Background & Aims Pancreatitis is the most important risk factor for pancreatic ductal adenocarcinoma (PDAC). Pancreatitis predisposes to PDAC because it induces a process of acinar cell ...reprogramming known as acinar-to-duct metaplasia (ADM)—a precursor of pancreatic intraepithelial neoplasia lesions that can progress to PDAC. Mutations in KRAS are found at the earliest stages of pancreatic tumorigenesis, and it appears to be a gatekeeper to cancer progression. We investigated how mutations in KRAS cooperate with pancreatitis to promote pancreatic cancer progression in mice. Methods We generated mice carrying conditional alleles of Yap1 and Taz and disrupted Yap1 and Taz using a Cre-lox recombination strategy in adult mouse pancreatic acinar cells (Yap1fl/fl;Tazfl/fl;Ela1-CreERT2). We crossed these mice with LSL-KrasG12D mice, which express a constitutively active form of KRAS after Cre recombination. Pancreatic tumor initiation and progression were analyzed after chemically induced pancreatitis. We analyzed pancreatic tissues from patients with pancreatitis or PDAC by immunohistochemistry. Results Oncogenic activation of KRAS in normal, untransformed acinar cells in the pancreatic tissues of mice resulted in increased levels of pancreatitis-induced ADM. Expression of the constitutive active form of KRAS in this system led to activation of the transcriptional regulators YAP1 and TAZ; their function was required for pancreatitis-induced ADM in mice. The JAK–STAT3 pathway was a downstream effector of KRAS signaling via YAP1 and TAZ. YAP1 and TAZ directly mediated transcriptional activation of several genes in the JAK–STAT3 signaling pathway; this could be a mechanism by which acinar cells that express activated KRAS become susceptible to inflammation. Conclusions We identified a mechanism by which oncogenic KRAS facilitates ADM and thereby generates the cells that initiate neoplastic progression. This process involves activation of YAP1 and TAZ in acinar cells, which up-regulate JAK–STAT3 signaling to promote development of PDAC in mice.
Neutrophil recruitment is an important early step in controlling tissue infections or injury. Here, we report that this influx depends on both tissue-resident mast cells and macrophages. Mice with ...mast cell deficiency recruit reduced numbers of neutrophils in the first few hours of intraperitoneal lipopolysaccharide (LPS) stimulation. Conversely, in mice with clodronate-ablated macrophages, neutrophils extravasate, but have limited ability to reach the peritoneal fluid. Tissue macrophages synthesize neutrophil chemoattractants CXCL1/CXCL2 (CXC chemokine ligands 1/2) in response to LPS. Mast cells also produce these chemokines of which a proportion are preformed in granules. Release of the granules and new CXCL1/CXCL2 synthesis is Toll-like receptor 4–dependent. Both in vivo studies with blocking monoclonal antibodies and in vitro chemotaxis experiments show the neutrophil response to mast cells and macrophages to be CXCL1/CXCL2-dependent. The data are in keeping with the model that mast cells, optimally positioned in close proximity to the vasculature, initiate an early phase of neutrophil recruitment by releasing the chemoattractants CXCL1/CXCL2. Having arrived within the stimulated tissue, neutrophils penetrate further in a macrophage-dependent manner. Therefore, we demonstrate a positive role for mast cells in tissue inflammation and define how this comes about with contribution from a second tissue cell, the macrophage.
• Mast cells contribute to early neutrophil recruitment.• Mast cells and macrophages both make CXCL1 and CXCL2.
The epithelium and immune compartment in the intestine are constantly exposed to a fluctuating external environment. Defective communication between these compartments at this barrier surface ...underlies susceptibility to infections and chronic inflammation. Environmental factors play a significant, but mechanistically poorly understood, role in intestinal homeostasis. We found that regeneration of intestinal epithelial cells (IECs) upon injury through infection or chemical insults was profoundly influenced by the environmental sensor aryl hydrocarbon receptor (AHR). IEC-specific deletion of Ahr resulted in failure to control C. rodentium infection due to unrestricted intestinal stem cell (ISC) proliferation and impaired differentiation, culminating in malignant transformation. AHR activation by dietary ligands restored barrier homeostasis, protected the stem cell niche, and prevented tumorigenesis via transcriptional regulation of of Rnf43 and Znrf3, E3 ubiquitin ligases that inhibit Wnt-β-catenin signaling and restrict ISC proliferation. Thus, activation of the AHR pathway in IECs guards the stem cell niche to maintain intestinal barrier integrity.
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•AHR deficiency in IECs promotes inflammation and colon tumorigenesis•AHR activation regulates intestinal crypt stem cell differentiation•AHR controls negative regulators of the Wnt-β-catenin pathway•A diet rich in AHR ligands can prevent or halt tumorigenesis
Metidji et al. show that deletion of Ahr in intestinal epithelial cells results in a defective barrier and unrestricted proliferation of intestinal stem cells (ISCs), culminating in malignant transformation. Activation of AHR by dietary ligands guards the ISC niche and maintains intestinal barrier homeostasis.
The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer ...cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3′ UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers.
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•Oncogenic RAS signaling via MEK increases PD-L1 expression•RAS regulates PD-L1 through AU-rich elements (AREs) in the 3′ UTR of PD-L1 mRNA•The ARE-binding protein tristetraprolin (TTP) negatively regulates PD-L1 expression•Restoration of tumor cell TTP activity enhances anti-tumor immunity
Coelho et al. demonstrate a post-transcriptional mechanism whereby oncogenic RAS signaling increases PD-L1 expression. Mechanistically, PD-L1 mRNA is targeted by TTP through AU-rich elements in the 3′ UTR, making it unstable. Oncogenic RAS signaling reduces TTP activity and stabilizes the PD-L1 transcript. Restoring TTP activity reduces PD-L1 expression and enhances anti-tumor immunity.
Clear-cell renal cell carcinoma (ccRCC) exhibits a broad range of metastatic phenotypes that have not been systematically studied to date. Here, we analyzed 575 primary and 335 metastatic biopsies ...across 100 patients with metastatic ccRCC, including two cases sampledat post-mortem. Metastatic competence was afforded by chromosome complexity, and we identify 9p loss as a highly selected event driving metastasis and ccRCC-related mortality (p = 0.0014). Distinct patterns of metastatic dissemination were observed, including rapid progression to multiple tissue sites seeded by primary tumors of monoclonal structure. By contrast, we observed attenuated progression in cases characterized by high primary tumor heterogeneity, with metastatic competence acquired gradually and initial progression to solitary metastasis. Finally, we observed early divergence of primitive ancestral clones and protracted latency of up to two decades as a feature of pancreatic metastases.
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•Evolutionary study of matched primary metastasis biopsies from 100 ccRCC cases•Metastasis competence is afforded by chromosome complexity, but not driver mutation load•The hallmark genomic drivers of ccRCC metastasis are loss of 9p and 14q•Punctuated and branched evolution result in distinct patterns of metastases
A multi-center prospective study and two validation cohorts of matched primary metastasis biopsies from 100 patients with clear-cell renal cell carcinoma provides a comprehensive picture of the genetic underpinnings and the evolutionary patterns of metastasis.
The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the ...multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with >10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance.
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•ccRCC evolutionary subtypes correlate with clinical phenotypes•Genetic diversity and chromosome complexity contribute to patient outcomes•Early fixation of multiple driver events leads to rapid growth and metastases•Subclonal diversification is linked with slower growth and attenuated metastases
A multi-center prospective study on 101 patients with clear-cell renal cell carcinoma resolves the evolutionary features and subtypes underpinning the diverse clinical phenotypes of the disease and suggests these features as potential biomarkers for guiding intervention and surveillance.
Sexual and gender minorities (SGMs) are individuals with sexual orientations, gender identities, or expressions (or a combination of these) that differ from cultural norms. Sexual and gender ...minorities often face workplace discrimination and report decreased physical and emotional well-being from discrimination.
To explore the workplace climate of SGM athletic trainers (ATs).
Sequential mixed-methods study.
Web-based survey and interviews.
Criterion sampling of SGM ATs (117 survey participants and 12 interview participants).
We modified the LGBTQ Inclusion Assessment and the Organizational Self-Assessment for the survey and developed a semistructured interview script (scale-level content validity index = 0.94). We used means ± SDs, frequencies (%), and the consensual qualitative research tradition to characterize participant responses. Trustworthiness was established through reflexivity (researchers checking bias throughout the research process), member-checking, multianalyst triangulation, and internal and external auditing.
Participants indicated their workplace was inclusive (24 20.5%), somewhat inclusive (29 24.8%), or not inclusive (14 12.0%) or did not indicate at all (50 42.7%). Respondents most often noted they were unsure of which stage of change their organizations and organizational units were in addressing lesbian, gay, bisexual, transgender, queer, questioning, pansexual, intersex, asexual, 2-spirit, and all within the community of queer and transspectrum identities (LGBTQPIA+) concerns in the workplace as well as specific actions taken for inclusion. Two domains emerged from the interview data: safety and inclusion. The safety domain represented aspects of the workplace climate that made participants feel safe and includes organizational initiatives (12/12), patient-centered policies (7/12), local and federal regulations (7/12), and signaling (12/12). The inclusion domain represented how participants felt a sense of belonging to the organization through their own experience (12/12), through the experiences of their patients (9/12), and through an infrastructure designed for inclusion (12/12). Participants expressed both affirmative and negative feelings of safety and inclusion throughout their responses.
Organizations must take both structural and cultural actions to address the concerns of exclusion and lack of safety.
Pancreatic ductal adenocarcinoma (PDAC) shows great cellular heterogeneity, with pronounced epithelial and mesenchymal cancer cell populations. However, the cellular hierarchy underlying PDAC cell ...diversity is unknown. Here we identify the tetraspanin CD9 as a marker of PDAC tumour-initiating cells. CD9
cells had increased organoid formation capability, and generated tumour grafts in vivo at limiting dilutions. Tumours initiated from CD9
cells recapitulated the cellular heterogeneity of primary PDAC, whereas CD9
cells produced only duct-like epithelial progeny. CD9 knockdown decreased the growth of PDAC organoids, and heterozygous CD9 deletion in Pdx1-Cre; LSL-KRas
; p53
mice prolonged overall survival. Mechanistically, CD9 promoted the plasma membrane localization of the glutamine transporter ASCT2, enhancing glutamine uptake in PDAC cells. Thus, our study identifies a PDAC subpopulation capable of initiating PDAC and giving rise to PDAC heterogeneity, suggesting that the cellular diversity of PDAC is generated by PDAC stem cell differentiation.
Tumors comprise functionally diverse subpopulations of cells with distinct proliferative potential. Here, we show that dynamic epigenetic states defined by the linker histone H1.0 determine which ...cells within a tumor can sustain the long-term cancer growth. Numerous cancer types exhibit high inter- and intratumor heterogeneity of H1.0, with H1.0 levels correlating with tumor differentiation status, patient survival, and, at the single-cell level, cancer stem cell markers. Silencing of H1.0 promotes maintenance of self-renewing cells by inducing derepression of megabase-sized gene domains harboring downstream effectors of oncogenic pathways. Self-renewing epigenetic states are not stable, and reexpression of H1.0 in subsets of tumor cells establishes transcriptional programs that restrict cancer cells' long-term proliferative potential and drive their differentiation. Our results uncover epigenetic determinants of tumor-maintaining cells.
Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide; nearly half contain mutations in the receptor tyrosine kinase/RAS pathway. Here we show that RAS-pathway ...mutant NSCLC cells depend on the transcription factor GATA2. Loss of GATA2 reduced the viability of NSCLC cells with RAS-pathway mutations, whereas wild-type cells were unaffected. Integrated gene expression and genome occupancy analyses revealed GATA2 regulation of the proteasome, and IL-1-signaling, and Rho-signaling pathways. These pathways were functionally significant, as reactivation rescued viability after GATA2 depletion. In a Kras-driven NSCLC mouse model, Gata2 loss dramatically reduced tumor development. Furthermore, Gata2 deletion in established Kras mutant tumors induced striking regression. Although GATA2 itself is likely undruggable, combined suppression of GATA2-regulated pathways with clinically approved inhibitors caused marked tumor clearance. Discovery of the nononcogene addiction of KRAS mutant lung cancers to GATA2 presents a network of druggable pathways for therapeutic exploitation.
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▸ GATA2 is essential for RAS-pathway mutant non-small cell lung cancer (NSCLC) ▸ Gene expression and ChIP-seq studies reveal a GATA2 transcriptional network in NSCLC ▸ Gata2 loss suppresses tumor growth in autochthonous models of Kras mutant NSCLC ▸ Combined drug inhibition of GATA2-regulated pathways suppresses Kras mutant tumors
Lung tumors with mutations in the RAS pathway are dependent on the transcription factor GATA2. Analysis of the GATA2 transcriptional network reveals components that when targeted in combination cause regression of KRAS-driven lung tumors in mice.
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