Evidence of steady-state fibroblast subtypes in the normal human breast as cells-of-origin for perturbed-state fibroblasts in breast cancer.

BACKGROUND: Human breast cancer most frequently originates within a well-defined anatomical structure referred to as the terminal duct lobular unit (TDLU). This structure is endowed with its very own lobular fibroblasts representing one out of two steady-state fibroblast subtypes-the other being interlobular fibroblasts. While cancer-associated fibroblasts (CAFs) are increasingly appreciated as covering a spectrum of perturbed states, we lack a coherent understanding of their relationship-if any-with the steady-state fibroblast subtypes. To address this, we here established two autologous CAF lines representing inflammatory CAFs (iCAFs) and myofibroblast CAFs (myCAFs) and compared them with already established interlobular- and lobular fibroblasts with respect to their origin and impact on tumor formation. METHODS: Primary breast tumor-derived CAFs were transduced to express human telomerase reverse transcriptase (hTERT) and sorted into CD105low and CD105high populations using fluorescence-activated cell sorting (FACS). The two populations were tested for differentiation similarities to iCAF and myCAF states through transcriptome-wide RNA-Sequencing (RNA-Seq) including comparison to an available iCAF-myCAF cell state atlas. Inference of origin in interlobular and lobular fibroblasts relied on RNA-Seq profiles, immunocytochemistry and growth characteristics. Osteogenic differentiation and bone formation assays in culture and in vivo were employed to gauge for origin in bone marrow-derived mesenchymal stem cells (bMSCs). Functional characteristics were assessed with respect to contractility in culture and interaction with tumor cells in mouse xenografts. The cells' gene expression signatures were tested for association with clinical outcome of breast cancer patients using survival data from The Cancer Genome Atlas database. RESULTS: We demonstrate that iCAFs have properties in common with interlobular fibroblasts while myCAFs and lobular fibroblasts are related. None of the CAFs qualify as bMSCs as revealed by lack of critical performance in bone formation assays. Functionally, myCAFs and lobular fibroblasts are almost equally tumor promoting as opposed to iCAFs and interlobular fibroblasts. A myCAF gene signature is found to associate with poor breast cancer-specific survival. CONCLUSIONS: We propose that iCAFs and myCAFs originate in interlobular and lobular fibroblasts, respectively, and more importantly, that the tumor-promoting properties of lobular fibroblasts render the TDLU an epicenter for breast cancer evolution.

Infection of Brain Pericytes Underlying Neuropathology of COVID-19 Patients.

A wide range of neurological manifestations have been associated with the development of COVID-19 following SARS-CoV-2 infection. However, the etiology of the neurological symptomatology is still largely unexplored. Here, we used state-of-the-art multiplexed immunostaining of human brains (n = 6 COVID-19, median age = 69.5 years; n = 7 control, median age = 68 years) and demonstrated that expression of the SARS-CoV-2 receptor ACE2 is restricted to a subset of neurovascular pericytes. Strikingly, neurological symptoms were exclusive to, and ubiquitous in, patients that exhibited moderate to high ACE2 expression in perivascular cells. Viral dsRNA was identified in the vascular wall and paralleled by perivascular inflammation, as signified by T cell and macrophage infiltration. Furthermore, fibrinogen leakage indicated compromised integrity of the blood-brain barrier. Notably, cerebrospinal fluid from additional 16 individuals (n = 8 COVID-19, median age = 67 years; n = 8 control, median age = 69.5 years) exhibited significantly lower levels of the pericyte marker PDGFRß in SARS-CoV-2-infected cases, indicative of disrupted pericyte homeostasis. We conclude that pericyte infection by SARS-CoV-2 underlies virus entry into the privileged central nervous system space, as well as neurological symptomatology due to perivascular inflammation and a locally compromised blood-brain barrier.

Activin receptor-like kinase 1 is associated with immune cell infiltration and regulates CLEC14A transcription in cancer.

Cancer cells sustain their metabolic needs through nutrients and oxygen supplied by the bloodstream. The requirement for tumor angiogenesis has been therapeutically exploited in the clinical setting mainly by means of inhibition of the vascular endothelial growth factor family of ligands and receptors. Despite promising results in preclinical models, the benefits for patients proved to be limited. Inadequate efficacy similarly halted the development of agents impinging on the activity of the activin receptor-like kinase (ALK)1, a member of the transforming growth factor-ß superfamily. Notwithstanding its characterization as an endothelial cell marker, the full spectrum of biological processes associated with ALK1 is essentially unexplored. Here, we present data revealing the genetic network associated with ACVRL1 (the gene encoding for ALK1) expression in human cancer tissues. Computational analysis unveiled a hitherto unknown role for ACVRL1 in relation to genes modulating the functionality of the immune cell compartment. Moreover, we generated a signature of 8 genes co-expressed with ACVRL1 across different tumor types and characterized the c-type lectin domain containing protein (CLEC)14A as a potential downstream target of ACVRL1. Considering the lack of reagents for ALK1 detection that has hampered the field to date, our work provides the opportunity to validate the 8-gene signature and CLEC14A as biomarkers for ALK1 activity. Ultimately, this may help revisit the clinical development of already existing ALK1-blocking compounds as precision medicines for cancer.

Identification of Hipk2 as an essential regulator of white fat development.

Homeodomain-interacting protein kinase 2 (Hipk2) has previously been implicated in the control of several transcription factors involved in embryonic development, apoptosis, cell proliferation, and tumor development, but very little is understood about the exact mechanisms through which Hipk2 influences these processes. Analysis of gene expression in normal tissues from genetically heterogeneous mouse or human populations can reveal network motifs associated with the structural or functional components of the tissue, and may predict roles for genes of unknown function. Here we have applied this network strategy to uncover a role for the Hipk2 gene in the transcriptional system controlling adipogenesis. Both in vitro and in vivo models were used to show that knockdown or loss of Hipk2 specifically inhibits white adipose cell differentiation and tissue development. In addition, loss of Hipk2 leads to induction of pockets of multilocular brown fat-like cells in remaining white adipose depots, which express markers of brown and beige fat such as uncoupling protein 1 and transmembrane protein 26. These changes are accompanied by increased insulin sensitivity in Hipk2 knockout mice and reduced high-fat diet-induced weight gain, highlighting a potential role for this kinase in diseases such as diabetes and obesity. Our study underscores the versatility and power of a readily available tissue, such as skin, for network modeling of systemic transcriptional programs involved in multiple pathways, including lipid metabolism and adipogenesis.

Cellular plasticity in the breast cancer ecosystem.

The complex interplay between genetically diverse tumor cells and their microenvironment significantly influences cancer progression and therapeutic responses. This review highlights recent findings on cellular plasticity and heterogeneity within the breast cancer ecosystem, focusing on the roles of cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). We discuss evidence suggesting that breast cancer cells exhibit phenotypic plasticity driven by both intrinsic genetic factors and external microenvironmental cues, impacting treatment responses and disease recurrence. Moreover, single-cell RNA sequencing studies reveal diverse subtypes of CAFs and TAMs, each with distinct functional gene expression programs and spatial organization within the tumor microenvironment. Understanding the hierarchical relationships and niche cues governing cellular phenotypes offers new opportunities for targeted therapeutic interventions. By elucidating the organizational principles of the tumor ecosystem, future therapies may target phenotypic states or entire cellular niches, advancing precision medicine approaches in breast cancer treatment.

Cancer-associated fibroblasts rewire the estrogen receptor response in luminal breast cancer, enabling estrogen independence.

Advanced breast cancers represent a major therapeutic challenge due to their refractoriness to treatment. Cancer-associated fibroblasts (CAFs) are the most abundant constituents of the tumor microenvironment and have been linked to most hallmarks of cancer. However, the influence of CAFs on therapeutic outcome remains largely unchartered. Here, we reveal that spatial coincidence of abundant CAF infiltration with malignant cells was associated with reduced estrogen receptor (ER)-α expression and activity in luminal breast tumors. Notably, CAFs mediated estrogen-independent tumor growth by selectively regulating ER-α signaling. Whereas most prototypical estrogen-responsive genes were suppressed, CAFs maintained gene expression related to therapeutic resistance, basal-like differentiation, and invasion. A functional drug screen in co-cultures identified effector pathways involved in the CAF-induced regulation of ER-α signaling. Among these, the Transforming Growth Factor-ß and the Janus kinase signaling cascades were validated as actionable targets to counteract the CAF-induced modulation of ER-α activity. Finally, genes that were downregulated in cancer cells by CAFs were predictive of poor response to endocrine treatment. In conclusion, our work reveals that CAFs directly control the luminal breast cancer phenotype by selectively modulating ER-α expression and transcriptional function, and further proposes novel targets to disrupt the crosstalk between CAFs and tumor cells to reinstate treatment response to endocrine therapy in patients.

Fibroblast subsets in non-small cell lung cancer: Associations with survival, mutations, and immune features.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are molecularly heterogeneous mesenchymal cells that interact with malignant cells and immune cells and confer anti- and protumorigenic functions. Prior in situ profiling studies of human CAFs have largely relied on scoring single markers, thus presenting a limited view of their molecular complexity. Our objective was to study the complex spatial tumor microenvironment of non-small cell lung cancer (NSCLC) with multiple CAF biomarkers, identify novel CAF subsets, and explore their associations with patient outcome. METHODS: Multiplex fluorescence immunohistochemistry was employed to spatially profile the CAF landscape in 2 population-based NSCLC cohorts (n = 636) using antibodies against 4 fibroblast markers: platelet-derived growth factor receptor-alpha (PDGFRA) and -beta (PDGFRB), fibroblast activation protein (FAP), and alpha-smooth muscle actin (αSMA). The CAF subsets were analyzed for their correlations with mutations, immune characteristics, and clinical variables as well as overall survival. RESULTS: Two CAF subsets, CAF7 (PDGFRA-/PDGFRB+/FAP+/αSMA+) and CAF13 (PDGFRA+/PDGFRB+/FAP-/αSMA+), showed statistically significant but opposite associations with tumor histology, driver mutations (tumor protein p53 [TP53] and epidermal growth factor receptor [EGFR]), immune features (programmed death-ligand 1 and CD163), and prognosis. In patients with early stage tumors (pathological tumor-node-metastasis IA-IB), CAF7 and CAF13 acted as independent prognostic factors. CONCLUSIONS: Multimarker-defined CAF subsets were identified through high-content spatial profiling. The robust associations of CAFs with driver mutations, immune features, and outcome suggest CAFs as essential factors in NSCLC progression and warrant further studies to explore their potential as biomarkers or therapeutic targets. This study also highlights multiplex fluorescence immunohistochemistry-based CAF profiling as a powerful tool for the discovery of clinically relevant CAF subsets.

Isolation and characterization of progenitor-like cells from human renal proximal tubules.

The tubules of the kidney display a remarkable capacity for self-renewal on damage. Whether this regeneration is mediated by dedifferentiating surviving cells or, as recently suggested, by stem cells has not been unequivocally settled. Herein, we demonstrate that aldehyde dehydrogenase (ALDH) activity may be used for isolation of cells with progenitor characteristics from adult human renal cortical tissue. Gene expression profiling of the isolated ALDH(high) and ALDH(low) cell fractions followed by immunohistochemical interrogation of renal tissues enabled us to delineate a tentative progenitor cell population scattered through the proximal tubules (PTs). These cells expressed CD24 and CD133, previously described markers for renal progenitors of Bowman's capsule. Furthermore, we show that the PT cells, and the glomerular progenitors, are positive for KRT7, KRT19, BCL2, and vimentin. In addition, tubular epithelium regenerating on acute tubular necrosis displayed long stretches of CD133(+)/VIM(+) cells, further substantiating that these cells may represent a progenitor cell population. Furthermore, a potential association of these progenitor cells with papillary renal cell carcinoma was discovered. Taken together, our data demonstrate the presence of a previously unappreciated subset of the PT cells that may be endowed with a more robust phenotype, allowing increased resistance to acute renal injury, enabling rapid repopulation of the tubules.

HIF-2alpha maintains an undifferentiated state in neural crest-like human neuroblastoma tumor-initiating cells.

High hypoxia-inducible factor-2alpha (HIF-2alpha) protein levels predict poor outcome in neuroblastoma, and hypoxia dedifferentiates cultured neuroblastoma cells toward a neural crest-like phenotype. Here, we identify HIF-2alpha as a marker of normoxic neural crest-like neuroblastoma tumor-initiating/stem cells (TICs) isolated from patient bone marrows. Knockdown of HIF-2alpha reduced VEGF expression and induced partial sympathetic neuronal differentiation when these TICs were grown in vitro under stem cell-promoting conditions. Xenograft tumors of HIF-2alpha-silenced cells were widely necrotic, poorly vascularized, and resembled the bulk of tumor cells in clinical neuroblastomas by expressing additional sympathetic neuronal markers, whereas control tumors were immature, well-vascularized, and stroma-rich. Thus, HIF-2alpha maintains an undifferentiated state of neuroblastoma TICs. Because low differentiation is associated with poor outcome and angiogenesis is crucial for tumor growth, HIF-2alpha is an attractive target for neuroblastoma therapy.

Upregulated functional gene expression programmes in tumour pericytes mark progression in patients with low-grade glioma.

Pericytes conceivably play important roles in the tumour microenvironment of glioblastoma multiforme (GBM) by allowing for an aberrant vasculature and acting as a component in the perivascular niche that supports glioma stem-like cells. However, a lack of specific markers has hampered in-depth elucidation of the functional contribution of pericytes to GBM. This study provides a comprehensive computational biology approach to annotate pericyte marker genes in the GBM vasculature through integration of data from single-cell RNA-sequencing studies of both mouse and human tissue, as well as bulk tumour and healthy tissue gene expression data from patients with GBM. We identified distinct vascular- and immune-related gene expression programmes in tumour pericytes that we assessed for association with GBM characteristics and patient survival. Most compellingly, pericyte gene signatures that were upregulated in tumours compared with normal brain tissue were indicative of progression of low-grade gliomas into high-grade glioma, suggested by a markedly shorter overall survival. Our results underline the functional importance of tumour pericytes in low-grade glioma and may serve as a starting point for efforts for precision targeting of pericytes.

Suppression of renal cell carcinoma growth by inhibition of Notch signaling in vitro and in vivo.

Loss of the tumor suppressor gene von Hippel-Lindau (VHL) plays a key role in the oncogenesis of clear cell renal cell carcinoma (CCRCC). The loss leads to stabilization of the HIF transcription complex, which induces angiogenic and mitogenic pathways essential for tumor formation. Nonetheless, additional oncogenic events have been postulated to be required for the formation of CCRCC tumors. Here, we show that the Notch signaling cascade is constitutively active in human CCRCC cell lines independently of the VHL/HIF pathway. Blocking Notch signaling resulted in attenuation of proliferation and restrained anchorage-independent growth of CCRCC cell lines. Using siRNA targeting the different Notch receptors established that the growth-promoting effects of the Notch signaling pathway were attributable to Notch-1 and that Notch-1 knockdown was accompanied by elevated levels of the negative cell-cycle regulators p21 Cip1 and/or p27 Kip1. Treatment of nude mice with an inhibitor of Notch signaling potently inhibited growth of xenotransplanted CCRCC cells. Moreover, Notch-1 and the Notch ligand Jagged-1 were expressed at significantly higher levels in CCRCC tumors than in normal human renal tissue, and the growth of primary CCRCC cells was attenuated upon inhibition of Notch signaling. These findings indicate that the Notch cascade may represent a novel and therapeutically accessible pathway in CCRCC.

Anti-tumor effects of rigosertib in high-risk neuroblastoma.

High-risk neuroblastoma has a poor prognosis despite intense treatment, demonstrating the need for new therapeutic strategies. Here we evaluated the effects of rigosertib (ON-01910.Na) in preclinical models of high-risk neuroblastoma. Among several hundred cancer cell lines representing 24 tumor types, neuroblastoma was the most sensitive to rigosertib. Treatment of MYCN-amplified neuroblastoma organoids resulted in organoid disintegration, decreased cell viability, and increased apoptotic cell death. Neuroblastoma response to rigosertib involved G2M cell cycle arrest and decreased phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204). Rigosertib delayed tumor growth and prolonged survival of mice carrying neuroblastoma MYCN-amplified PDX tumors (median survival: 31 days, treated; 22 days, vehicle) accompanied with increased apoptosis in treated tumors. We further identified vincristine and rigosertib as a potential promising drug combination treatment. Our results show that rigosertib might be a useful therapeutic agent for MYCN-amplified neuroblastomas, especially in combination with existing agents.

Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer.

BACKGROUND: Cancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment. CAFs exhibit both tumour-promoting and tumour-suppressing functions, making them exciting targets for improving cancer treatments. Careful isolation, identification, and characterisation of CAF heterogeneity is thus necessary for ex vivo validation and future implementation of CAF-targeted strategies in cancer. METHODS: Murine 4T1 (metastatic) and 4T07 (poorly/non-metastatic) orthotopic triple negative breast cancer tumours were collected after 7, 14, or 21 days. The tumours were analysed via flow cytometry for the simultaneous expression of six CAF markers: alpha smooth muscle actin (αSMA), fibroblast activation protein alpha (FAPα), platelet derived growth factor receptor alpha and beta (PDGFRα and PDGFRß), CD26/DPP4 and podoplanin (PDPN). All non-CAFs were excluded from the analysis using a lineage marker cocktail (CD24, CD31, CD45, CD49f, EpCAM, LYVE-1, and TER-119). In total 128 murine tumours and 12 healthy mammary fat pads were analysed. RESULTS: We have developed a multicolour flow cytometry strategy based on exclusion of non-CAFs and successfully employed this to explore the temporal heterogeneity of freshly isolated CAFs in the 4T1 and 4T07 mouse models of triple-negative breast cancer. Analysing 128 murine tumours, we identified 5-6 main CAF populations and numerous minor ones based on the analysis of αSMA, FAPα, PDGFRα, PDGFRß, CD26, and PDPN. All markers showed temporal changes with a distinct switch from primarily PDGFRα+ fibroblasts in healthy mammary tissue to predominantly PDGFRß+ CAFs in tumours. CD26+ CAFs emerged as a large novel subpopulation, only matched by FAPα+ CAFs in abundance. CONCLUSION: We demonstrate that multiple subpopulations of CAFs co-exist in murine triple negative breast cancer, and that the abundance and dynamics for each marker differ depending on tumour type and time. Our results form the foundation needed to isolate and characterise specific CAF populations, and ultimately provide an opportunity to therapeutically target specific CAF subpopulations.

Therapeutic targeting of KSP in preclinical models of high-risk neuroblastoma.

Neuroblastoma is a childhood malignancy with often dismal prognosis; relapse is common despite intense treatment. Here, we used human tumor organoids representing multiple MYCN-amplified high-risk neuroblastomas to perform a high-throughput drug screen with approved or emerging oncology drugs. Tumor-selective effects were calculated using drug sensitivity scores. Several drugs with previously unreported anti-neuroblastoma effects were identified by stringent selection criteria. ARRY-520, an inhibitor of kinesin spindle protein (KSP), was among those causing reduced viability. High expression of the KSP-encoding gene KIF11 was associated with poor outcome in neuroblastoma. Genome-scale loss-of-function screens in hundreds of human cancer cell lines across 22 tumor types revealed that KIF11 is particularly important for neuroblastoma cell viability. KSP inhibition in neuroblastoma patient-derived xenograft (PDX) cells resulted in the formation of abnormal monoastral spindles, mitotic arrest, up-regulation of mitosis-associated genes, and apoptosis. In vivo, KSP inhibition caused regression of MYCN-amplified neuroblastoma PDX tumors. Furthermore, treatment of mice harboring orthotopic neuroblastoma PDX tumors resulted in increased survival. Our results suggested that KSP inhibition could be a promising treatment strategy in children with high-risk neuroblastoma.

CRIM1 is localized to the podocyte filtration slit diaphragm of the adult human kidney.

BACKGROUND: CRIM1 is a plasma membrane bound protein containing six cysteine-rich repeats (CRR). Through these, CRIM1 has been shown to interact with a subgroup of the TGF-beta superfamily, the bone morphogenic proteins (BMP) isoforms 2, 4 and 7. The probable action is to modulate the signalling properties of these factors. CRIM1 has also been shown to regulate the release of VEGFA by podocytes during renal organogenesis. Knock-out studies in mice have shown that CRIM1 is critically involved in the development of the central nervous system, eye and kidney. Replacement of CRIM1 with a defective version leads to renal dysgenesis and perinatal death. We have analysed the distribution of CRIM1 in adult human renal tissue. METHODS: To this end, we have used immunofluorescence, immunohistochemistry and immunoelectron microscopy. We performed western blotting for the CRIM1 protein, using lysates from isolated glomerular podocytes and human renal tissue homogenate. By using quantitative PCR, we compared the CRIM1 mRNA levels in podocytes, human renal tissue homogenate, primary human renal proximal tubular epithelial cells and primary human pulmonary artery smooth muscle cells. RESULTS: The results show that in the human adult kidney, CRIM1 is mainly expressed in the glomerular podocytes and is associated with the insertional region of the filtration slit diaphragm (SD) of the podocyte pedicles. CONCLUSIONS: CRIM1 is a protein that should be added to the list of proteins associated with the podocyte filtration SD and with the probable action of modulating BMP and VEGFA signalling.

PAK4 suppresses RELB to prevent senescence-like growth arrest in breast cancer.

Overcoming cellular growth restriction, including the evasion of cellular senescence, is a hallmark of cancer. We report that PAK4 is overexpressed in all human breast cancer subtypes and associated with poor patient outcome. In mice, MMTV-PAK4 overexpression promotes spontaneous mammary cancer, while PAK4 gene depletion delays MMTV-PyMT driven tumors. Importantly, PAK4 prevents senescence-like growth arrest in breast cancer cells in vitro, in vivo and ex vivo, but is not needed in non-immortalized cells, while PAK4 overexpression in untransformed human mammary epithelial cells abrogates H-RAS-V12-induced senescence. Mechanistically, a PAK4 - RELB - C/EBPß axis controls the senescence-like growth arrest and a PAK4 phosphorylation residue (RELB-Ser151) is critical for RELB-DNA interaction, transcriptional activity and expression of the senescence regulator C/EBPß. These findings establish PAK4 as a promoter of breast cancer that can overcome oncogene-induced senescence and reveal a selective vulnerability of cancer to PAK4 inhibition.

Tracing Renal Cell Carcinomas back to the Nephron.

Renal cell carcinomas (RCCs) are a heterogeneous group of tumors derived from the epithelial cells of the nephron. In recent years the genetic landscape of these tumors has been detailed, leading to progress in mouse modeling of the human disease. In parallel, substantial advancements have been made in describing the transcriptional programs of normal nephron cell types and how they respond to renal insults. Integrating these research fields may provide a deeper understanding of renal tumor initiation and progression, and provide leads that can be conveyed into mouse models that faithfully recapitulate the different RCC subtypes. We summarize here the genetic lesions and molecular pathways that define RCC subtypes and discuss how these relate to cell-of-origin and renal repair programs.

Microenvironmental control of breast cancer subtype elicited through paracrine platelet-derived growth factor-CC signaling.

Breast tumors of the basal-like, hormone receptor-negative subtype remain an unmet clinical challenge, as there is high rate of recurrence and poor survival in patients following treatment. Coevolution of the malignant mammary epithelium and its underlying stroma instigates cancer-associated fibroblasts (CAFs) to support most, if not all, hallmarks of cancer progression. Here we delineate a previously unappreciated role for CAFs as determinants of the molecular subtype of breast cancer. We identified paracrine crosstalk between cancer cells expressing platelet-derived growth factor (PDGF)-CC and CAFs expressing the cognate receptors in human basal-like mammary carcinomas. Genetic or pharmacological intervention of PDGF-CC activity in mouse models of cancer resulted in conversion of basal-like breast cancers into a hormone receptor-positive state that enhanced sensitivity to endocrine therapy in previously resistant tumors. We conclude that specification of breast cancer to the basal-like subtype is under microenvironmental control and is therapeutically actionable.

Cell-Type-Specific Gene Programs of the Normal Human Nephron Define Kidney Cancer Subtypes.

Comprehensive transcriptome studies of cancers often rely on corresponding normal tissue samples to serve as a transcriptional reference. In this study, we performed in-depth analyses of normal kidney tissue transcriptomes from the TCGA and demonstrate that the histological variability in cellularity, inherent in the kidney architecture, lead to considerable transcriptional differences between samples. This should be considered when comparing expression profiles of normal and cancerous kidney tissues. We exploited these differences to define renal-cell-specific gene signatures and used these as a framework to analyze renal cell carcinoma (RCC) ontogeny. Chromophobe RCCs express FOXI1-driven genes that define collecting duct intercalated cells, whereas HNF-regulated genes, specific for proximal tubule cells, are an integral part of clear cell and papillary RCC transcriptomes. These networks may be used as a framework for understanding the interplay between genomic changes in RCC subtypes and the lineage-defining regulatory machinery of their non-neoplastic counterparts.