in TNBC but not HR + BC patients. This finding highlights the prognostic significance of CIRBP in TNBC and suggests differential underlying mRNA targets bound and modulated by CIRBP in TNBC and HR + BC, respectively.

We have previously shown that lipoma preferred partner (LPP) mediates TGFβ-induced breast cancer cell migration and invasion. Herein, we demonstrate that diminished LPP expression reduces circulating tumour cell numbers, impairs cancer cell extravasation and diminishes lung metastasis. LPP localizes to invadopodia, along with Tks5/actin, at sites of matrix degradation and at the tips of extravasating breast cancer cells as revealed by intravital imaging of the chick chorioallantoic membrane (CAM). Invadopodia formation, breast cancer cell extravasation and metastasis require an intact LPP LIM domain and the ability of LPP to interact with α-actinin. Finally, we show that Src-mediated LPP phosphorylation at specific tyrosine residues (Y245/301/302) is critical for invadopodia formation, breast cancer cell invasion and metastasis. Together, these data define a previously unknown function for LPP in the formation of invadopodia and reveal a requirement for LPP in mediating the metastatic ability of breast cancer cells. Lipoma preferred partner (LPP) mediates TGFβ-induced breast cancer cell migration and invasion. Here the authors show that LPP is a Src-family kinase substrate that regulates the formation of protrusions of the plasma membrane -called invadopodia- required for breast cancer metastasis.

Chemotherapy resistance is a critical barrier in cancer treatment. Metabolic adaptations have been shown to fuel therapy resistance; however, little is known regarding the generality of these changes and whether specific therapies elicit unique metabolic alterations. Using a combination of metabolomics, transcriptomics, and functional genomics, we show that two anthracyclines, doxorubicin and epirubicin, elicit distinct primary metabolic vulnerabilities in human breast cancer cells. Doxorubicin-resistant cells rely on glutamine to drive oxidative phosphorylation and de novo glutathione synthesis, while epirubicin-resistant cells display markedly increased bioenergetic capacity and mitochondrial ATP production. The dependence on these distinct metabolic adaptations is revealed by the increased sensitivity of doxorubicin-resistant cells and tumor xenografts to buthionine sulfoximine (BSO), a drug that interferes with glutathione synthesis, compared with epirubicin-resistant counterparts that are more sensitive to the biguanide phenformin. Overall, our work reveals that metabolic adaptations can vary with therapeutics and that these metabolic dependencies can be exploited as a targeted approach to treat chemotherapy-resistant breast cancer.

Glycoprotein non-metastatic melanoma protein B (GPNMB)/Osteoactivin (OA) is a transmembrane protein expressed in approximately 40-75% of breast cancers. GPNMB/OA promotes the migration, invasion and metastasis of breast cancer cells; it is commonly expressed in basal/triple-negative breast tumors and is associated with shorter recurrence-free and overall survival times in patients with breast cancer. Thus, GPNMB/OA represents an attractive target for therapeutic intervention in breast cancer; however, little is known about the functions of GPNMB/OA within the primary tumor microenvironment. We have employed mouse and human breast cancer cells to investigate the effects of GPNMB/OA on tumor growth and angiogenesis. GPNMB/OA-expressing tumors display elevated endothelial recruitment and reduced apoptosis when compared to vector control-derived tumors. Primary human breast cancers characterized by high vascular density also display elevated levels of GPNMB/OA when compared to those with low vascular density. Using immunoblot and ELISA assays, we demonstrate the GPNMB/OA ectodomain is shed from the surface of breast cancer cells. Transient siRNA-mediated knockdown studies of known sheddases identified ADAM10 as the protease responsible for GPNMB/OA processing. Finally, we demonstrate that the shed extracellular domain (ECD) of GPNMB/OA can promote endothelial migration in vitro. GPNMB/OA expression promotes tumor growth, which is associated with enhanced endothelial recruitment. We identify ADAM10 as a sheddase capable of releasing the GPNMB/OA ectodomain from the surface of breast cancer cells, which induces endothelial cell migration. Thus, ectodomain shedding may serve as a novel mechanism by which GPNMB/OA promotes angiogenesis in breast cancer.

Background While primary breast cancer (BC) is often effectively managed, metastasis remains the primary cause of BC-related fatalities. Gaps remain in our understanding of the mechanisms regulating cancer cell organotropism with predilection to specific organs. Unraveling mediators of site-specific metastasis could enhance early detection and enable more tailored interventions. Liquid biopsy represents an innovative approach in cancer involving the analysis of biological materials such as circulating tumor DNA and tumor-derived extracellular vesicles (EV) found in body fluids like blood or urine. This offers valuable insights for characterizing and monitoring tumor genomes to advance personalized medicine in metastatic cancers. Methods We performed in-depth analyses of EV cargo associated with BC metastasis using eight murine cell line models with distinct metastatic potentials and organotropism to the lung, the bone, the liver, and the brain. We characterized the secretome of these cells to identify unique biomarkers specific to metastatic sites. Results Small EVs isolated from all cell lines were quantified and validated for established EV markers. Tracking analysis and electron microscopy revealed EV secretion patterns that differed according to cell line. Cell-free (cf)DNA and EV-associated DNA (EV-DNA) were detected from all cell lines with varying concentrations. We detected a TP53 mutation in both EV-DNA and cfDNA. Mass spectrometry-based proteomics analyses identified 698 EV-associated proteins, which clustered according to metastatic site. This analysis highlighted both common EV signatures and proteins involved in cancer progression and organotropism unique to metastatic cell lines. Among these, 327 significantly differentially enriched proteins were quantified with high confidence levels across BC and metastatic BC cells. We found enrichment of specific integrin receptors in metastatic cancer EVs compared to EVs secreted from non-transformed epithelial cells and matched tumorigenic non-metastatic cells. Pathway analyses revealed that EVs derived from parental cancer cells display a cell adhesion signature and are enriched with proteins involved in cancer signaling pathways. Conclusion Taken together, the characterization of EV cargo in a unique model of BC organotropism demonstrated that EV-DNA and EV proteomes were informative of normal and cancer states. This work could help to identify BC biomarkers associated with site-specific metastasis and new therapeutic targets.