Tumour-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression.

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.

When nature's robots go rogue: exploring protein homeostasis dysfunction and the implications for understanding human aging disease pathologies.

INTRODUCTION: Proteins have been historically regarded as 'nature's robots': Molecular machines that are essential to cellular/extracellular physical mechanical properties and catalyze key reactions for cell/system viability. However, these robots are kept in check by other protein-based machinery to preserve proteome integrity and stability. During aging, protein homeostasis is challenged by oxidation, decreased synthesis, and increasingly inefficient mechanisms responsible for repairing or degrading damaged proteins. In addition, disruptions to protein homeostasis are hallmarks of many neurodegenerative diseases and diseases disproportionately affecting the elderly. Areas covered: Here we summarize age- and disease-related changes to the protein machinery responsible for preserving proteostasis and describe how both aging and disease can each exacerbate damage initiated by the other. We focus on alteration of proteostasis as an etiological or phenomenological factor in neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's, along with Down syndrome, ophthalmic pathologies, and cancer. Expert commentary: Understanding the mechanisms of proteostasis and their dysregulation in health and disease will represent an essential breakthrough in the treatment of many (senescence-associated) pathologies. Strides in this field are currently underway and largely attributable to the introduction of high-throughput omics technologies and their combination with novel approaches to explore structural and cross-link biochemistry.

Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization.

The extracellular matrix (ECM) is readily enriched by decellularizing tissues with nondenaturing detergents to solubilize and deplete the vast majority of cellular components. This approach has been used extensively to generate ECM scaffolds for regenerative medicine technologies and in 3D cell culture to model how the ECM contributes to disease progression. A highly enriched ECM fraction can then be generated using a strong chaotrope buffer that is compatible with downstream bottom-up proteomic analysis or 3D cell culture experiments after extensive dialysis. With most tissues, an insoluble pellet remains after chaotrope extraction that is rich in structural ECM components. Previously, we showed that this understudied fraction represented approximately 80% of total fibrillar collagen from the lung and other ECM fiber components that are known to be covalently cross-linked. Here, we present a hydroxylamine digestion approach for chaotrope-insoluble ECM analysis with comparison to an established CNBr method for matrisome characterization. Because ECM characteristics vary widely among tissues, we chose five tissues that represent unique and diverse ECM abundances, composition, and biomechanical properties. Hydroxylamine digestion is compatible with downstream proteomic workflows, yields high levels of ECM peptides from the insoluble ECM fraction, and reduces analytical variability when compared to CNBr digestion. Data are available via ProteomeXchange with identifier PXD006428.

Targeting HER2/HER3 co-mutations in metastatic breast cancer: Case reports of exceptional responders to trastuzumab and pertuzumab therapy.

BACKGROUND: Overexpression of HER2 plays an important role in cancer progression and is the target of multiple therapies in HER2-positive breast cancer. Recent studies have also highlighted the presence of activating mutations in HER2, and HER3 that are predicted to enhance HER2 downstream pathway activation in a HER2-dependent manner. METHODS: In this report, we present two exceptional responses in hormone receptor-positive, HER2-nonamplified, HER2/HER3 co-mutated metastatic breast cancer patients who were treated with the anti-HER2-directed monoclonal antibodies, trastuzumab and pertuzumab. RESULTS: Both patients acheived exceptional responses to treatment, suggesting that combined trastuzumab, pertuzumab, and endocrine therapy could be a highly effective therapy for these patients and our observations could help prioritize trastuzumab deruxtecan as an early therapeutic choice for patients whose cancers have activating mutations in HER2.

Mammary collagen is under reproductive control with implications for breast cancer.

Mammographically-detected breast density impacts breast cancer risk and progression, and fibrillar collagen is a key component of breast density. However, physiologic factors influencing collagen production in the breast are poorly understood. In female rats, we analyzed gene expression of the most abundantly expressed mammary collagens and collagen-associated proteins across a pregnancy, lactation, and weaning cycle. We identified a triphasic pattern of collagen gene regulation and evidence for reproductive state-dependent composition. An initial phase of collagen deposition occurred during pregnancy, followed by an active phase of collagen suppression during lactation. The third phase of collagen regulation occurred during weaning-induced mammary gland involution, which was characterized by increased collagen deposition. Concomitant changes in collagen protein abundance were confirmed by Masson's trichrome staining, second harmonic generation (SHG) imaging, and mass spectrometry. We observed similar reproductive-state dependent collagen patterns in human breast tissue obtained from premenopausal women. SHG analysis also revealed structural variation in collagen across a reproductive cycle, with higher packing density and more collagen fibers arranged perpendicular to the mammary epithelium in the involuting rat mammary gland compared to nulliparous and lactating glands. Involution was also characterized by high expression of the collagen cross-linking enzyme lysyl oxidase, which was associated with increased levels of cross-linked collagen. Breast cancer relevance is suggested, as we found that breast cancer diagnosed in recently postpartum women displayed gene expression signatures consistent with increased collagen deposition and crosslinking compared to breast cancers diagnosed in age-matched nulliparous women. Using publicly available data sets, we found this involution-like, collagen gene signature correlated with poor progression-free survival in breast cancer patients overall and in younger women. In sum, these findings of physiologic collagen regulation in the normal mammary gland may provide insight into normal breast function, the etiology of breast density, and inform breast cancer risk and outcomes.

Landscape of KRASG12C, Associated Genomic Alterations, and Interrelation With Immuno-Oncology Biomarkers in KRAS-Mutated Cancers.

PURPOSE: Promising single-agent activity from sotorasib and adagrasib in KRASG12C-mutant tumors has provided clinical evidence of effective KRAS signaling inhibition. However, comprehensive analysis of KRAS-variant prevalence, genomic alterations, and the relationship between KRAS and immuno-oncology biomarkers is lacking. MATERIALS AND METHODS: Retrospective analysis of deidentified records from 79,004 patients with various cancers who underwent next-generation sequencing was performed. Fisher's exact test evaluated the association between cancer subtypes and KRAS variants. Logistic regression assessed KRASG12C comutations with other oncogenes and the association between KRAS variants and immuno-oncology biomarkers. RESULTS: Of the 79,004 samples assessed, 13,758 (17.4%) harbored KRAS mutations, with 1,632 (11.9%) harboring KRASG12C and 12,126 (88.1%) harboring other KRAS variants (KRASnon-G12C). Compared with KRASnon-G12C across all tumor subtypes, KRASG12C was more prevalent in females (56% v 51%, false discovery rate-adjusted P value [FDR-P] = .0006), current or prior smokers (85% v 56%, FDR-P < .0001), and patients age > 60 years (73% v 63%, FDR-P ≤ .0001). The most frequent KRAS variants across all subtypes were G12D (29.5%), G12V (23.0%), G12C (11.9%), G13D (6.5%), and G12R (6.2%). KRASG12C was most prevalent in patients with non-small-cell lung cancer (9%), appendiceal (3.9%), colorectal (3.2%), tumor of unknown origin (1.6%), small bowel (1.43%), and pancreatic (1.3%) cancers. Compared with KRASnon-G12C-mutated, KRASG12C-mutated tumors were significantly associated with tumor mutational burden-high status (17.9% v 8.4%, odds ratio [OR] = 2.38; FDR-P < .0001). KRASG12C-mutated tumors exhibited a distinct comutation profile from KRASnon-G12C-mutated tumors, including higher comutations of STK11 (20.59% v 5.95%, OR = 4.10; FDR-P < .01) and KEAP1 (15.38% v 4.61%, OR = 3.76; FDR-P < .01). CONCLUSION: This study presents the first large-scale, pan-cancer genomic characterization of KRASG12C. The KRASG12C mutation was more prevalent in females and older patients and appeared to be associated with smoking status. KRASG12C tumors exhibited a distinct comutation profile and were associated with tumor mutational burden-high status.

Fibroblast subtypes define a metastatic matrisome in breast cancer.

Small primary breast cancers can show surprisingly high potential for metastasis. Clinical decision-making for tumor aggressiveness, including molecular profiling, relies primarily on analysis of the cancer cells. Here we show that this analysis is insufficient - that the stromal microenvironment of the primary tumor plays a key role in tumor cell dissemination and implantation at distant sites. We previously described 2 cancer-associated fibroblasts (CAFs) that either express (CD146+) or lack (CD146-) CD146 (official symbol MCAM, alias MUC18). We now find that when mixed with human breast cancer cells, each fibroblast subtype determines the fate of cancer cells: CD146- fibroblasts promoted increased metastasis compared with CD146+ fibroblasts. Potentially novel quantitative and qualitative proteomic analyses showed that CD146+ CAFs produced an environment rich in basement membrane proteins, while CD146- CAFs exhibited increases in fibronectin 1, lysyl oxidase, and tenascin C, all overexpressed in aggressive disease. We also show clinically that CD146- CAFs predicted for likelihood of lymph node involvement even in small primary tumors (<5 cm). Clearly small tumors enriched for CD146- CAFs require aggressive treatments.

Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217.

Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness- and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographic density.

Compartment resolved proteomics reveals a dynamic matrisome in a biomechanically driven model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a severe fibrotic component that compromises treatment, alters the immune cell profile and contributes to patient mortality. It has been shown that early on in this process, dynamic changes in tissue biomechanics play an integral role in supporting pancreatic cancer development and progression. Despite the acknowledgement of its importance, a granular view of how stromal composition changes during the course of PDAC progression remains largely unknown. To mimic the quasi-mesenchymal phenotype and pronounced desmoplastic response observed clinically, we utilized a genetically engineered mouse model of PDAC that is driven by a KrasG12D mutation and loss of Tgfbr2 expression. Application of compartment resolved proteomics revealed that PDAC progression in this KTC model is associated with dynamic stromal alterations that are indicative of a wound healing program. We identified an early provisional matricellular fibrosis that was accompanied by markers of macrophage activation and infiltration, consistent with the inflammatory phase of wound healing. At 20 weeks a proliferative phenotype was observed with increased fibroblast markers, further collagen deposition and loss of basement membrane and native cell markers.

A tension-mediated glycocalyx-integrin feedback loop promotes mesenchymal-like glioblastoma.

Glioblastoma multiforme (GBMs) are recurrent lethal brain tumours. Recurrent GBMs often exhibit mesenchymal, stem-like phenotypes that could explain their resistance to therapy. Analyses revealed that recurrent GBMs have increased tension and express high levels of glycoproteins that increase the bulkiness of the glycocalyx. Studies showed that a bulky glycocalyx potentiates integrin mechanosignalling and tissue tension and promotes a mesenchymal, stem-like phenotype in GBMs. Gain- and loss-of-function studies implicated integrin mechanosignalling as an inducer of GBM growth, survival, invasion and treatment resistance, and a mesenchymal, stem-like phenotype. Mesenchymal-like GBMs were highly contractile and expressed elevated levels of glycoproteins that expanded their glycocalyx, and they were surrounded by a stiff extracellular matrix that potentiated integrin mechanosignalling. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignalling and a bulky glycocalyx, implying a causal link towards a mesenchymal, stem-like phenotype in GBMs. Strategies to ameliorate GBM tissue tension offer a therapeutic approach to reduce mortality due to GBM.

Structural Insight into Recognition of Methylated Histone H3K4 by Set3.

The plant homeodomain (PHD) finger of Set3 binds methylated lysine 4 of histone H3 in vitro and in vivo; however, precise selectivity of this domain has not been fully characterized. Here, we explore the determinants of methyllysine recognition by the PHD fingers of Set3 and its orthologs. We use X-ray crystallographic and spectroscopic approaches to show that the Set3 PHD finger binds di- and trimethylated states of H3K4 with comparable affinities and employs similar molecular mechanisms to form complexes with either mark. Composition of the methyllysine-binding pocket plays an essential role in determining the selectivity of the PHD fingers. The finding that the histone-binding activity is not conserved in the PHD finger of Set4 suggests different functions for the Set3 and Set4 paralogs.