Enhancer Reprogramming Promotes Pancreatic Cancer Metastasis.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancies, owing in part to its propensity for metastasis. Here, we used an organoid culture system to investigate how transcription and the enhancer landscape become altered during discrete stages of disease progression in a PDA mouse model. This approach revealed that the metastatic transition is accompanied by massive and recurrent alterations in enhancer activity. We implicate the pioneer factor FOXA1 as a driver of enhancer activation in this system, a mechanism that renders PDA cells more invasive and less anchorage-dependent for growth in vitro, as well as more metastatic in vivo. In this context, FOXA1-dependent enhancer reprogramming activates a transcriptional program of embryonic foregut endoderm. Collectively, our study implicates enhancer reprogramming, FOXA1 upregulation, and a retrograde developmental transition in PDA metastasis.

Membrane structure and internalization dynamics of human Flower isoforms hFWE3 and hFWE4 indicate a conserved endocytic role for hFWE4.

Human Flower (hFWE) isoforms hFWE1-4 are putative transmembrane (TM) proteins that reportedly mediate fitness comparisons during cell competition through extracellular display of their C-terminal tails. Isoform topology, subcellular localization, and duration of plasma membrane presentation are essential to this function. However, disagreement persists regarding the structure of orthologous fly and mouse FWEs, and experimental evidence for hFWE isoform subcellular localization or membrane structure is lacking. Here, we used AlphaFold2 and subsequent molecular dynamics-based structural predictions to construct epitope-tagged hFWE3 and hFWE4, the most abundant human isoforms, for experimental determination of their structure and internalization dynamics. We demonstrate that hFWE3 resides in the membrane of the endoplasmic reticulum (ER), while hFWE4 partially colocalizes with Rab4-, Rab5-, and Rab11-positive vesicles as well as with the plasma membrane. An array of imaging techniques revealed that hFWE4 positions both N- and C-terminal tails and a loop between second and third TM segments within the cytosol, while small (4-12aa) loops between the first and second and the third and fourth TM segments are either exposed to the extracellular space or within the lumen of cytoplasmic vesicles. Similarly, we found hFWE3 positions both N- and C-terminal tails in the cytosol, while a short loop between TM domains extends into the ER lumen. Finally, we demonstrate that hFWE4 exists only transiently at the cell surface and is rapidly internalized in an AP-2- and dynamin-1-dependent manner. Collectively, these data are consistent with a conserved role for hFWE4 in endocytic processes.

Isoforms of MUC16 activate oncogenic signaling through EGF receptors to enhance the progression of pancreatic cancer.

Aberrant expression of CA125/MUC16 is associated with pancreatic ductal adenocarcinoma (PDAC) progression and metastasis. However, knowledge of the contribution of MUC16 to pancreatic tumorigenesis is limited. Here, we show that MUC16 expression is associated with disease progression, basal-like and squamous tumor subtypes, increased tumor metastasis, and short-term survival of PDAC patients. MUC16 enhanced tumor malignancy through the activation of AKT and GSK3ß oncogenic signaling pathways. Activation of these oncogenic signaling pathways resulted in part from increased interactions between MUC16 and epidermal growth factor (EGF)-type receptors, which were enhanced for aberrant glycoforms of MUC16. Treatment of PDAC cells with monoclonal antibody (mAb) AR9.6 significantly reduced MUC16-induced oncogenic signaling. mAb AR9.6 binds to a unique conformational epitope on MUC16, which is influenced by O-glycosylation. Additionally, treatment of PDAC tumor-bearing mice with either mAb AR9.6 alone or in combination with gemcitabine significantly reduced tumor growth and metastasis. We conclude that the aberrant expression of MUC16 enhances PDAC progression to an aggressive phenotype by modulating oncogenic signaling through ErbB receptors. Anti-MUC16 mAb AR9.6 blocks oncogenic activities and tumor growth and could be a novel immunotherapeutic agent against MUC16-mediated PDAC tumor malignancy.

Combination of mAb-AR20.5, anti-PD-L1 and PolyICLC inhibits tumor progression and prolongs survival of MUC1.Tg mice challenged with pancreatic tumors.

A substantial body of evidence suggests the existence of MUC1-specific antibodies and cytotoxic T cell activities in pancreatic cancer patients. However, tumor-induced immunosuppression renders these responses ineffective. The current study explores a novel therapeutic combination wherein tumor-bearing hosts can be immunologically primed with their own antigen, through opsonization with a tumor antigen-targeted antibody, mAb-AR20.5. We evaluated the efficacy of immunization with this antibody in combination with PolyICLC and anti-PD-L1. The therapeutic combination of mAb-AR20.5 + anti-PD-L1 + PolyICLC induced rejection of human MUC1 expressing tumors and provided a long-lasting, MUC1-specific cellular immune response, which could be adoptively transferred and shown to provide protection against tumor challenge in human MUC1 transgenic (MUC.Tg) mice. Furthermore, antibody depletion studies revealed that CD8 cells were effectors for the MUC1-specific immune response generated by the mAb-AR20.5 + anti-PD-L1 + PolyICLC combination. Multichromatic flow cytometry data analysis demonstrated a significant increase over time in circulating, activated CD8 T cells, CD3+CD4-CD8-(DN) T cells, and mature dendritic cells in mAb-AR20.5 + anti-PD-L1 + PolyICLC combination-treated, tumor-bearing mice, as compared to saline-treated control counterparts. Our study provides a proof of principle that an effective and long-lasting anti-tumor cellular immunity can be achieved in pancreatic tumor-bearing hosts against their own antigen (MUC1), which can be further potentiated using a vaccine adjuvant and an immune checkpoint inhibitor.

Transcriptomic changes and gene fusions during the progression from Barrett's esophagus to esophageal adenocarcinoma.

The incidence of esophageal adenocarcinoma (EAC) has surged by 600% in recent decades, with a dismal 5-year survival rate of just 15%. Barrett's esophagus (BE), affecting about 2% of the population, raises the risk of EAC by 40-fold. Despite this, the transcriptomic changes during the BE to EAC progression remain unclear. Our study addresses this gap through comprehensive transcriptomic profiling to identify key mRNA signatures and genomic alterations, such as gene fusions. We performed RNA-sequencing on BE and EAC tissues from 8 individuals, followed by differential gene expression, pathway and network analysis, and gene fusion prediction. We identified mRNA changes during the BE-to-EAC transition and validated our results with single-cell RNA-seq datasets. We observed upregulation of keratin family members in EAC and confirmed increased levels of keratin 14 (KRT14) using immunofluorescence. More differentiated BE marker genes are downregulated during progression to EAC, suggesting undifferentiated BE subpopulations contribute to EAC. We also identified several gene fusions absent in paired BE and normal esophagus but present in EAC. Our findings are critical for the BE-to-EAC transition and have the potential to promote early diagnosis, prevention, and improved treatment strategies for EAC.

Engrailed-1 Promotes Pancreatic Cancer Metastasis.

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, it is reported that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating roles in the activation of MAPK pathways and the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. The findings nominate the targeting of EN1 and downstream pathways in aggressive PDA.

IgE-Based Therapeutic Combination Enhances Antitumor Response in Preclinical Models of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents 3% of all cancer cases and 7% of all cancer deaths in the United States. Late diagnosis and inadequate response to standard chemotherapies contribute to an unfavorable prognosis and an overall 5-year survival rate of less than 10% in PDAC. Despite recent advances in tumor immunology, tumor-induced immunosuppression attenuates the immunotherapy response in PDAC. To date, studies have focused on IgG-based therapeutic strategies in PDAC. With the recent interest in IgE-based therapies in multiple solid tumors, we explored the MUC1-targeted IgE potential against pancreatic cancer. Our study demonstrates the notable expression of FceRI (receptor for IgE antibody) in tumors from PDAC patients. Our study showed that administration of MUC1 targeted-IgE (mouse/human chimeric anti-MUC1.IgE) antibody at intermittent levels in combination with checkpoint inhibitor (anti-PD-L1) and TLR3 agonist (PolyICLC) induces a robust antitumor response that is dependent on NK and CD8 T cells in pancreatic tumor-bearing mice. Subsequently, our study showed that the antigen specificity of the IgE antibody plays a vital role in executing the antitumor response as nonspecific IgE, induced by ovalbumin (OVA), failed to restrict tumor growth in pancreatic tumor-bearing mice. Utilizing the OVA-induced allergic asthma-PDAC model, we demonstrate that allergic phenotype induced by OVA cannot restrain pancreatic tumor growth in orthotopic tumor-bearing mice. Together, our data demonstrate the novel tumor protective benefits of tumor antigen-specific IgE-based therapeutics in a preclinical model of pancreatic cancer, which can open new avenues for future clinical interventions.

Mucin-1 is required for Coxsackie Virus B3-induced inflammation in pancreatitis.

The Muc-1 oncoprotein is a tumor-associated mucin often overexpressed in pancreatic cancer. We report that knockout of Muc-1 reduced the degree of pancreatic inflammation that resulted from infection with Coxsackievirus B3 (CVB3) in a mouse model. CVB3-infected Muc-1-deficient (Muc-1KO) mice had significantly reduced infiltration of macrophages into the murine pancreas. We found that Muc-1 signaling through NF-κB increased expression of ICAM-1, a pro-inflammatory mediator that recruits macrophages. Further investigation revealed that bone marrow derived macrophages (BMDM) from the Muc-1KO mice exhibited defective migration properties, in part due to low expression of the C-C motif chemokine receptor (CCR2) and the integrin Very Late Antigen 4 (VLA-4). The results presented here provide novel insight into the role of Muc-1 in regulating the inflammatory response and the cellular microenvironment in pancreatitis.

SOX2 functions as a molecular rheostat to control the growth, tumorigenicity and drug responses of pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) is a highly deadly malignancy. Expression of the stem cell transcription factor SOX2 increases during progression of PDAC. Knockdown of SOX2 in PDAC cell lines decreases growth in vitro; whereas, stable overexpression of SOX2 in one PDAC cell line reportedly increases growth in vitro. Here, we reexamined the role of SOX2 in PDAC cells, because inducible SOX2 overexpression in other tumor cell types inhibits growth. In this study, four PDAC cell lines were engineered for inducible overexpression of SOX2 or inducible knockdown of SOX2. Remarkably, inducible overexpression of SOX2 in PDAC cells inhibits growth in vitro and reduces tumorigenicity. Additionally, inducible knockdown of SOX2 in PDAC cells reduces growth in vitro and in vivo. Thus, growth and tumorigenicity of PDAC cells is highly dependent on the expression of optimal levels of SOX2 - a hallmark of molecular rheostats. We also determined that SOX2 alters the responses of PDAC cells to drugs used in PDAC clinical trials. Increasing SOX2 reduces growth inhibition mediated by MEK and AKT inhibitors; whereas knockdown of SOX2 further reduces growth when PDAC cells are treated with these inhibitors. Thus, targeting SOX2, or its mode of action, could improve the treatment of PDAC.

Identification of FRA-1 as a novel player in pancreatic cancer in cooperation with a MUC1: ERK signaling axis.

The MUC1 glycoprotein is overexpressed and aberrantly glycosylated in >90% of pancreatic ductal adenocarcinoma cases and impacts tumor progression by initiating downstream signaling through phosphorylation of its cytoplasmic tail. Previous studies have demonstrated that MUC1 alters expression of known targets of activator protein 1 (AP-1); however, no studies have evaluated the precise impact of MUC1 signaling on the activity and formation of AP-1. Given the known role of these proteins in modulating migration, invasion, and tumor progression, we explored the effects of MUC1 on AP-1 dimer formation and function. We determined that MUC1 increased the protein levels of c-Jun, the major component of AP-1, and promoted dimerization of c-Jun with the Fos-protein FRA-1. We demonstrate that FRA-1 acts as a potent mediator of migration and invasion in a manner that is modulated by signals through MUC1, which acts as a dominant regulator of specific AP-1 and FRA-1 target genes. Our results provide the first in vivo evidence of a FRA-1 mediated expression profile that impacts pancreatic tumor growth properties. In summary, we show that MUC1 enhancement of ERK activation influences FRA-1 activity to modulate tumor migration, invasion and metastasis in a subset of pancreatic cancer cases.

CD2-associated protein (CD2AP) expression in podocytes rescues lethality of CD2AP deficiency.

Mice born without CD2-associated protein (CD2AP) develop renal failure and nephrotic syndrome about 4 weeks after birth and die around 6 weeks of age. Although CD2AP is widely expressed, the severity of the renal failure precludes a clear determination of the role of CD2AP in other tissues. Here we generated transgenic mice expressing CD2AP using a podocyte-specific promoter. Podocyte-specific expression of CD2AP prevented the development of proteinuria, demonstrating that the renal failure is solely due to loss of CD2AP in podocytes and not in other renal or in immune cells. CD2AP-deficient mice are long-lived and appear phenotypically normal. Histological analysis demonstrated testicular abnormalities that were age-related. CIN85, a paralog of CD2AP, is poorly expressed in both the podocyte and the basal seminiferous tubule, suggesting that the loss of CD2AP in specific tissues may be compensated for by CIN85.

Blocking angiotensin II synthesis/activity preserves glomerular nephrin in rats with severe nephrosis.

Angiotensin-converting enzyme inhibitors restore size-selective dysfunction of the glomerular barrier in experimental animals and humans with proteinuric nephropathies, although the structural and molecular determinants of such an effect are not completely understood. This study used an accelerated model of experimental nephrosis to assess nephrin gene and protein expression in the kidney and the possible modulating effect of drugs that block angiotensin II (AII) synthesis/activity. Passive Heymann nephritis (PHN) and control animals were studied at day 7, month 4, and month 8. Additional PHN rats were treated with lisinopril or AII receptor blocker L-158,809 and studied at 8 mo. Lisinopril and L-158,809 controlled BP, prevented proteinuria, and protected PHN animals from renal injury. An intense signal of nephrin mRNA was detected in glomeruli of control animals mainly restricted to podocytes. In PHN rats, nephrin staining progressively and remarkably decreased with time. Lisinopril and L-158,809 fully prevented the decrease in nephrin transcripts to levels comparable to those of control rats. Consistent with nephrin mRNA expression, immunostaining of the protein showed a progressive decrease in kidneys from PHN rats that was completely abolished by lisinopril and L-158,809. In summary, progressive renal injury was associated with downregulation of nephrin gene that was totally prevented by angiotensin-converting enzyme inhibitor and AII receptor blocker, suggesting that renoprotection afforded by drugs that interfere with AII synthesis/activity was related to an effect on nephrin assembly.

Determinants of vascular permeability in the kidney glomerulus.

The human kidneys filter 70 liters of blood plasma every day. The hallmark of almost all kidney diseases, whether acquired or genetic, is the leakage of plasma proteins into the urine because of alterations in the glomerular filtration unit of the kidney. In this regard, the human mutations in nephrin, podocin, alpha-actinin-4, COL4A3, and COL4A5 genes expressed in the glomeruli have been implicated to cause alterations in glomerular filtration apparatus. Nevertheless, the expression of these proteins in relation to each other in mouse models for glomerular vascular leak is unknown. Additionally, within the glomerulus, the central question of whether the primary filtration barrier is the basement membrane or the epithelial slit diaphragm remains ambiguous. Therefore, in this study, we examined the localization and expression of glomerular epithelial slit diaphragm and glomerular basement membrane proteins implicated in glomerular vascular leak using mice deficient in either the alpha3 chain of type IV collagen, the major constituent of glomerular basement membrane, or LMX1B transcription factor, which regulates the expression of key glomerular type IV collagen genes COL4A3 and COL4A4 or nephrin, a glomerular epithelial slit diaphragm-associated protein. This study demonstrates that decreased expression of slit diaphragm protein, nephrin, correlates with a loss of glomerular filter integrity. Additionally, we demonstrate that defects induced by proteins of glomerular basement membrane lead to an insidious plasma protein leak, whereas the defects induced by proteins in the glomerular epithelial slit diaphragms lead to a precipitous plasma protein leak.