ABSTRACT
Early diagnosis and intervention of esophageal squamous cell carcinoma (ESCC) can improve the prognosis. The purpose of this study was to identify biomarkers for ESCC and esophageal precancerous lesions (intraepithelial neoplasia, IEN). Based on the proteomic and genomic data of esophageal tissue including previously reported data, up-regulated proteins with copy number amplification in esophageal cancer were screened as candidate biomarkers. Five proteins, including KDM2A, RAD9A, ECT2, CYHR1 and TONSL, were confirmed by immunohistochemistry on ESCC and normal esophagus (NE). Then, we investigated the expression of 5 proteins in 236 participants (60 NEs, 93 IENs and 83 ESCCs) which were randomly divided into training set and test set. When distinguishing ESCC from NE, the area under curve (AUC) of the multiprotein model was 0.940 in the training set, while the lowest AUC of a protein was 0.735. In the test set, the results were similar. When distinguishing ESCC from IEN or distinguishing IEN from NE, the diagnostic efficiency of the multi-protein models were also improved compared with that of single protein. Our findings suggest that combined detection of KDM2A, RAD9A, ECT2, CYHR1 and TONSL can be used as potential biomarkers for the early diagnosis of ESCC and precancerous lesion development prediction. SIGNIFICANCE: Candidate biomarkers including KDM2A, RAD9A, ECT2, CYHR1 and TONSL screened by integrating genomic and proteomic data from the esophagus can be used as potential biomarkers for the early diagnosis of esophageal squamous cell carcinoma and precancerous lesion development prediction.
Subject(s)
Biomarkers, Tumor , Esophageal Neoplasms , Esophageal Squamous Cell Carcinoma , Humans , Esophageal Neoplasms/diagnosis , Esophageal Neoplasms/metabolism , Biomarkers, Tumor/metabolism , Esophageal Squamous Cell Carcinoma/diagnosis , Esophageal Squamous Cell Carcinoma/metabolism , Male , Female , Middle Aged , Carcinoma in Situ/diagnosis , Carcinoma in Situ/metabolism , Neoplasm Proteins/metabolism , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/metabolism , Proteomics/methods , AgedABSTRACT
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is marked by a dismal survival rate, lacking effective therapeutics due to its aggressive growth, late-stage diagnosis, and chemotherapy resistance. Despite debates on NF-κB targeting for PDAC treatment, no successful approach has emerged. METHODS: To elucidate the role of NF-κB, we ablated NF-κB essential modulator (NEMO), critical for conventional NF-κB signaling, in the pancreata of mice that develop precancerous lesions (KC mouse model). Secretagogue-induced pancreatitis by cerulein injections was utilized to promote inflammation and accelerate PDAC development. RESULTS: NEMO deletion reduced fibrosis and inflammation in young KC mice, resulting in fewer pancreatic intraepithelial neoplasias (PanINs) at later stages. Paradoxically, however, NEMO deletion accelerated the progression of these fewer PanINs to PDAC and reduced median lifespan. Further, analysis of tissue microarrays from human PDAC sections highlighted the correlation between reduced NEMO expression in neoplastic cells and poorer prognosis, supporting our observation in mice. Mechanistically, NEMO deletion impeded oncogene-induced senescence (OIS), which is normally active in low-grade PanINs. This blockage resulted in fewer senescence-associated secretory phenotype (SASP) factors, reducing inflammation. However, blocked OIS fostered replication stress and DNA damage accumulation which accelerated PanIN progression to PDAC. Finally, treatment with the DNA damage-inducing reagent etoposide resulted in elevated cell death in NEMO-ablated PDAC cells compared to their NEMO-competent counterparts, indicative of a synthetic lethality paradigm. CONCLUSIONS: NEMO exhibited both oncogenic and tumor-suppressive properties during PDAC development. Caution is suggested in therapeutic interventions targeting NF-κB, which may be detrimental during PanIN progression but beneficial post-PDAC development.
Subject(s)
Carcinoma, Pancreatic Ductal , Disease Progression , NF-kappa B , Pancreatic Neoplasms , Signal Transduction , Animals , Humans , Mice , Carcinoma in Situ/pathology , Carcinoma in Situ/genetics , Carcinoma in Situ/metabolism , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/genetics , Cell Line, Tumor , Disease Models, Animal , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice, Knockout , NF-kappa B/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/etiologyABSTRACT
Borderline Brenner tumors (BBT) have a range of morphology that shows considerable overlap with that of malignant Brenner tumors (MBT). In particular, two histological patterns of BBT can be particularly challenging: 1) BBT with intraepithelial carcinoma (BBT-IEC) and 2) BBT with a small nested pattern (BBT-SNP). BBT-IEC is characterized by a tumor with the low-power non-infiltrative silhouette of a conventional BBT, but with increased cytological atypia and mitotic activity similar to that of MBT. Conversely, BBT-SNP is characterized by a complex proliferation of small tumor nests that closely resemble the infiltrative growth pattern of MBT, but without the obligate cytologic atypia and mitotic activity of MBT. We suggest that the combination of p16, p53 and Ki-67 may be helpful in distinguishing these 2 patterns of BBT from both conventional BBT and from MBT. While both conventional BBT and BBT-IEC show a null pattern of p16 expression, our case of BBT-IEC showed aberrant p53 overexpression, albeit with a maturation pattern similar to that described for TP53 mutant mucinous ovarian carcinoma and differentiated vulvar intraepithelial neoplasia (dVIN). Similarly, while BBT-SNP shows an infiltrative-like growth pattern similar to that of MBT, our case also showed a wild-type pattern of p53 expression and a Ki-67 proliferative index similar to areas with conventional BBT histology. In conclusion, in our small case series, we show that the use of immunohistochemistry for p53 and Ki-67 may help to distinguish challenging patterns of BBT from MBT. Further studies are needed to validate this finding in a larger case cohort.
Subject(s)
Biomarkers, Tumor , Brenner Tumor , Immunohistochemistry , Ki-67 Antigen , Humans , Female , Immunohistochemistry/methods , Brenner Tumor/pathology , Brenner Tumor/metabolism , Brenner Tumor/diagnosis , Middle Aged , Biomarkers, Tumor/metabolism , Ki-67 Antigen/metabolism , Ovarian Neoplasms/pathology , Ovarian Neoplasms/metabolism , Tumor Suppressor Protein p53/metabolism , Aged , Adult , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Carcinoma in Situ/pathology , Carcinoma in Situ/metabolism , Carcinoma in Situ/diagnosisABSTRACT
BACKGROUND/AIM: Although several studies in some neoplasms have reported correlation between the expression levels of Doublecortin-like kinase1(DCLK1) and carcinogenesis, its role in cholangiocarcinoma remains unknown. MATERIALS AND METHODS: DCLK1 expression in normal epithelium (NE), biliary intraepithelial neoplasia (BilIN)1â¼3, and intrahepatic cholangiocarcinoma (ICC) were investigated immuno-histochemically. The molecular effects of DCLK1 were investigated by gene silencing using RNAi [DCLK1-tagrgeting (siDCLK1)]. The human ICC cell lines HuCCT1 and HuH28 were transfected with these siRNAs, and used for assays in the presence or absence of DCLK1 inhibitors. RESULTS: The positive ratio of DCLK1 expression in ICC was higher than that in NE, and equally distributed among BilIN1â¼3 (NE: BilIN1: BilIN2: BilIN3: ICC=62%: 91%: 97%: 100%: 95%, p<0.05). In the wound healing assay, the migration of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the cell invasion assay, the invasion of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the presence of the DCLK1 inhibitor, cell proliferative capacity at 24 hours was decreased in a concentration-dependent manner. CONCLUSION: DCLK1 was highly expressed in the early stage of ICC carcinogenesis. Human ICC cell growth was suppressed in vitro by siRNA silencing of DCLK1 or after treatment with the DCLK1 inhibitor, indicating DCLK1 may be molecular target for ICC therapy.
Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Doublecortin-Like Kinases , Intracellular Signaling Peptides and Proteins , Protein Serine-Threonine Kinases , Humans , Cholangiocarcinoma/genetics , Cholangiocarcinoma/pathology , Cholangiocarcinoma/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Bile Duct Neoplasms/pathology , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/genetics , Gene Expression Regulation, Neoplastic , Neoplasm Staging , Male , Cell Proliferation , Middle Aged , Female , RNA, Small Interfering/genetics , Carcinoma in Situ/pathology , Carcinoma in Situ/genetics , Carcinoma in Situ/metabolismABSTRACT
Because the mechanotransduction by stromal stiffness stimulates the rupture and repair of the nuclear envelope in pancreatic progenitor cells, accumulated genomic aberrations are under selection in the tumor microenvironment. Analysis of cell growth, micronuclei, and phosphorylated Ser-139 residue of the histone variant H2AX (γH2AX) foci linked to mechanotransduction pressure in vivo during serial orthotopic passages of mouse KrasLSL-G12D/+;Trp53flox/flox;Pdx1-Cre (KPC) cancer cells in the tumor and in migrating through the size-restricted 3-µm micropores. To search for pancreatic cancer cell-of-origin, analysis of single-cell data sets revealed that the extracellular matrix shaped an alternate route of acinar-ductal transdifferentiation of acinar cells into topoisomerase II α (TOP2A)-overexpressing cancer cells and derived subclusters with copy number amplifications in MYC-PTK2 (protein tyrosine kinase 2) locus and PIK3CA. High-PTK2 expression is associated with 171 differentially methylated CpG loci, 319 differentially expressed genes, and poor overall survival in The Cancer Genome Atlas-Pancreatic Adenocarcinoma cohort. Abolished RGD-integrin signaling by disintegrin KG blocked the PTK2 phosphorylation, increased cancer apoptosis, decreased vav guanine nucleotide exchange factor 1 (VAV1) expression, and prolonged overall survival in the KPC mice. Reduction of α-smooth muscle actin deposition in the CD248 knockout KPC mice remodeled the tissue stroma and down-regulated TOP2A expression in the epithelium. In summary, stromal stiffness induced the onset of cancer cells-of-origin by ectopic TOP2A expression, and the genomic amplification of MYC-PTK2 locus via alternative transdifferentiation of pancreatic progenitor cells is the vulnerability useful for disintegrin KG treatment.
Subject(s)
Chromosomal Instability , Disease Progression , Pancreatic Neoplasms , Animals , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Mice , Humans , Carcinoma in Situ/pathology , Carcinoma in Situ/genetics , Carcinoma in Situ/metabolism , Stromal Cells/metabolism , Stromal Cells/pathology , Tumor Microenvironment , Mechanotransduction, Cellular , Focal Adhesion Kinase 1ABSTRACT
We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The KrasG12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated KrasG12D. Mechanistically, KrasG12D expression strengthened sensitivity to NUPR1 inactivation, inducing cell death, activating caspase 3 and releasing LDH. Remarkably, ZZW-115-mediated SG-formation inhibition hampered the development of pancreatic intraepithelial neoplasia (PanINs) in Pdx1-cre;LSL-KrasG12D (KC) mice. ZZW-115-treatment of KC mice triggered caspase 3 activation, DNA fragmentation, and formation of the apoptotic bodies, leading to cell death, specifically in KrasG12D-expressing cells. We further demonstrated that, in developed PanINs, short-term ZZW-115 treatment prevented NUPR1-associated SGs presence. Lastly, a four-week ZZW-115 treatment significantly reduced the number and size of PanINs in KC mice. This study proposes that targeting NUPR1-dependent SGs formation could be a therapeutic approach to induce cell death in KrasG12D-dependent tumors.
Subject(s)
Carcinoma in Situ , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Piperazines , Thiazines , Animals , Mice , Carcinoma in Situ/genetics , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Carcinoma, Pancreatic Ductal/genetics , Caspase 3/genetics , Caspase 3/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Stress Granules , Synthetic Lethal MutationsABSTRACT
AIMS: Verruciform acanthotic vulvar intra-epithelial neoplasia (vaVIN) is an HPV-independent, p53 wild-type lesion with distinct morphology and documented risk of recurrence and cancer progression. vaVIN is rare, and prospective distinction from non-neoplastic hyperplastic lesions can be difficult. CK17, SOX2 and GATA3 immunohistochemistry has emerging value in the diagnosis of HPV-independent lesions, particularly differentiated VIN. We aimed to test the combined value of these markers in the diagnosis of vaVIN versus its non-neoplastic differentials in the vulva. METHODS AND RESULTS: CK17, SOX2 and GATA3 immunohistochemistry was evaluated on 16 vaVINs and 34 mimickers (verruciform xanthoma, lichen simplex chronicus, lichen sclerosus, psoriasis, pseudo-epitheliomatous hyperplasia). CK17 was scored as 3+ = full-thickness, 2+ = partial-thickness, 1+ = patchy, 0 = absent; SOX2 as 3+ = strong staining ≥ 10% cells, 2+ = moderate, 1 + =weak, 0 = staining in < 10% cells; and GATA3 as pattern 0 = loss in < 25% basal cells, 1 = loss in 25-75% basal cells, 2 = loss in > 75% basal cells. For analysis, results were recorded as positive (CK17 = 3+, SOX2 = 3+, GATA3 = patterns 1/2) or negative (CK17 = 2+/1+/0, SOX2 = 2+/1+/0, GATA3 = pattern 0). CK17, SOX2 and GATA3 positivity was documented in 81, 75 and 58% vaVINs, respectively, versus 32, 17 and 22% of non-neoplastic mimickers, respectively; ≥ 2 marker positivity conferred 83 sensitivity, 88 specificity and 86% accuracy in vaVIN diagnosis. Compared to vaVIN, SOX2 and GATA3 were differentially expressed in lichen sclerosus, lichen simplex chronicus and pseudo-epitheliomatous hyperplasia, whereas CK17 was differentially expressed in verruciform xanthoma and adjacent normal mucosa. CONCLUSIONS: CK17, SOX2 and GATA3 can be useful in the diagnosis of vaVIN and its distinction from hyperplastic non-neoplastic vulvar lesions. Although CK17 has higher sensitivity, SOX2 and GATA3 are more specific, and the combination of all markers shows optimal diagnostic accuracy.
Subject(s)
Biomarkers, Tumor , GATA3 Transcription Factor , Immunohistochemistry , Keratin-17 , SOXB1 Transcription Factors , Vulvar Neoplasms , Adult , Aged , Aged, 80 and over , Female , Humans , Middle Aged , Biomarkers, Tumor/analysis , Biomarkers, Tumor/metabolism , Carcinoma in Situ/diagnosis , Carcinoma in Situ/pathology , Carcinoma in Situ/metabolism , Diagnosis, Differential , GATA3 Transcription Factor/analysis , GATA3 Transcription Factor/immunology , GATA3 Transcription Factor/metabolism , Immunohistochemistry/methods , Keratin-17/analysis , Keratin-17/immunology , Keratin-17/metabolism , SOXB1 Transcription Factors/analysis , SOXB1 Transcription Factors/immunology , SOXB1 Transcription Factors/metabolism , Vulvar Neoplasms/pathology , Vulvar Neoplasms/diagnosis , Vulvar Neoplasms/metabolismABSTRACT
At the early stage of tumor progression, fibroblasts are located at the outer edges of the tumor, forming an encasing layer around it. In this work, we have developed a 3D in vitro model where fibroblasts' layout resembles the structure seen in carcinoma in situ. We use a microfluidic encapsulation technology to co-culture fibroblasts and cancer cells within hollow, permeable, and elastic alginate shells. We find that in the absence of spatial constraint, fibroblasts and cancer cells do not mix but segregate into distinct aggregates composed of individual cell types. However, upon confinement, fibroblasts enwrap cancer cell spheroid. Using a combination of biophysical methods and live imaging, we find that buildup of compressive stress is required to induce fibroblasts spreading over the aggregates of tumor cells. We propose that compressive stress generated by the tumor growth might be a mechanism that prompts fibroblasts to form a capsule around the tumor.
Subject(s)
Carcinoma in Situ , Fibroblasts , Humans , Cell Line, Tumor , Fibroblasts/metabolism , Spheroids, Cellular , Coculture Techniques , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathologyABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Shuangshen granules (SSG), a nationally patented Chinese medicinal formula, including Panax quinquefolium L., Panax notoginseng (Burkill) F. H. Chen, and Cordyceps sinensis (Berk.) Sacc., has demonstrated remarkable therapeutic effects on pancreatic cancer in clinical treatment for nearly 10 years. Previous pharmacological researches have found that its main components, including ginsenosides and cordycepin have anticancer or preventive effects on pancreatic ductal adenocarcinoma (PDAC), which may be associated with immune metabolism. However, the underlying pharmacological mechanism of SSG in the truncation effect of PDAC progression is still unclear. AIM OF THE STUDY: To comprehensively understand the infiltrating immune cells during the different stages of the PDAC development chain and search for immune-related biomarkers that could potentially serve as drug targets through bioinformatic analysis. Meanwhile, the truncation effect of SSG on PDAC progression was also investigated. MATERIALS AND METHODS: The gene expression profiles at different PDAC developmental stages, including normal pancreas, pancreatic intraepithelial neoplasia (PanIN), and PDAC, were retrieved from the GEO database. The GEO2R tool was used to identify differentially expressed genes among the three groups. Functional enrichment analysis was performed with the GSEA software and Metascape platform. The CIBERSORT algorithm evaluated immune cell infiltration in the three groups, and immune-related biomarkers were identified. Correlation analysis was employed to examine the association between immune cells and the biomarkers. One of these biomarkers was selected for immunohistochemistry validation in human samples. Lastly, the effectiveness of SSG against PDAC progression and the influence on the selected biomarker were validated in vivo. The underlying pharmacological mechanisms were also explored. RESULTS: One dataset was obtained, where the functional enrichment of DEGs primarily involved immune effector processes and cytokine production of immune cells. The differential immune cells reflected during the progression from PanIN to PDAC were B memory cells, monocytes, M2 macrophages, and activated dendritic cells. The upregulation of ACTA2 was closely associated with M2 macrophage regulation. The immunohistochemistry on human samples validated significant differences in ACTA2 expression levels as the PDAC progressed. Moreover, animal experiments revealed that the national patented drug SSG ameliorated the pathological changes, decreased the expression of ACTA2 and its functional protein α-smooth muscle actin during PDAC progression. The underlying pharmacological mechanism was related to the regulation of macrophage polarization and downregulation of TGF-ß/Smad signaling pathway. CONCLUSIONS: The immunosuppressive environment changes during the PDAC progression. ACTA2 is a potential immuned-target for drug prevention of PDAC, while SSG could be a promising drug candidate.
Subject(s)
Carcinoma in Situ , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Animals , Humans , Biomarkers, Tumor/metabolism , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma in Situ/genetics , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Computational Biology , Drugs, Chinese HerbalABSTRACT
Background: Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). Materials and methods: CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. Results: IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. Conclusion: We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.
Subject(s)
Adenocarcinoma , Carcinoma in Situ , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Mice , Animals , Mice, Transgenic , Mice, Inbred C57BL , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Galectins , Immunotherapy , Tumor MicroenvironmentABSTRACT
The rising pancreatic cancer incidence due to obesity and type 2 diabetes is closely tied to hyperinsulinemia, an independent cancer risk factor. Previous studies demonstrated reducing insulin production suppressed pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in Kras-mutant mice. However, the pathophysiological and molecular mechanisms remained unknown, and in particular it was unclear whether hyperinsulinemia affected PanIN precursor cells directly or indirectly. Here, we demonstrate that insulin receptors (Insr) in KrasG12D-expressing pancreatic acinar cells are dispensable for glucose homeostasis but necessary for hyperinsulinemia-driven PanIN formation in the context of diet-induced hyperinsulinemia and obesity. Mechanistically, this was attributed to amplified digestive enzyme protein translation, triggering of local inflammation, and PanIN metaplasia in vivo. In vitro, insulin dose-dependently increased acinar-to-ductal metaplasia formation in a trypsin- and Insr-dependent manner. Collectively, our data shed light on the mechanisms connecting obesity-driven hyperinsulinemia and pancreatic cancer development.
Subject(s)
Carcinoma in Situ , Diabetes Mellitus, Type 2 , Hyperinsulinism , Insulins , Pancreatic Neoplasms , Mice , Animals , Proto-Oncogene Proteins p21(ras)/metabolism , Receptor, Insulin/metabolism , Diabetes Mellitus, Type 2/metabolism , Pancreatic Neoplasms/metabolism , Acinar Cells/metabolism , Acinar Cells/pathology , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Inflammation/metabolism , Hyperinsulinism/complications , Metaplasia/metabolism , Metaplasia/pathology , Obesity/metabolism , Insulins/metabolismABSTRACT
BACKGROUND AND AIMS: Development of pancreatic ductal adenocarcinoma (PDAC) is a multistep process intensively studied; however, precocious diagnosis and effective therapy still remain unsatisfactory. The role for Notch signaling in PDAC has been discussed controversially, as both cancer-promoting and cancer-antagonizing functions have been described. Thus, an improved understanding of the underlying molecular mechanisms is necessary. Here, we focused on RBPJ, the receiving transcription factor in the Notch pathway, examined its expression pattern in PDAC, and characterized its function in mouse models of pancreatic cancer development and in the regeneration process after acute pancreatitis. METHODS: Conditional transgenic mouse models were used for functional analysis of RBPJ in the adult pancreas, initiation of PDAC precursor lesions, and pancreatic regeneration. Pancreata and primary acinar cells were tested for acinar-to-ductal metaplasia together with immunohistology and comprehensive transcriptional profiling by RNA sequencing. RESULTS: We identified reduced RBPJ expression in a subset of human PDAC specimens. Ptf1α-CreERT-driven depletion of RBPJ in transgenic mice revealed that its function is dispensable for the homeostasis and maintenance of adult acinar cells. However, primary RBPJ-deficient acinar cells underwent acinar-to-ductal differentiation in ex vivo. Importantly, oncogenic KRAS expression in the context of RBPJ deficiency facilitated the development of pancreatic intraepithelial neoplasia lesions with massive fibrotic stroma formation. Interestingly, RNA-sequencing data revealed a transcriptional profile associated with the cytokine/chemokine and extracellular matrix changes. In addition, lack of RBPJ delays the course of acute pancreatitis and critically impairs it in the context of KRASG12D expression. CONCLUSIONS: Our findings imply that downregulation of RBPJ in PDAC patients derepresses Notch targets and promotes KRAS-mediated pancreatic acinar cells transformation and desmoplasia development.
Subject(s)
Carcinoma in Situ , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Pancreatitis , Animals , Humans , Mice , Acinar Cells/metabolism , Acute Disease , Carcinoma in Situ/metabolism , Carcinoma, Pancreatic Ductal/pathology , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Mice, Transgenic , Pancreatic Neoplasms/pathology , Pancreatitis/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Pancreatic NeoplasmsABSTRACT
Ras plays an essential role in the development of acinar-to-ductal metaplasia (ADM) and pancreatic ductal adenocarcinoma (PDAC). However, mutant Kras is an inefficient driver for PDAC development. The mechanisms of the switching from low Ras activity to high Ras activity that are required for development and progression of pancreatic intraepithelial neoplasias (PanINs) are unclear. In this study, we found that hematopoietic progenitor kinase 1 (HPK1) was upregulated during pancreatic injury and ADM. HPK1 interacted with the SH3 domain and phosphorylated Ras GTPase-activating protein (RasGAP) and upregulated RasGAP activity. Using transgenic mouse models of HPK1 or M46, a kinase-dead mutant of HPK1, we showed that HPK1 inhibited Ras activity and its downstream signaling and regulated acinar cell plasticity. M46 promoted the development of ADM and PanINs. Expression of M46 in KrasG12D Bac mice promoted the infiltration of myeloid-derived suppressor cells and macrophages, inhibited the infiltration of T cells, and accelerated the progression of PanINs to invasive and metastatic PDAC, while HPK1 attenuated mutant Kras-driven PanIN progression. Our results showed that HPK1 plays an important role in ADM and the progression of PanINs by regulating Ras signaling. Loss of HPK1 kinase activity promotes an immunosuppressive tumor microenvironment and accelerates the progression of PanINs to PDAC.
Subject(s)
Carcinoma in Situ , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Mice , Animals , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Pancreatic Neoplasms/pathology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma in Situ/genetics , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Mice, Transgenic , Tumor Microenvironment , Pancreatic NeoplasmsABSTRACT
The adult healthy human pancreas has been poorly studied given the lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors, thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathologic analysis of the samples revealed pancreatic intraepithelial neoplasia (PanIN) lesions in most individuals irrespective of age. Using a combination of multiplex IHC, single-cell RNA sequencing, and spatial transcriptomics, we provide the first-ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis. SIGNIFICANCE: Precursor lesions to pancreatic cancer are poorly characterized. We analyzed donor pancreata and discovered that precursor lesions are detected at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell-intrinsic factors that restrain or, conversely, promote malignant progression. See related commentary by Hoffman and Dougan, p. 1288. This article is highlighted in the In This Issue feature, p. 1275.
Subject(s)
Carcinoma in Situ , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Adult , Humans , Transcriptome , Pancreas/pathology , Pancreatic Neoplasms/pathology , Carcinoma in Situ/genetics , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Carcinoma, Pancreatic Ductal/pathology , Tumor Microenvironment/geneticsABSTRACT
BACKGROUND: Pagetoid urothelial intraepithelial neoplasia (PUIN) is a form of secondary Extramammary Paget Disease (EMPD). It is a rare malignant condition seen on the female genitalia synchronous or metachronous with bladder cancer (BC). CASE PRESENTATION: A 66-year-old female presented with PUIN at the labia minora 2 years after an open anterior pelvic exenteration with ileal conduit urinary diversion for carcinoma in situ (CIS) of the bladder. PUIN of the vulva and vagina was confirmed by a punch biopsy and the patient underwent a radical vaginectomy with urethrectomy and inguinal sentinel node procedure. Immunohistochemically EMPD was identified by the expression tumor protein 63 (p63), cytokeratin 7, and cytokeratin 20 (CK20). CONCLUSIONS: PUIN is a rare but distinct clinical entity as a form of secondary EMPD which can be differentiated from primary EMPD based on medical history, histology, and immunohistochemistry.
Subject(s)
Carcinoma in Situ , Carcinoma, Squamous Cell , Paget Disease, Extramammary , Urinary Bladder Neoplasms , Vulvar Neoplasms , Humans , Female , Aged , Biomarkers, Tumor , Vulvar Neoplasms/diagnosis , Vulvar Neoplasms/surgery , Vulvar Neoplasms/metabolism , Carcinoma in Situ/surgery , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Urinary Bladder Neoplasms/surgery , Urinary Bladder Neoplasms/pathology , Carcinoma, Squamous Cell/pathology , Paget Disease, Extramammary/diagnosis , Paget Disease, Extramammary/surgeryABSTRACT
SAG/RBX2 is an E3 ligase, whereas SHOC2 is a RAS-RAF positive regulator. In this study, we address how Sag-Shoc2 crosstalk regulates pancreatic tumorigenesis induced by KrasG12D. Sag deletion increases the size of pancreas and causes the conversion of murine pancreatic intraepithelial neoplasms (mPanINs) to neoplastic cystic lesions with a mechanism involving Shoc2 accumulation, suggesting that Sag determines the pathological process via targeting Shoc2. Shoc2 deletion significantly inhibits pancreas growth, mPanIN formation, and acinar cell transdifferentiation, indicating that Shoc2 is essential for KrasG12D-induced pancreatic tumorigenesis. Likewise, in a primary acinar 3D culture, Sag deletion inhibits acinar-to-ductal transdifferentiation, while Shoc2 deletion significantly reduces the duct-like structures. Mechanistically, SAG is an E3 ligase that targets SHOC2 for degradation to affect both Mapk and mTorc1 pathways. Shoc2 deletion completely rescues the phenotype of neoplastic cystic lesions induced by Sag deletion, indicating physiological relevance of the Sag-Shoc2 crosstalk. Thus, the Sag-Shoc2 axis specifies the pancreatic tumor types induced by KrasG12D.
Subject(s)
Carcinoma in Situ , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Mice , Animals , Signal Transduction , Pancreatic Neoplasms/pathology , Pancreas/metabolism , Carcinoma in Situ/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Carcinogenesis , Carcinoma, Pancreatic Ductal/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Cell Transformation, Neoplastic/pathologyABSTRACT
Skeletal muscle wasting critically impairs the survival and quality of life in patients with pancreatic ductal adenocarcinoma (PDAC). To identify the local factors initiating muscle wasting, we studied inflammation, fiber cross-sectional area (CSA), composition, amino acid metabolism and capillarization, as well as the integrity of neuromuscular junctions (NMJ, pre-/postsynaptic co-staining) and mitochondria (electron microscopy) in the hindlimb muscle of LSL-KrasG12D/+; LSL-TrP53R172H/+; Pdx1-Cre mice with intraepithelial-neoplasia (PanIN) 1-3 and PDAC, compared to wild-type mice (WT). Significant decreases in fiber CSA occurred with PDAC but not with PanIN 1-3, compared to WT: These were found in the gastrocnemius (type 2x: −20.0%) and soleus (type 2a: −21.0%, type 1: −14.2%) muscle with accentuation in the male soleus (type 2a: −24.8%, type 1: −17.4%) and female gastrocnemius muscle (−29.6%). Significantly higher densities of endomysial CD68+ and cyclooxygenase-2+ (COX2+) cells were detected in mice with PDAC, compared to WT mice. Surprisingly, CD68+ and COX2+ cell densities were also higher in mice with PanIN 1-3 in both muscles. Significant positive correlations existed between muscular and hepatic CD68+ or COX2+ cell densities. Moreover, in the gastrocnemius muscle, suppressor-of-cytokine-3 (SOCS3) expressions was upregulated >2.7-fold with PanIN 1A-3 and PDAC. The intracellular pools of proteinogenic amino acids and glutathione significantly increased with PanIN 1A-3 compared to WT. Capillarization, NMJ, and mitochondrial ultrastructure remained unchanged with PanIN or PDAC. In conclusion, the onset of fiber atrophy coincides with the manifestation of PDAC and high-grade local (and hepatic) inflammatory infiltration without compromised microcirculation, innervation or mitochondria. Surprisingly, muscular and hepatic inflammation, SOCS3 upregulation and (proteolytic) increases in free amino acids and glutathione were already detectable in mice with precancerous PanINs. Studies of initial local triggers and defense mechanisms regarding cachexia are warranted for targeted anti-inflammatory prevention.
Subject(s)
Carcinoma in Situ , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Amino Acids , Animals , Cachexia , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Carcinoma, Pancreatic Ductal/complications , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Cyclooxygenase 2/metabolism , Disease Progression , Female , Glutathione/metabolism , Humans , Inflammation , Male , Mice , Muscle, Skeletal/metabolism , Pancreatic Neoplasms/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Quality of Life , Tumor Suppressor Protein p53 , Pancreatic NeoplasmsABSTRACT
BACKGROUND: The differential diagnosis between flat urothelial lesions [reactive urothelial atypia (RUA), atypia of unknown significance (AUS), urothelial dysplasia (UD) and carcinoma in situ (CIS)] has relevant prognostic and therapeutic implications. This crucial distinction could be very challenging but it is currently performed on hematoxylin and eosin (H&E) slides, with a great amount of partially discordant and/or not conclusive findings of the potential adjunctive role of immunohistochemistry. Herein, we tested double staining (DS) for p53/CK20 to verify if p53(+) cells, CK20(+) cells and double-positive cells (DPCs) are differentially expressed among these lesions and if p53/CK20 could be a useful tool in this diagnostic setting. METHODS: We tested 50, 9, 36 and 29 consecutive and retrospectively enrolled cases of RUA, AUS, UD and CIS, respectively. p53(+) cells, CK20(+) cells and DPCs were evaluated and compared by adopting the appropriate statistic tests (Mann-Whitney U and Kruskal-Wallis tests). RESULTS: We found that p53(+) cells (p = 0.000), CK20(+) cells (p = 0.000) and DPCs (p = 0.000) showed statistically significant differences among the different flat urothelial lesions. Besides, when dichotomized, both CIS and RUA are easily differentiable from their histological mimickers adopting all these markers; by contrast, AUS and UD did not reach statistically significant differences able to differentiate them from each other [p53(+) cells, p = 0.123; CK20(+) cells, p = 0.567; DPCs, p = 0.409], except if compared to CIS [AUS VS CIS: p53(+) cells, p = 0.013; CK20(+) cells, p = 0.000; DPCs, p = 0.000; UD vs CIS: p53(+) cells, p = 0.000; CK20(+) cells, p = 0.000; DPCs, p = 0.000]. CONCLUSIONS: p53(+) cells, CK20(+) cells and DPCs are differently expressed by flat urothelial lesions and p53/CK20 could be a time- and money-saving tool for the appropriate management of these lesions if applied to a routine scenario.
Subject(s)
Carcinoma in Situ , Carcinoma, Transitional Cell , Urinary Bladder Neoplasms , Biomarkers, Tumor/analysis , Biomarkers, Tumor/metabolism , Carcinoma in Situ/diagnosis , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Carcinoma, Transitional Cell/diagnosis , Carcinoma, Transitional Cell/metabolism , Carcinoma, Transitional Cell/pathology , Coloring Agents , Humans , Keratin-20/analysis , Keratin-20/metabolism , Retrospective Studies , Staining and Labeling , Tumor Suppressor Protein p53/analysis , Tumor Suppressor Protein p53/metabolism , Urinary Bladder Neoplasms/diagnosis , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathologyABSTRACT
Molecular events occurring in stepwise progression from pre-malignant lesions (pancreatic intraepithelial neoplasia; PanIN) to the development of pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Thus, characterization of early PanIN lesions may reveal markers that can help in diagnosing PDAC at an early stage and allow understanding the pathology of the disease. We performed the molecular and histological assessment of patient-derived PanINs, tumor tissues and pancreas from mouse models with PDAC (KC mice that harbor K-RAS mutation in pancreatic tissue), where we noted marked upregulation of gastrokine (GKN) proteins. To further understand the role of gastrokine proteins in PDAC development, GKN-deficient KC mice were developed by intercrossing gastrokine-deficient mice with KC mice. Panc-02 (pancreatic cancer cells of mouse origin) were genetically modified to express GKN1 for further in vitro and in vivo analysis. Our results show that gastrokine proteins were absent in healthy pancreas and invasive cancer, while its expression was prominent in low-grade PanINs. We could detect these proteins in pancreatic juice and serum of KC mice. Furthermore, accelerated PanIN and tumor development were noted in gastrokine deficient KC mice. Loss of gastrokine 1 protein delayed apoptosis during carcinogenesis leading to the development of desmoplastic stroma while loss of gastrokine 2 increased the proliferation rate in precursor lesions. In summary, we identified gastrokine proteins in early pancreatic precursor lesions, where gastrokine proteins delay pancreatic carcinogenesis.
Subject(s)
Carcinoma in Situ , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Peptide Hormones , Animals , Carcinogenesis , Carcinoma in Situ/genetics , Carcinoma in Situ/metabolism , Carcinoma in Situ/pathology , Carcinoma, Pancreatic Ductal/pathology , Humans , Mice , Pancreas/pathology , Pancreatic Neoplasms/pathology , Pancreatic NeoplasmsABSTRACT
BACKGROUND: Preinvasive cancer conditions are often actively treated to minimize progression to life-threatening invasive cancers, but this creates challenges for analysis of invasive cancer risk. Conventional methods of treating preinvasive conditions as censoring events or targeting at the composite outcome could both lead to bias. METHODS: We propose two solutions: one that provides exact estimates of risk based on distributional assumptions about progression, and one that provides risk bounds corresponding to extreme cases of no or complete progression. We compare these approaches through simulations and an analysis of the Sister Study data in the context of ductal carcinoma in situ (DCIS) and invasive breast cancer. RESULTS: Simulations suggested important biases with conventional approaches, whereas the proposed estimate is consistent when progression parameters are correctly specified, and the risk bounds are robust in all scenarios. With Sister Study, the estimated lifetime risks for invasive breast cancer are 0.220 and 0.269 with DCIS censored or combined. Without detailed progression information, a sensitivity analysis suggested lifetime risk falls between the bounds of 0.214 and 0.269 across assumptions of 10%-95% of DCIS patients progressing to invasive cancer in an average of 1-10 years. CONCLUSIONS: When estimating invasive cancer risk while preinvasive conditions are actively treated, it is important to consider the implied assumptions and potential biases of conventional approaches. Although still not perfect, we proposed two practical solutions that provide improved understanding of the underlying mechanism of invasive cancer.