Mucin-producing urothelial-type adenocarcinoma of the prostate with sarcomatoid features and novel molecular phenotype.

Introduction: Mucin-producing urothelial-type adenocarcinoma of the prostate is a rare tumor that may not elevate serum prostate-specific antigen, creating significant diagnostic and monitoring challenges. We evaluate our case in detail and review prior studies to demonstrate that the pathologic and molecular features of this tumor are distinct from conventional prostate adenocarcinoma. Case presentation: Our patient had a remote history of radiation-treated conventional prostate adenocarcinoma and presented many years later with an abscess-like prostate mass leading to urinary obstruction and hematuria. Biopsy revealed mucin-producing urothelial-type adenocarcinoma of the prostate with concurrent sarcomatoid features. Molecular studies showed a unique phenotype involving alterations in the KRAS, PTEN, RAD21, and TP53 genes. Conclusions: To our knowledge, this is the first report that describes sarcomatoid features and molecular mutations in mucin-producing urothelial-type adenocarcinoma of the prostate.

Initial experience with an electron FLASH research extension (FLEX) for the Clinac system.

PURPOSE: Radiotherapy delivered at ultra-high-dose-rates (≥40 Gy/s), that is, FLASH, has the potential to effectively widen the therapeutic window and considerably improve the care of cancer patients. The underlying mechanism of the FLASH effect is not well understood, and commercial systems capable of delivering such dose rates are scarce. The purpose of this study was to perform the initial acceptance and commissioning tests of an electron FLASH research product for preclinical studies. METHODS: A linear accelerator (Clinac 23EX) was modified to include a non-clinical FLASH research extension (the Clinac-FLEX system) by Varian, a Siemens Healthineers company (Palo Alto, CA) capable of delivering a 16 MeV electron beam with FLASH and conventional dose rates. The acceptance, commissioning, and dosimetric characterization of the FLEX system was performed using radiochromic film, optically stimulated luminescent dosimeters, and a plane-parallel ionization chamber. A radiation survey was conducted for which the shielding of the pre-existing vault was deemed sufficient. RESULTS: The Clinac-FLEX system is capable of delivering a 16 MeV electron FLASH beam of approximately 1 Gy/pulse at isocenter and reached a maximum dose rate >3.8 Gy/pulse near the upper accessory mount on the linac gantry. The percent depth dose curves of the 16 MeV FLASH and conventional modes for the 10 × 10 cm2 applicator agreed within 0.5 mm at a range of 50% of the maximum dose. Their respective profiles agreed well in terms of flatness but deviated for field sizes >10 × 10 cm2 . The output stability of the FLASH system exhibited a dose deviation of <1%. Preliminary cell studies showed that the FLASH dose rate (180 Gy/s) had much less impact on the cell morphology of 76N breast normal cells compared to the non-FLASH dose rate (18 Gy/s), which induced large-size cells. CONCLUSION: Our studies characterized the non-clinical Clinac-FLEX system as a viable solution to conduct FLASH research that could substantially increase access to ultra-high-dose-rate capabilities for scientists.

The Development of Thermoresponsive Polymeric Simvastatin Prodrug for the Treatment of Experimental Periodontitis in Rats.

Periodontitis (PD) is a severe inflammatory gum pathology that damages the periodontal soft tissue and bone. It is highly prevalent in the US, affecting more than 47% of adults. Besides routine scaling and root planing, there are few effective treatments for PD. Developed as an effective treatment for hyperlipidemia, simvastatin (SIM) is also known for its well-established anti-inflammatory and osteogenic properties, suggesting its potential utility in treating PD. Its clinical translation, however, has been impeded by its poor water-solubility, lack of osteotropicity, and side effects (e.g., hepatoxicity) associated with systemic exposure. To address these challenges, an N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based thermoresponsive polymeric prodrug of SIM (ProGel-SIM) was developed as a local therapy for PD. Its aqueous solution is free-flowing at 4 °C and transitions into a hydrogel at ∼30 °C, allowing for easy local application and retention. After a thorough characterization of its physicochemical properties, ProGel-SIM was administered weekly into the periodontal pocket of an experimental rat model of PD. At 3 weeks post initiation of the treatment, the animals were euthanized with palate isolated for µ-CT and histological analyses. When compared to dose equivalent simvastatin acid (SMA, active form of SIM) treatment, the rats in the ProGel-SIM treated group showed significantly higher periodontal bone volume (0.34 mm3 vs 0.20 mm3, P = 0.0161) and less neutrophil (PMN) infiltration (P < 0.0001) and IL-1ß secretion (P = 0.0036). No measurable side effect was observed. Collectively, these results suggest that ProGel-SIM may be developed as a promising drug candidate for the effective clinical treatment of PD.

Left Axilla Adenocarcinoma of Unknown Origin.

A 76-year-old woman presents with a palpable left axillary mass, yet no breast lesions are found. What is your diagnosis?

MUC16 promotes triple-negative breast cancer lung metastasis by modulating RNA-binding protein ELAVL1/HUR.

BACKGROUND: Triple-negative breast cancer (TNBC) is highly aggressive with an increased metastatic incidence compared to other breast cancer subtypes. However, due to the absence of clinically reliable biomarkers and targeted therapy in TNBC, outcomes are suboptimal. Hence, there is an urgent need to understand biological mechanisms that lead to identifying novel therapeutic targets for managing metastatic TNBC. METHODS: The clinical significance of MUC16 and ELAVL1 or Hu antigen R (HuR) was examined using breast cancer TCGA data. Microarray was performed on MUC16 knockdown and scramble TNBC cells and MUC16-associated genes were identified using RNA immunoprecipitation and metastatic cDNA array. Metastatic properties of MUC16 were evaluated using tail vein experiment. MUC16 and HuR downstream pathways were confirmed by ectopic overexpression of MUC16-carboxyl-terminal (MUC16-Cter), HuR and cMyc as well as HuR inhibitors (MS-444 and CMLD-2) in TNBC cells. RESULTS: MUC16 was highly expressed in TNBC and correlated with its target HuR. Depletion of MUC16 showed decreased invasion, migration, and colony formation abilities of human and mouse TNBC cells. Mice injected with MUC16 depleted cells were less likely to develop lung metastasis (P = 0.001). Notably, MUC16 and HuR were highly expressed in the lung tropic TNBC cells and lung metastases. Mechanistically, we identified cMyc as a HuR target in TNBC using RNA immunoprecipitation and metastatic cDNA array. Furthermore, MUC16 knockdown and pharmacological inhibition of HuR (MS-444 and CMLD-2) in TNBC cells showed a reduction in cMyc expression. MUC16-Cter or HuR overexpression models indicated MUC16/HuR/cMyc axis in TNBC cell migration. CONCLUSIONS: Our study identified MUC16 as a TNBC lung metastasis promoter that acts through HuR/cMyc axis. This study will form the basis of future studies to evaluate the targeting of both MUC16 and HuR in TNBC patients.

Role of GD2 and its biosynthetic enzyme GD3 synthase in prostate cancer tumorigenesis.

While better management of loco-regional prostate cancer (PC) has greatly improved survival, advanced PC remains a major cause of cancer deaths. Identification of novel, targetable, pathways that contribute to tumor progression of PC could open new therapeutic options. The di-ganglioside GD2 is a target of FDA-approved antibody therapies in neuroblastoma, but the role of GD2 in PC has been only little explored. Here, we show that GD2 is expressed on a small subpopulation of PC cells in a subset of patients, especially in metastatic PC. Variable levels of cell surface GD2 expression are seen in most PC cell lines, and the expression is highly upregulated by experimental induction of lineage progression or enzalutamide resistance in CRPC cell models. GD2high cell fraction is enriched upon growth of PC cells as tumorspheres and GD2high fraction is enriched in tumorsphere growth. CRISPR-Cas9 knockout (KO) of the rate-limiting GD2 biosynthetic enzyme GD3 Synthase (GD3S) in GD2-high CRPC cell models led to marked impairment of their in vitro oncogenic traits, reduced cancer stem cell (CSC) and epithelial-mesenchymal transition (EMT) marker expression and growth as bone-implanted xenograft tumors. Our results support the potential role of GD3S and its product GD2 in promoting PC tumorigenesis by maintaining cancer stem cells and suggest the potential for GD2 targeting in advanced PC.

A case of metastatic adenoid cystic (basal cell) carcinoma of the prostate: Systemic therapy for a rare disease.

BACKGROUND: Metastatic adenoid cystic (basal cell) carcinoma of the prostate is an exceedingly rare disease entity. As a result, no current consensus exists for optimal systemic therapy. METHODS: We present a patient with metastatic adenoid cystic (basal cell) carcinoma of the prostate who subsequently received systemic treatment, including chemotherapy and immunotherapy. We comprehensively reviewed all published data on therapy outcomes in advanced disease. RESULTS: Our patient benefited from combination chemotherapy (carboplatin and paclitaxel), with objective radiographic response and reduction in cancer-related pain. However, chemotherapy was stopped due to cumulative neurotoxicity, and subsequent immunotherapy with atezolizumab did not produce any response. Our literature review revealed inconsistent outcomes with various treatments but showed most promise with chemotherapy. Targeted therapy and immunotherapy seem to benefit specific cases, and androgen deprivation therapy had minimal evidence of benefit. CONCLUSION: Based on the findings of our case report and literature review, we suggest platinum-based chemotherapy doublets as first-line treatment for metastatic cases of adenoid cystic (basal cell) carcinoma of the prostate, reserving targeted therapy or immunotherapy for select cases based upon molecular profiles.

Pathology Data-Based Risk Group Stratification Is Equivalent to That Obtained by Oncotype DX Testing in Prostatic Adenocarcinoma.

CONTEXT.­: Low-risk (Gleason score 3 + 3 = 6) and intermediate-risk (Gleason score 3 + 4 = 7) prostate carcinoma cases diagnosed on needle biopsies are frequently referred for gene expression studies such as Oncotype DX to help validate the risk. Risk assessment helps in determining prognosis and therapeutic decision making. OBJECTIVE.­: To determine if addition of molecular testing is necessary, by evaluating its correlation with risk stratification provided by pathology report (Gleason score, Grade Group, proportion of positive cores) and serum prostate-specific antigen (PSA) level. DESIGN.­: Our institutional database was searched for cases that had Oncotype DX testing after prostate biopsy. The final risk category determined by molecular testing was compared to the risk stratification predicted by the pathology report and serum PSA levels. Cases were classified as concordant if they fell under the same National Comprehensive Cancer Network risk and recommended initial therapy group. Follow-up information on discordant cases was obtained and used to determine if risk stratification by molecular testing was superior to that obtained from the clinicopathologic data. RESULTS.­: A total of 4967 prostate biopsies (2015-2020) were screened. Of these, 131 prostate carcinoma cases (2.6%) had Oncotype DX testing and 111 of 131 cases (85%) had follow-up information. There was risk stratification concordance in 93 of 111 cases (84%). All 18 of 111 cases (16%) that were discordant had a follow-up course that matched the risk predicted by pathology data and serum PSA. CONCLUSIONS.­: Risk stratification provided by information in the pathology report on routine biopsy assessment coupled with the serum PSA level is equivalent to that obtained by Oncotype DX testing.

PAF1 cooperates with YAP1 in metaplastic ducts to promote pancreatic cancer.

Acinar-to-ductal metaplasia (ADM) is a precursor lesion of pancreatic ductal adenocarcinoma (PDAC); however, the regulators of the ADM-mediated PDAC development and its targeting are poorly understood. RNA polymerase II-associated factor 1 (PAF1) maintains cancer stem cells leading to the aggressiveness of PDAC. In this study, we investigated whether PAF1 is required for the YAP1-mediated PDAC development and whether CA3 and verteporfin, small molecule inhibitors of YAP1/TEAD transcriptional activity, diminish pancreatic cancer (PC) cell growth by targeting the PAF1/YAP1 axis. Here, we demonstrated that PAF1 co-expresses and interacts with YAP1 specifically in metaplastic ducts of mouse cerulein- or KrasG12D-induced ADM and human PDAC but not in the normal pancreas. PAF1 knockdown (KD) reduced SOX9 in PC cells, and the PC cells showed elevated PAF1/YAP1 complex recruitment to the promoter of SOX9. The PAF1 KD reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in the mouse KC (KrasG12D; Pdx-1 Cre) cells and human PC cells, indicating that the PAF1 is required for the YAP1-mediated development of ADM and PC. Moreover, treatment with CA3 or verteporfin reduced the expressions of PAF1, YAP1, TEAD4, and SOX9 and decreased colony formation and stemness in KC and PC cells. CA3 treatment also reduced the viability and proliferation of PC cells and diminished the duct-like structures in KC acinar explants. CA3 or verteporfin treatment decreased the recruitment of the PAF1/YAP1 complex to the SOX9 promoter in PC cells and reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in KC and PC cells. Overall, PAF1 cooperates with YAP1 during ADM and PC development, and verteporfin and CA3 inhibit ADM and PC cell growth by targeting the PAF1/YAP1/SOX9 axis in vitro and ex vivo models. This study identified a regulatory axis of PDAC initiation and its targeting, paving the way for developing targeted therapeutic strategies for pancreatic cancer patients.

NCCN Guidelines® Insights: Bladder Cancer, Version 2.2022.

The NCCN Guidelines for Bladder Cancer provide recommendations for the diagnosis, evaluation, treatment, and follow-up of patients with bladder cancer and other urinary tract cancers (upper tract tumors, urothelial carcinoma of the prostate, primary carcinoma of the urethra). These NCCN Guidelines Insights summarize the panel discussion behind recent important updates to the guidelines regarding the treatment of non-muscle-invasive bladder cancer, including how to treat in the event of a bacillus Calmette-Guérin (BCG) shortage; new roles for immune checkpoint inhibitors in non-muscle invasive, muscle-invasive, and metastatic bladder cancer; and the addition of antibody-drug conjugates for metastatic bladder cancer.

Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism.

Ecdysoneless (ECD) protein is essential for embryogenesis, cell-cycle progression, and cellular stress mitigation with an emerging role in mRNA biogenesis. We have previously shown that ECD protein as well as its mRNA are overexpressed in breast cancer and ECD overexpression predicts shorter survival in patients with breast cancer. However, the genetic evidence for an oncogenic role of ECD has not been established. Here, we generated transgenic mice with mammary epithelium-targeted overexpression of an inducible human ECD transgene (ECDTg). Significantly, ECDTg mice develop mammary hyperplasia, preneoplastic lesions, and heterogeneous tumors with occasional lung metastasis. ECDTg tumors exhibit epithelial to mesenchymal transition and cancer stem cell characteristics. Organoid cultures of ECDTg tumors showed ECD dependency for in vitro oncogenic phenotype and in vivo growth when implanted in mice. RNA sequencing (RNA-seq) analysis of ECDTg tumors showed a c-MYC signature, and alterations in ECD levels regulated c-MYC mRNA and protein levels as well as glucose metabolism. ECD knockdown-induced decrease in glucose uptake was rescued by overexpression of mouse ECD as well as c-MYC. Publicly available expression data analyses showed a significant correlation of ECD and c-MYC overexpression in breast cancer, and ECD and c-MYC coexpression exhibits worse survival in patients with breast cancer. Taken together, we establish a novel role of overexpressed ECD as an oncogenesis driver in the mouse mammary gland through upregulation of c-MYC-mediated glucose metabolism. IMPLICATIONS: We demonstrate ECD overexpression in the mammary gland of mice led to the development of a tumor progression model through upregulation of c-MYC signaling and glucose metabolism.

Thermoresponsive polymeric dexamethasone prodrug for arthritis pain.

Intra-articular (IA) glucocorticoids (GC) are commonly used for clinical management of both osteoarthritis and rheumatoid arthritis, but their efficacy is limited by the relatively short duration of action and associated side effects. To provide sustained efficacy and to improve the safety of GCs, we previously developed a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based dexamethasone (Dex) prodrug. Serendipitously, we discovered that, by increasing the Dex content of the prodrug to unusually high levels, the aqueous solution of the polymeric prodrug becomes thermoresponsive, transitioning from a free-flowing liquid at 4 °C to a hydrogel at 30 °C or greater. Upon IA injection, the prodrug solution forms a hydrogel (ProGel-Dex) that is retained in the joint for more than 1 month, where it undergoes gradual dissolution, releasing the water-soluble polymeric prodrug. The released prodrug is swiftly internalized and intracellularly processed by phagocytic synoviocytes to release free Dex, resulting in sustained amelioration of joint inflammation and pain in rodent models of inflammatory arthritis and osteoarthritis. The low molecular weight (6.8 kDa) of the ProGel-Dex ensures rapid renal clearance once it escapes the joint, limiting systemic GC exposure and risk of potential off-target side effects. The present study illustrates the translational potential of ProGel-Dex as a potent opioid-sparing, locally delivered adjuvant analgesic for sustained clinical management of arthritis pain and inflammation. Importantly, the observed thermoresponsive properties of the prodrug establishes ProGel as a platform technology for the local delivery of a broad spectrum of therapeutic agents to treat a diverse array of pathological conditions.

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.

PGC1α-Mediated Metabolic Reprogramming Drives the Stemness of Pancreatic Precursor Lesions.

PURPOSE: Metabolic reprogramming and cancer stem cells drive the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). However, the metabolic and stemness programs of pancreatic precursor lesions (PPL), considered early PDAC development events, have not been thoroughly explored. EXPERIMENTAL DESIGN: Meta-analyses using gene expression profile data from NCBI Gene Expression Omnibus and IHC on tissue microarrays (TMA) were performed. The following animal and cellular models were used: cerulean-induced KrasG12D; Pdx1 Cre (KC) acinar-to-ductal metaplasia (ADM) mice, KrasG12D; Smad4Loss; Pdx-1 Cre (KCSmad4-) intraductal papillary mucinous neoplasm (IPMN) mice, LGKC1 cell line derived from the doxycycline-inducible Gnas IPMN model, and human IPMN organoids. Flow cytometry, Seahorse extracellular flux analyzer, qRT-PCR, and sphere assay were used to analyze metabolic and stemness features. SR18292 was used to inhibit PGC1α, and short hairpin RNA was used to knockdown (KD) PGC1α. RESULTS: The meta-analysis revealed a significant upregulation of specific stemness genes in ADM-mediated pancreatic intraepithelial neoplasms (PanIN) and IPMN. Meta- and TMA analyses followed by in vitro and in vivo validation revealed that ADM/PanIN exhibit increased PGC1α and oxidative phosphorylation (OXPhos) but reduced CPT1A. IPMN showed elevated PGC1α, fatty acid ß-oxidation (FAO) gene expression, and FAO-OXPhos. PGC1α was co-overexpressed with its coactivator NRF1 in ADM/PanINs and with PPARγ in IPMN. PGC1α KD or SR18292 inhibited the specific metabolic and stemness features of PPLs and repressed IPMN organoid growth. CONCLUSIONS: ADM/PanINs and IPMNs show specific stemness signatures with unique metabolisms. Inhibition of PGC1α using SR18292 diminishes the specific stemness by targeting FAO-independent and FAO-dependent OXPhos of ADM/PanINs and IPMNs, respectively.

ST6GalNAc-I promotes lung cancer metastasis by altering MUC5AC sialylation.

Lung cancer (LC) is the leading cause of cancer-related mortality. However, the molecular mechanisms associated with the development of metastasis are poorly understood. Understanding the biology of LC metastasis is critical to unveil the molecular mechanisms for designing targeted therapies. We developed two genetically engineered LC mouse models KrasG12D/+ ; Trp53R172H/+ ; Ad-Cre (KPA) and KrasG12D/+ ; Ad-Cre (KA). Survival analysis showed significantly (P = 0.0049) shorter survival in KPA tumor-bearing mice as compared to KA, suggesting the aggressiveness of the model. Our transcriptomic data showed high expression of N-acetylgalactosaminide alpha-2, 6-sialyltransferase 1 (St6galnac-I) in KPA compared to KA tumors. ST6GalNAc-I is an O-glycosyltransferase, which catalyzes the addition of sialic acid to the initiating GalNAc residues forming sialyl Tn (STn) on glycoproteins, such as mucins. Ectopic expression of species-specific p53 mutants in the syngeneic mouse and human LC cells led to increased cell migration and high expression of ST6GalNAc-I, STn, and MUC5AC. Immunoprecipitation of MUC5AC in the ectopically expressing p53R175H cells exhibited higher affinity toward STn. In addition, ST6GalNAc-I knockout (KO) cells also showed decreased migration, possibly due to reduced glycosylation of MUC5AC as observed by low STn on the glycoprotein. Interestingly, ST6GalNAc-I KO cells injected mice developed less liver metastasis (P = 0.01) compared to controls, while colocalization of MUC5AC and STn was observed in the liver metastatic tissues of control mice. Collectively, our findings support the hypothesis that mutant p53R175H mediates ST6GalNAc-I expression, leading to the sialyation of MUC5AC, and thus contribute to LC liver metastasis.

Thermoresponsive Hydrogel-Based Local Delivery of Simvastatin for the Treatment of Periodontitis.

Except for routine scaling and root planing, there are few effective nonsurgical therapeutic interventions for periodontitis and associated alveolar bone loss. Simvastatin (SIM), one of the 3-hydroxy-3-methylglutaryl-cosenzyme A reductase inhibitors, which is known for its capacity as a lipid-lowering medication, has been proven to be an effective anti-inflammatory and bone anabolic agent that has shown promising benefits in mitigating periodontal bone loss. The local delivery of SIM into the periodontal pocket, however, has been challenging due to SIM's poor water solubility and its lack of osteotropicity. To overcome these issues, we report a novel SIM formulation of a thermoresponsive, osteotropic, injectable hydrogel (PF127) based on pyrophosphorolated pluronic F127 (F127-PPi). After mixing F127-PPi with F127 at a 1:1 ratio, the resulting PF127 was used to dissolve free SIM to generate the SIM-loaded formulation. The thermoresponsive hydrogel's rheologic behavior, erosion and SIM release kinetics, osteotropic property, and biocompatibility were evaluated in vitro. The therapeutic efficacy of SIM-loaded PF127 hydrogel on periodontal bone preservation and inflammation resolution was validated in a ligature-induced periodontitis rat model. Given that SIM is already an approved medication for hyperlipidemia, the data presented here support the translational potential of the SIM-loaded PF127 hydrogel for better clinical management of periodontitis and associated pathologies.

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.

Primary prostatic squamous cell carcinoma.

Primary prostate squamous cell malignancies are rare and very aggressive. A 65-year-old man presented to our institution with clinical and radiographic findings concerning for advanced prostatic malignancy. Surgical pathology of the tumor revealed poorly differentiated carcinoma with squamous differentiation, and histopathological markers were positive for markers of squamous differentiation negative for all urothelial and prostatic markers.

Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis.

Pancreatic cancer (PC) is difficult to defeat due to mechanism (s) driving metastasis and drug resistance. Cancer stemness is a major challenging phenomenon associated with PC metastasis and limiting therapy efficacy. In this study, we evaluated the pre-clinical and clinical significance of eradicating pancreatic cancer stem cells (PCSC) and its components using a pan-EGFR inhibitor afatinib in combination with gemcitabine. Afatinib in combination with gemcitabine significantly reduced KrasG12D/+; Pdx-1 Cre (KC) (P < 0.01) and KrasG12D/+; p53R172H/+; Pdx-1 Cre (KPC) (P < 0.05) derived mouse tumoroids and KPC-derived murine syngeneic cell line growth compared to gemcitabine/afatinib alone treatment. The drug combination also reduced PC xenograft tumor burden (P < 0.05) and the incidence of metastasis by affecting key stemness markers, as confirmed by co-localization studies. Moreover, the drug combination significantly decreases the growth of various PC patient-derived organoids (P < 0.001). We found that SOX9 is significantly overexpressed in high-grade PC tumors (P < 0.05) and in chemotherapy-treated patients compared to chemo-naïve patients (P < 0.05). These results were further validated using publicly available datasets. Moreover, afatinib alone or in combination with gemcitabine decreased stemness and tumorspheres by reducing phosphorylation of EGFR family proteins, ERK, FAK, and CSC markers. Mechanistically, afatinib treatment decreased CSC markers by downregulating SOX9 via FOXA2. Indeed, EGFR and FOXA2 depletion reduced SOX9 expression in PCSCs. Taken together, pan-EGFR inhibition by afatinib impedes PCSCs growth and metastasis via the EGFR/ERK/FOXA2/SOX9 axis. This novel mechanism of pan-EGFR inhibitor and its ability to eradicate CSC may serve as a tailor-made approach to enhance chemotherapeutic benefits in other cancer types.