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Khokha, R.

Publications and source records attributed to Khokha, R..

15 recordsLinked to original sources

Germline BRCA1 Mutation Deregulates Irradiation-Induced Cell Cycle Checkpoints in Human Mammary Luminal Progenitors

Mammary ductal tree comprises two epithelial lineages, basal and luminal, which harbour stem and progenitor cell subsets essential to breast physiology and are cancer-initiating precursors. Dysregulated cellular stress response is an early event in tumorigenesis, yet lineage-rooted study of primary breast epithelium pertinent to familial mutation carriers is limited. Here, we establish a lineage-resolved monolayer culture system that enables propagation of primary human mammary epithelial populations while preserving lineage identity across passages. We identify intrinsically distinct cell cycle response of luminal progenitors and basal cells to irradiation stress where the former engages both G1/S and G2/M checkpoints to achieve cell cycle arrest and the latter relies on the G2/M checkpoint. These lineage distinctions are diminished in germline BRCA1 mutation carriers with an attenuated G1/S checkpoint in luminal progenitors. Paired transcriptomic-proteomic profiling of acute irradiation response uncovers BRCA1+/- luminal progenitor population with sustained AKT-mTOR signaling and compromised cell cycle arrest marking an early deviation in the stress response of these purported cells-of-origin of aggressive breast cancers known to arise in BRCA1 germline mutation carriers. Our study provides a functional framework for determining critical events in the expansion of genomically altered mammary epithelial cells in the high-risk breast to enable future preventive interventions.

cell biology↗

Mapping lineage and functional diversity in the high-risk human mammary epithelium

BackgroundBreast cancer risk is shaped by the vast heterogeneity of mammary epithelial cells, comprising basal, luminal progenitor, and mature luminal populations. While transcriptional variation among these lineages has been extensively studied, protein-level features - particularly in high-risk women - remain underexplored, limiting insight into early cellular and molecular determinants of susceptibility. Moreover, little is known about how clinical covariates influence clonogenic capacity, proteomic states, and epithelial proportions, complicating the design of properly controlled human studies of early breast cancer risk. ResultsWe combined low-input proteomics with functional clonogenic assays to profile mammary epithelial cell subpopulations from a cohort of 21 breast tissues encompassing different germline mutation backgrounds, parity status and age. We quantified over 5,555 proteins and observed marked inter-donor variation in epithelial composition, proteomic programs, and colony-forming capacity. Multivariable modeling revealed that clinical covariates - including age, parity, and germline mutation status - modulate both global proteomic architecture and lineage-specific pathway activity. Parity was associated with reduced basal cell abundance, altered luminal progenitor and mature luminal proteomes, and changes in clonogenicity. Pathway analyses identified both conserved and lineage-restricted responses to shared risk factors. Projection of clonogenic signatures onto METABRIC and TCGA tumors further linked functional programs to tumor subtypes and clinical outcomes. ConclusionsThis study provides the most comprehensive proteomic atlas of cell-type resolved diversity in the high-risk breast to date. By defining how clinical covariates remodel epithelial composition and molecular state, it clarifies key sources of biological variability that challenge controlled study design and offers a resource for improving mechanistic insight, risk assessment, and prevention strategies.

cancer biology↗

Medium-Chain Fatty Acid exposure in non-transformed mammary glands leads to pro-tumorigenic alterations associated with aging

The local breast environment is a valuable research focus for identifying the etiological and biological factors that contribute to the development of breast cancer. Single-cell RNA sequencing shows that the increased availability of the medium-chain fatty acid octanoic acid induces changes typical of the aged mammary gland, including downregulation of cell-cell junctions, altered extracellular matrix (ECM)-receptor interactions, and upregulation of aging markers like MDK and GDF15. Ex vivo exposure to medium-chain fatty acids compromises cell-cell junctions leading to cell dissemination. As aging is associated with an increased risk of developing breast cancer, it is crucial to identify the factors driving these age-related changes in the mammary gland to develop effective cancer prevention strategies. Given that the proportion of breast adipocytes increases with age, we propose that the remodeling of the mammary gland associated with aging is partly due to the increase in adipocytes and fatty-acid release.

cancer biology↗

Progesterone Receptor Expressing Basal Cells and Progesterone Receptor-Primed Progenitors Redefine the Mammary Epithelial Hierarchy

The central role of progesterone in breast biology and cancer is undeniable. Progesterone is a potent mitogen for mammary stem cell expansion and essential for mammopoiesis, yet only a fraction of the luminal epithelium is described to express the progesterone receptor (PR). Progestins in contraceptives and HRT regimens increases breast cancer risk, whilst progesterone inhibition reduces mammary tumorigenesis. Understanding PR within mammary epithelial dynamics is imperative, especially given mammary stem/progenitors cells are the cells-of-origin in breast cancer. Here, we show PR-primed progenitors contribute to both mammary lineages and expose a novel PR+ basal population, challenging current dogma in the field. PR lineage-tracing yields an unprecedented contribution to the luminal and basal compartments. We uncover an asymmetrically dividing PR-primed subpopulation, which has unique bipotent clonogenic capacity and forms TEB-like outgrowths upon transplantation. We enumerate PR+ basal cells and dissect their proteomic landscape, establishing PR+ basal cells as a discrete basal subset, disparate from luminal PR+ cells. Finally, we identify PR+ basal cells in multiple proteomes, scRNAseq datasets and localize PR in clinical breast specimens. This forms a new foundation for comprehending hormone receptor patterning in the breast, not only based on lineage-identity but also the progenitor-progeny hierarchy. Our study shifts the current paradigm of breast biology with implications for breast cancer treatment given the growing interest in anti-progestin based primary prevention strategies.

cell biology↗

Stress Granules Underlie Acute Myeloid Leukemia Stem Cell Survival and Stress Adaptation

The link between cancer maintenance and an ability to sustain continued growth through stresses conferred by the cancer state itself is growing. However, there are significant gaps in our understanding of how this stress is managed, particularly at the level of cancer initiating cells. Here, we identify proteins comprising the dynamic, stress-adaptive ribonucleoprotein complexes known as stress granules (SG) to be enriched among the factors essential for leukemic stem cell (LSC)-driven leukemic propagation. Focusing on core SG nucleator G3BP1, we dissect the role of SGs in human acute myeloid leukemia (AML), their targetability, and the mechanisms they govern to uncover a novel propensity for AML, and in particular LSC-enriched fractions, to prime the expression of SG components, form SGs with greater fidelity and to be reliant on their establishment and continued integrity for LSC maintenance. We further unveil the transcript and protein interactome of G3BP1 in the AML context and show that consolidated control of innate immune signaling, and apoptosis repression is executed through regional binding specificity of G3BP1 to highly structured 3UTRs and cooperation with the RNA helicase UPF1 to mediate transcript decay in SGs. Altogether our findings advance novel fundamental principles of stress adaptation exploited in AML and LSCs that may extend to other cancers and uncover SGs as a novel axis for therapy development.

cancer biology↗

Pan-cancer N-glycoproteomic atlas of patient derived xenografts uncovers FAT2 as a therapeutic target for head and neck cancers

Cell surface proteins offer significant cancer therapeutic potential attributable to their accessible membrane localization and central role in cellular signaling. Despite this, their promise remains largely untapped due to the technical challenges inherent to profiling cell surface proteins. Here, we employed N-glycoproteomics to analyze 85 patient-derived xenografts (PDX), constructing Glyco PDXplorer - an in vivo pan-cancer atlas of cancer-derived cell surface proteins. We developed a target discovery pipeline to prioritize proteins with favorable expression profiles for immunotherapeutic targeting and validated FAT2 as a head and neck squamous cancer (HNSC) enriched surface protein with limited expression in normal tissue. Functional studies revealed that FAT2 is essential for HNSC growth and adhesion through regulation of surface architecture and integrin-PI3K signaling. Chimeric antigen receptor (CAR) T cells targeting FAT2 demonstrated potent anti-tumor activity in HNSC models. This work lays the foundation for developing FAT2-targeted therapies and represents a pivotal resource to inform therapeutic target discovery for multiple cancers. HIGHLIGHTSO_LIPan-cancer landscape of cancer-derived cell surface proteins detected in vivo C_LIO_LIDevelopment of a multi-omic discovery pipeline to prioritize proteins with optimal expression profiles as immunotherapy targets C_LIO_LIIdentification and validation of FAT2 as a head and neck squamous cancer enriched surface protein with minimal expression in normal tissues C_LIO_LIFAT2 coordinates cell surface organization, adhesion, growth and survival through the integrin-PI3K-AKT pathway C_LIO_LIFAT2 CAR T cells demonstrate anti-tumour activity in pre-clinical models C_LI

cancer biology↗

Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells

A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increasing flux through various metabolic reactions; but little is known about the mechanism(s) linking lipids and epigenetic reprogramming with the genesis of ER-BC. Here we show that the metabolism of the medium-chain fatty acid Octanoic Acid (OA) in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM, glutathione, and 2-HG, with implications for oncogenesis: SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express Neural, EMT and BC related genes. 2-HG exposure results in appearance of DNA breaks, potentially consequent to the inhibition of essential demethylases for HR repair. ROS increases shortly after OA exposure and is mitigated by antioxidant defenses, which favors/enables the survival of specific cell subtypes.

cancer biology↗

FABP7 Progenitors are a Targetable Metabolic Root in the BRCA1 Breast

It has been nearly 3 decades since the discovery of the BRCA1/2 genes and their link to breast cancer risk, with prophylactic mastectomy remaining the primary management option for these high-risk mutation carriers. The current paucity of interception strategies is due to undefined, targetable cancer precursor populations in the high-risk breast. Despite known cellular alterations in the BRCA1 breast, epithelial populations at the root of unwarranted cell state transitions remain unresolved. Here, we identify a root progenitor population that is dysregulated in BRCA1 carriers stemming from the metabolic role of BRCA1. This fatty-acid binding protein 7 (FABP7) expressing luminal progenitor population is spatially confined to the mammary ducts, has enhanced clonogenic capacity, and is the predicted origin of mixed basal-luminal differentiation in the BRCA1 but not BRCA2 breast. We show global H3K27 acetylation is reduced within ductal FABP7 cells in BRCA1 carriers in situ, linking to a non-canonical metabolic role of BRCA1 in regulating acetyl-CoA pools and de novo fatty acid synthesis. We demonstrate FABP7 progenitor capacity is preferentially ablated in BRCA1 carriers through inhibition of fatty acid metabolism using an FDA-approved fatty acid synthase (FASN) inhibitor. This study lays the foundation for metabolic control of breast progenitor dynamics to mitigate breast cancer risk in the BRCA1 breast.

cell biology↗

Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response via AP1 dichotomy in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) displays a high degree of spatial subtype heterogeneity. This intratumoral co-existence of classical and basal-like programs is evident in multi-scale transcriptomic and spatial analyses of resected, advanced-stage and chemotherapy-treated specimens and reciprocally linked to a diverse stromal immune microenvironment as well as worse clinical outcome. However, the underlying mechanisms of intratumoral subtype heterogeneity remain largely unclear. Here, by combining preclinical models, multi-center clinical, bulk and compartment-specific transcriptomic, proteomic, and bioimaging data from human specimens, we identified an interplay between neoplastic intrinsic AP1 transcription factor dichotomy and extrinsic CD68+ macrophages as a driver of intratumoral subtype co-existence along with an immunosuppressive tumor microenvironment with T cell exclusion. Our ATAC-, ChIP-, and RNA-seq analyses revealed that JUNB/AP1- and HDAC-mediated epigenetic programs repress pro-inflammatory immune signatures in tumor cells, antagonizing cJUN/AP1 signaling to favor a therapy-responsive classical neoplastic identity. Through the tumor microenvironment, this dichotomous regulation was further amplified via regional macrophage populations. Moreover, CD68+/TNF-+ cells associated with a reactive phenotype and reduced CD8+ T cell infiltration in human PDAC tumors. Consequently, combined anti-TNF- immunotherapy and chemotherapy resulted in reduced macrophage counts and promoted CD3+/CD8+ T cell infiltration in basal-like PDAC, leading to improved survival in preclinical murine models. We conclude that tumor cell intrinsic epigenetic programs, together with extrinsic microenvironmental cues, facilitate intratumoral subtype heterogeneity and disease progression.

cancer biology↗

Droplet based low input proteomic platform for rare cell populations

Deep proteomic profiling of rare cell populations has been constrained by sample input requirements. Here, we present DROPPS, an accessible low-input platform that generates high-fidelity proteomic profiles of 100 - 2,500 cells. By applying DROPPS within the mammary epithelium, we elucidated the connection between mitochondrial activity and clonogenicity, discovering and validating CD36 as a marker of progenitor capacity in the basal cell compartment. We anticipate DROPPS will accelerate biology-driven proteomic research for a multitude of rare cell populations.

biochemistry↗

Hourglass, a tool to mine bioimaging data, uncovers sex-disparities in the IL-6-associated T cell response in pancreatic tumors

Recent advances in digital pathology have led to an explosion in high-content multidimensional imaging approaches. Yet, our ability to gainfully process, visualize, integrate and mine the resulting mass of bioimaging data remains a challenge. We have developed Hourglass, an open access user-friendly software that streamlines complex biology-driven post-processing and visualization of multiparametric data. Directed at datasets derived from tissue microarrays or imaging methods that analyze multiple regions of interest per patient specimen, Hourglass systematically organizes observations across spatial and global levels as well as within patient subgroups. Application of Hourglass to our large and complex pancreatic cancer bioimaging dataset (540,617 datapoints derived from 26 bioimaging analyses applied to 596 specimens from 165 patients) consolidated a breadth of known IL-6 functions in a well-annotated human pancreatic cancer cohort and uncovered new unprecedented insights into a sex-linked Interleukin-6 (IL-6) association with immune phenotypes. Specifically, regional effects of IL-6 on the intratumoral T cell response were restricted to male patients only. In conclusion, Hourglass facilitates multi-layered knowledge extraction from complex multiparametric bioimaging datasets and provides tailored analytical means to productively harness heterogeneity at the sample and patient level.

cancer biology↗

Glycoproteomics Identifies Plexin-B3 as Targetable Cell Surface Protein Required for Growth and Invasion of Triple Negative Breast Cancer Cells

Driven by the lack of targeted therapies, triple negative breast cancers (TNBC) have the worst overall survival of all breast cancer subtypes. Considering cell surface proteins are favorable drug targets and are predominantly glycosylated, glycoproteome profiling has significant potential to facilitate the identification of much-needed drug targets for TNBC. Here, we performed N-glycoproteomics on six TNBC and five normal control (NC) cell lines using hydrazide-based enrichment. Quantitative proteomics and integrative data mining led to the discovery of Plexin-B3 (PLXNB3), a previously undescribed TNBC-enriched cell surface protein. Furthermore, siRNA knock-down and CRISPR-Cas9 editing of in vitro and in vivo models show that PLXNB3 is required for TNBC cell line growth, invasion, and migration. Altogether, we provide insight into N-glycoproteome remodeling associated with TNBC and functional evaluation of an extracted target, which indicate the surface protein PLXNB3 as a potential therapeutic target for TNBC. HighlightsO_LIIn-depth N-glycoproteomic profiles of six TNBC and five NC cell line models C_LIO_LIIdentification of PLXNB3 as a novel TNBC-enriched cell surface protein C_LIO_LIPLXNB3 affects growth, invasion, and migration in TNBC models C_LIO_LIPLXNB3 inhibition represents a targeted treatment option for TNBC C_LI

cancer biology↗

Mammary lineage dictates homologous recombination repair and PARP inhibitor vulnerability

It has long been assumed that all normal cells have the same capacity to engage homologous recombination (HR) and non-homologous end joining (NHEJ) to repair DNA double-strand breaks (DSBs), a concept exploited for DNA-damaging chemotherapeutics. We show that mammary epithelial lineage dictates the DSB repair pathway choice. Primary mammary proteomes and DSB repair enumeration by {gamma}-H2AX, Rad51 and DNA-PKc foci reveal that NHEJ operates in all epithelial cells, but high-fidelity HR is restricted to the luminal lineage. This translates to divergent poly (ADP-ribose) polymerase inhibitor (PARPi) vulnerability of mammary epithelial progenitor activity in both mouse and human, irrespective of the BRCA1/2 status. Proteome-defined lineage-specific signatures correlate to breast cancer subtypes and predict PARPi response of triple-negative human breast cancer xenografts. These intrinsically divergent HR characteristics of mammary cell types underpin a new strategy for identifying PARPi responders.

cancer biology↗

Spatially confined sub-tumor microenvironments orchestrate pancreatic cancer pathobiology

Pancreatic ductal adenocarcinoma (PDAC) remains resistant to most treatments and demonstrates a complex pathobiology. Here, we deconvolute regional heterogeneity in the human PDAC tumor microenvironment (TME), a long-standing obstacle, to define precise stromal contributions to PDAC progression. Large scale integration of histology-guided multiOMICs with clinical data sets and functional in vitro models uncovers two microenvironmental programs in PDAC that were anchored in fibroblast differentiation states. These sub-tumor microenvironments (subTMEs) co-occurred intratumorally and were spatially confined, producing patient-specific cellular and molecular heterogeneity associated with shortened patient survival. Each subTME was uniquely structured to support discrete aspects of tumor biology: reactive regions rich in activated fibroblast communities were immune-hot and promoted aggressive tumor progression while deserted regions enriched in extracellular matrix supported tumor differentiation yet were markedly chemoprotective. In conclusion, PDAC regional heterogeneity derives from biologically distinct reactive and protective TME elements with a defined, active role in PDAC progression. Graphical Abstract & Key findings O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/431890v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@17adabdorg.highwire.dtl.DTLVardef@aee778org.highwire.dtl.DTLVardef@deae00org.highwire.dtl.DTLVardef@1718272_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIPDAC regional heterogeneity originates in sub-tumor microenvironments (subTMEs) C_LIO_LISubTMEs exhibit distinct immune phenotypes and CAF differentiation states C_LIO_LIDifferent subTMEs are either tumor-promoting or chemoprotective C_LIO_LIIntratumoral subTME co-occurrence links stromal heterogeneity to patient outcome C_LI

cancer biology↗

Mammary epithelial cells have lineage-restricted metabolic identities

Cancer metabolism adapts the metabolic network of its tissue-of-origin. However, breast cancer is not a disease of a singular origin. Multiple epithelial populations serve as the culprit cell-of-origin for specific breast cancer subtypes, yet knowledge surrounding the metabolic network of normal mammary epithelial cells is limited. Here, we show that mammary populations have cell type-specific metabolic programs. Primary human breast cell proteomes of basal, luminal progenitor, and mature luminal populations revealed their unique enrichment of metabolic proteins. Luminal progenitors had higher abundance of electron transport chain subunits and capacity for oxidative phosphorylation, whereas basal cells were more glycolytic. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed distinct metabolic vulnerabilities of the mammary lineages. Computational analysis indicated that breast cancer subtypes retain metabolic features of their putative cell-of-origin. Lineage-restricted metabolic identities of normal mammary cells partly explain breast cancer metabolic heterogeneity and rationalize targeting subtype-specific metabolic vulnerabilities to advance breast cancer therapy.

cell biology↗