Cancer-Associated Fibroblast Induces Acinar-to-Ductal Cell Transdifferentiation and Pancreatic Cancer Initiation Via LAMA5/ITGA4 Axis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplastic stroma surrounding most tumors. Activated stromal fibroblasts, namely cancer-associated fibroblasts (CAFs), play a major role in PDAC progression. We analyzed whether CAFs influence acinar cells and impact PDAC initiation, that is, acinar-to-ductal metaplasia (ADM). ADM connection with PDAC pathophysiology is indicated, but not yet established. We hypothesized that CAF secretome might play a significant role in ADM in PDAC initiation. METHODS: Mouse and human acinar cell organoids, acinar cells cocultured with CAFs and exposed to CAF-conditioned media, acinar cell explants, and CAF cocultures were examined by means of quantitative reverse transcription polymerase chain reaction, RNA sequencing, immunoblotting, and confocal microscopy. Data from liquid chromatography with tandem mass spectrometry analysis of CAF-conditioned medium and RNA sequencing data of acinar cells post-conditioned medium exposure were integrated using bioinformatics tools to identify the molecular mechanism for CAF-induced ADM. Using confocal microscopy, immunoblotting, and quantitative reverse transcription polymerase chain reaction analysis, we validated the depletion of a key signaling axis in the cell line, acinar explant coculture, and mouse cancer-associated fibroblasts (mCAFs). RESULTS: A close association of acino-ductal markers (Ulex europaeus agglutinin 1, amylase, cytokeratin-19) and mCAFs (α-smooth muscle actin) in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1Cre (KPC) and LSL-KrasG12D/+; Pdx1Cre (KC) autochthonous progression tumor tissue was observed. Caerulein treatment-induced mCAFs increased cytokeratin-19 and decreased amylase in wild-type and KC pancreas. Likewise, acinar-mCAF cocultures revealed the induction of ductal transdifferentiation in cell line, acinar-organoid, and explant coculture formats in WT and KC mice pancreas. Proteomic and transcriptomic data integration revealed a novel laminin α5/integrinα4/stat3 axis responsible for CAF-mediated acinar-to-ductal cell transdifferentiation. CONCLUSIONS: Results collectively suggest the first evidence for CAF-influenced acino-ductal phenotypic switchover, thus highlighting the tumor microenvironment role in pancreatic carcinogenesis inception.

Reduction in O-glycome induces differentially glycosylated CD44 to promote stemness and metastasis in pancreatic cancer.

Aberrant protein glycosylation has been shown to have a significant contribution in aggressive cancer, including pancreatic cancer (PC). Emerging evidence has implicated the involvement of cancer stem cells (CSCs) in PC aggressiveness; however, the contribution of glycosylation on self-renewal properties and maintenance of CSC is understudied. Here, using several in vitro and in vivo models lacking C1GALT1 expression, we identified the role of aberrant O-glycosylation in stemness properties and aggressive PC metastasis. A loss in C1GALT1 was found to result in the truncation of O-glycosylation on several glycoproteins with an enrichment of Tn carbohydrate antigen. Mapping of Tn-bearing glycoproteins in C1GALT1 KO cells identified significant Tn enrichment on CSC glycoprotein CD44. Notably, a loss of C1GALT1 in PC cells was found to enhance CSC features (side population-SP, ALDH1+, and tumorspheres) and self-renewal markers NANOG, SOX9, and KLF4. Furthermore, a loss of CD44 in existing C1GALT1 KO cells decreased NANOG expression and CSC features. We determined that O-glycosylation of CD44 activates ERK/NF-kB signaling, which results in increased NANOG expression in PC cells that facilitated the alteration of CSC features, suggesting that NANOG is essential for PC stemness. Finally, we identified that loss of C1GALT1 expression was found to augment tumorigenic and metastatic potential, while an additional loss of CD44 in these cells reversed the effects. Overall, our results identified that truncation of O-glycans on CD44 increases NANOG activation that mediates increased CSC activation.

Three regulatory compliant test systems show no signs of MDMA-related genotoxicity.

3,4 Methylenedioxymethamphetamine (MDMA)-assisted therapy has been recently found to be highly effective for treatment of posttraumatic stress disorder (PTSD). Previous studies have been inconclusive in elucidating potential MDMA genotoxicity. We performed three regulatory compliant studies to investigate the potential of genotoxic effects of MDMA treatment in humans: (1) an in vitro bacterial reverse mutation (Ames) assay, (2) an in vitro chromosome aberration test in Chinese hamster ovary cells, and (3) an in vivo micronucleus study in male Sprague Dawley rats. MDMA was found to not have genotoxic effects in any of the assays at or above clinically relevant concentrations.

Pancreatic Tumor Microenvironment Factor Promotes Cancer Stemness via SPP1-CD44 Axis.

BACKGROUND & AIMS: Tumor-microenvironment factors and cancer stem cells (CSCs) play a critical role in the aggressiveness of pancreatic cancer (PC). However, the degree to which tumor-microenvironment factors promote stemness remains unexplored. Here, we examined whether cancer-associated fibroblasts (CAFs) promote CSC features in PC. METHODS: PC cells were treated long-term (30, 60, and 90 days) with conditioned media (CM)-derived from normal human fibroblasts (NFs) and CAFs. The stemness features of tumorsphere formation and stemness populations, along with CSCs markers, were analyzed using 2-dimensional and 3-dimensional sodium alginate bead-based co-culture models. Immunohistochemistry and immunofluorescence staining were performed for CSCs and fibroblast markers in autochthonous KrasG12D/+; Trp53R172H/+; Pdx1-Cre mice and human pancreatic tumors. Polymerase chain reaction array and gene knockdown were performed to identify the mechanism of stemness enrichment. RESULTS: Long-term treatment of PC cells with CAF-CM enriched stemness, as indicated by significantly higher CD44+, ALDH+, and AF+ populations in PC cells. Increased tumorsphere formation and elevated CSC, self-renewal, and drug-resistance markers in CAF-CM-treated PC cells were observed. In addition, CAFs co-cultured with PC cells in the 3-dimensional model showed a substantial increase in stemness features. CD44 and α-smooth muscle actin were positively correlated and their expressions progressively increased from the early to late stages of KrasG12D/+; Trp53R172H/+; Pdx1-Cre mouse and human pancreatic tumors. Osteopontin/secreted phosphoprotein 1 was identified as the top differentially overexpressed gene in CAF-CM-treated PC cells and knockdown of osteopontin/secreted phosphoprotein 1 significantly reduced stemness characteristics in CAF-CM-treated PC cells. CONCLUSIONS: Our data uncovered novel insight into the interplay between CAF and enrichment of stemness population through the osteopontin/secreted phosphoprotein 1-CD44 axis in PC.

Metabolic programming of distinct cancer stem cells promotes metastasis of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) metastasizes to distant organs, which is the primary cause of mortality; however, specific features mediating organ-specific metastasis remain unexplored. Emerging evidence demonstrates that cancer stem cells (CSCs) and cellular metabolism play a pivotal role in metastasis. Here we investigated the role of distinct subtypes of pancreatic CSCs and their metabolomic signatures in organ-specific metastatic colonization. We found that PDAC consists of ALDH+/CD133+ and drug-resistant (MDR1+) subtypes of CSCs with specific metabolic and stemness signatures. Human PDAC tissues with gemcitabine treatment, autochthonous mouse tumors from KrasG12D; Pdx1-Cre (KC) and KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) mice, and KPC- Liver/Lung metastatic cells were used to evaluate the CSC, EMT (epithelial-to-mesenchymal transition), and metabolic profiles. A strong association was observed between distinct CSC subtypes and organ-specific colonization. The liver metastasis showed drug-resistant CSC- and EMT-like phenotype with aerobic glycolysis and fatty acid ß-oxidation-mediated oxidative (glyco-oxidative) metabolism. On the contrary, lung metastasis displayed ALDH+/CD133+ and MET-like phenotype with oxidative metabolism. These results were obtained by evaluating FACS-based side population (SP), autofluorescence (AF+) and Alde-red assays for CSCs, and Seahorse-based oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and fatty acid ß-oxidation (FAO)-mediated OCR assays for metabolic features along with specific gene signatures. Further, we developed in vitro human liver and lung PDAC metastasis models by using a combination of liver or lung decellularized scaffolds, a co-culture, and a sphere culture methods. PDAC cells grown in the liver-mimicking model showed the enrichment of MDR1+ and CPT1A+ populations, whereas the PDAC cells grown in the lung-mimicking environment showed the enrichment of ALDH+/CD133+ populations. In addition, we observed significantly elevated expression of ALDH1 in lung metastasis and MDR1/LDH-A expression in liver metastasis compared to human primary PDAC tumors. Our studies elucidate that distinct CSCs adapt unique metabolic signatures for organotropic metastasis, which will pave the way for the development of targeted therapy for PDAC metastasis.

RNA Polymerase II-Associated Factor 1 Regulates Stem Cell Features of Pancreatic Cancer Cells, Independently of the PAF1 Complex, via Interactions With PHF5A and DDX3.

BACKGROUND & AIMS: It is not clear how pancreatic cancer stem cells (CSCs) are regulated, resulting in ineffective treatments for pancreatic cancer. PAF1, a RNA polymerase II-associated factor 1 complex (PAF1C) component, maintains pluripotency of stem cells, by unclear mechanisms, and is a marker of CSCs. We investigated mechanisms by which PAF1 maintains CSCs and contributes to development of pancreatic tumors. METHODS: Pancreatic cancer cell lines were engineered to knockdown PAF1 using inducible small hairpin RNAs. These cells were grown as orthotopic tumors in athymic nude mice and PAF1 knockdown was induced by administration of doxycycline in drinking water. Tumor growth and metastasis were monitored via IVIS imaging. CSCs were isolated from pancreatic cancer cell populations using flow cytometry and characterized by tumor sphere formation, tumor formation in nude mice, and expression of CSC markers. Isolated CSCs were depleted of PAF1 using the CRISPR/Cas9 system. PAF1-regulated genes in CSCs were identified via RNA-seq and PCR array analyses of cells with PAF1 knockdown. Proteins that interact with PAF1 in CSCs were identified by immunoprecipitations and mass spectrometry. We performed chromatin immunoprecipitation sequencing of CSCs to confirm the binding of the PAF1 sub-complex to target genes. RESULTS: Pancreatic cancer cells depleted of PAF1 formed smaller and fewer tumor spheres in culture and orthotopic tumors and metastases in mice. Isolated CSCs depleted of PAF1 downregulated markers of self-renewal (NANOG, SOX9, and ß-CATENIN), of CSCs (CD44v6, and ALDH1), and the metastasis-associated gene signature, compared to CSCs without knockdown of PAF1. The role of PAF1 in CSC maintenance was independent of its RNA polymerase II-associated factor 1 complex component identity. We identified DDX3 and PHF5A as proteins that interact with PAF1 in CSCs and demonstrated that the PAF1-PHF5A-DDX3 sub-complex bound to the promoter region of Nanog, whose product regulates genes that control stemness. Levels of the PAF1-DDX3 and PAF1-PHF5A were increased and co-localized in human pancreatic tumor specimens, human pancreatic tumor-derived organoids, and organoids derived from tumors of KPC mice, compared with controls. Binding of DDX3 and PAF1 to the Nanog promoter, and the self-renewal capacity of CSCs, were decreased in cells incubated with the DDX3 inhibitor RK-33. CSCs depleted of PAF1 downregulated genes that regulate stem cell features (Flot2, Taz, Epcam, Erbb2, Foxp1, Abcc5, Ddr1, Muc1, Pecam1, Notch3, Aldh1a3, Foxa2, Plat, and Lif). CONCLUSIONS: In pancreatic CSCs, PAF1 interacts with DDX3 and PHF5A to regulate expression of NANOG and other genes that regulate stemness. Knockdown of PAF1 reduces the ability of orthotopic pancreatic tumors to develop and progress in mice and their numbers of CSCs. Strategies to target the PAF1-PHF5A-DDX3 complex might be developed to slow or inhibit progression of pancreatic cancer.

Correction: Global analysis of human glycosyltransferases reveals novel targets for pancreatic cancer pathogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Global analysis of human glycosyltransferases reveals novel targets for pancreatic cancer pathogenesis.

BACKGROUND: Several reports have shown the role of glycosylation in pancreatic cancer (PC), but a global systematic screening of specific glycosyltransferases (glycoTs) in its progression remains unknown. METHODS: We demonstrate a rigorous top-down approach using TCGA-based RNA-Seq analysis, multi-step validation using RT-qPCR, immunoblots and immunohistochemistry. We identified six unique glycoTs (B3GNT3, B4GALNT3, FUT3, FUT6, GCNT3 and MGAT3) in PC pathogenesis and studied their function using CRISPR/Cas9-based KD systems. RESULTS: Serial metastatic in vitro models using T3M4 and HPAF/CD18, generated in house, exhibited decreases in B3GNT3, FUT3 and GCNT3 expression on increasing metastatic potential. Immunohistochemistry identified clinical significance for GCNT3, B4GALNT3 and MGAT3 in PC. Furthermore, the effects of B3GNT3, FUT3, GCNT3 and MGAT3 were shown on proliferation, migration, EMT and stem cell markers in CD18 cell line. Talniflumate, GCNT3 inhibitor, reduced colony formation and migration in T3M4 and CD18 cells. Moreover, we found that loss of GCNT3 suppresses PC progression and metastasis by downregulating cell cycle genes and ß-catenin/MUC4 axis. For GCNT3, proteomics revealed downregulation of MUC5AC, MUC1, MUC5B including many other proteins. CONCLUSIONS: Collectively, we demonstrate a critical role of O- and N-linked glycoTs in PC progression and delineate the mechanism encompassing the role of GCNT3 in PC.

A Systematic Review on the Implications of O-linked Glycan Branching and Truncating Enzymes on Cancer Progression and Metastasis.

Glycosylation is the most commonly occurring post-translational modifications, and is believed to modify over 50% of all proteins. The process of glycan modification is directed by different glycosyltransferases, depending on the cell in which it is expressed. These small carbohydrate molecules consist of multiple glycan families that facilitate cell-cell interactions, protein interactions, and downstream signaling. An alteration of several types of O-glycan core structures have been implicated in multiple cancers, largely due to differential glycosyltransferase expression or activity. Consequently, aberrant O-linked glycosylation has been extensively demonstrated to affect biological function and protein integrity that directly result in cancer growth and progression of several diseases. Herein, we provide a comprehensive review of several initiating enzymes involved in the synthesis of O-linked glycosylation that significantly contribute to a number of different cancers.

Growth factor-responsive progenitors in the postnatal mammalian retina.

It is thought that the adult mammalian retina lacks the regenerative capacity of fish and amphibians retina because it does not harbor a progenitor population. However, recent observations suggest that another derivative of the optic neuroepithelium, the ciliary body, contains a mitotically quiescent population of neural progenitors that proliferate in the presence of growth factors and demonstrate properties of neural stem cells. Examination of the hypothesis that similar mitotically quiescent and growth factor-responsive progenitors may exist in the postnatal retina revealed a population of cells located in the periphery of the retina that displayed proliferative responsiveness to growth factors and possessed potential to support neurogenesis. Given their marginal position and neural properties and potential, these cells may represent a residual population of retinal progenitors, analogous to those found in the ciliary marginal zone of fish and amphibians. Their progressive decrease in proliferative potential and number in postnatal stages suggests a temporal decline in regulatory signaling that supports their maintenance during retinal neurogenesis.

Experiencia en tumores malignos en el Complejo Científico Ortopédico Internacional ôFrank Paísö / Malignant tumor experience in Frank País International Scientific Orthopedic Institution

Se presenta una muestra estadística retrospectiva de los pacientes afectados de tumores malignos del sistema osteomioarticular, diagnosticados en Servicio de Investigación y Desarrollo del Complejo Científico Ortopédico Internacional ôFrank Paísö en el período comprendido entre enero de 1990 y diciembre de 1999. Se estudió la variedad histológica, localización, edad y sexo. En el trabajo se halló que los tumores malignos más frecuentes fueron el osteosarcoma, el tumor de células gigantes y el condrosarcoma, los cuales predominaron en pacientes menores de 25 años

Experiencia en tumores malignos en el Complejo Científico Ortopédico Internacional “Frank País” / Malignant tumor experience in “Frank País” International Scientific Orthopedic Institution

Se presenta una muestra estadística retrospectiva de los pacientes afectados de tumores malignos del sistema osteomioarticular, diagnosticados en Servicio de Investigación y Desarrollo del Complejo Científico Ortopédico Internacional “Frank País” en el período comprendido entre enero de 1990 y diciembre de 1999. Se estudió la variedad histológica, localización, edad y sexo. En el trabajo se halló que los tumores malignos más frecuentes fueron el osteosarcoma, el tumor de células gigantes y el condrosarcoma, los cuales predominaron en pacientes menores de 25 años(AU)

Incidencia de tumores óseos y de partes blandas, malignos / Incidence of malignant bone and soft tissue tumors

Se realiza un estudio retrospectivo descriptivo, de los pacientes portadores de tumores óseos y de partes blandas, malignos, diagnosticados y tratados en nuestro servicio, entre enero de 1990 y noviembre de 1998. El objetivo fue conocer la incidencia de estas enfermedades malignas y sus características, así como las variables clínicas de las formas de presentación: edad, sexo, localización de la lesión y el tipo y variedad del tumor. De 370 historias con diagnóstico de un tumor esquelético se encontraron 177 tumores malignos. De éstos 158 fueron primitivos (126 óseos y 32 en partes blandas) y 19 se diagnosticaron como metastásicos. Se encontró que el osteosarcoma fue el tumor óseo más frecuente en 51 pacientes (32 porciento), siguiendo en frecuencia el tumor de células gigantes en 36 pacientes (23 porciento) y el condrosarcoma 24 pacientes (15 porciento), donde predominaron menores de 25 años. Sólo hubo 4 casos de sarcoma de Ewing (2 porciento). Hubo 32 pacientes con sarcomas de partes blandas, lo que representa el 20 porciento del total. De éstos, los más frecuentes fueron el sarcoma sinovial con 9 pacientes (6 porciento), el liposarcoma con 8 pacientes (5 porciento) y el rabdomiosarcoma con 5 pacientes (3 porciento). También se encontraron 19 pacientes con lesiones secundarias en huesos. Estas lesiones metastásicas secundarias a tumores primarios de la próstata en 11, y la mama en 5 predominaron en pacientes de más de 50 años, y sobre todo se localizaron en el fémur en 14 casos

Incidencia de tumores óseos y de partes blandas, malignos / Incidence of malignant bone and soft tissue tumors

Se realiza un estudio retrospectivo descriptivo, de los pacientes portadores de tumores óseos y de partes blandas, malignos, diagnosticados y tratados en nuestro servicio, entre enero de 1990 y noviembre de 1998. El objetivo fue conocer la incidencia de estas enfermedades malignas y sus características, así como las variables clínicas de las formas de presentación: edad, sexo, localización de la lesión y el tipo y variedad del tumor. De 370 historias con diagnóstico de un tumor esquelético se encontraron 177 tumores malignos. De éstos 158 fueron primitivos (126 óseos y 32 en partes blandas) y 19 se diagnosticaron como metastásicos. Se encontró que el osteosarcoma fue el tumor óseo más frecuente en 51 pacientes (32 porciento), siguiendo en frecuencia el tumor de células gigantes en 36 pacientes (23 porciento) y el condrosarcoma 24 pacientes (15 porciento), donde predominaron menores de 25 años. Sólo hubo 4 casos de sarcoma de Ewing (2 porciento). Hubo 32 pacientes con sarcomas de partes blandas, lo que representa el 20 porciento del total. De éstos, los más frecuentes fueron el sarcoma sinovial con 9 pacientes (6 porciento), el liposarcoma con 8 pacientes (5 porciento) y el rabdomiosarcoma con 5 pacientes (3 porciento). También se encontraron 19 pacientes con lesiones secundarias en huesos. Estas lesiones metastásicas secundarias a tumores primarios de la próstata en 11, y la mama en 5 predominaron en pacientes de más de 50 años, y sobre todo se localizaron en el fémur en 14 casos(AU)

Triángulo de Codman vs. signos de malignidad. Presentación de 2 casos / Codman triangle versus malignancy signs. Report of 2 cases

Las reacciones periostáticas son comunes en las lesiones óseas. A pesar de la facilidad con que se las reconoce radiográficamente, existen considerables aprehensiones infundadas sobre su diagnóstico. El triángulo de Codman es una imagen descrita por primera vez en 1914 por Ribbert, quien la atribuyó a una elevación perióstica causada por una masa de expansión. Considerado desde tiempo atrás como una manifestación de osteopatía maligna, en la actualidad se admite que puede deberse a cualquier bulto que levante el periostio, sea benigno o maligno. Se presentan 2 casos en los cuales aparece en las radiografías, desde las etapas iniciales, reacción perióstica tipo triángulo de Codman cuyo diagnóstico de anatomía patológica corresponde a lesiones tumorales benignas

Presentación atípica de un miofibroma aponeurótico calcificante / Atypical presentation of a calcifying aponeurotic myofibroma / Présentation atypique d´un myofibrome aponéurotique calcifiante

Se presenta un paciente con miofibroma aponeurótico juvenil de la pierna izquierda, localización atípica de este tumor derivado del tejido fibroso. Se expone el cuadro clínico y la terapéutica efectuada. Se revisa la literatura.

A patient with a juvenile aponeurotic myofibroma of the left leg, an atypical localization of this tumor derived from the fibrous tissue, is presented. The clinical picture and the therapeutics used with these patients are exposed. A literature review is made.

Un patient atteint d´un myofibrome aponévrotique jeune de la jambe gauche, une localisation atypique de cette tumeur résultant du tissu fibreux, est présenté. Le tableau clinique et la thérapeutique effectuée sont exposés. La littérature est mise en revue.