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TP53 mutations are frequent in esophageal squamous cell carcinoma (ESCC) and other SCCs and are associated with a proclivity for metastasis. Here, we report that colony-stimulating factor-1 (CSF-1) expression is upregulated significantly in a p53-R172H-dependent manner in metastatic lung lesions of ESCC. The p53-R172H-dependent CSF-1 signaling, through its cognate receptor CSF-1R, increases tumor cell invasion and lung metastasis, which in turn is mediated in part through Stat3 phosphorylation and epithelial-to-mesenchymal transition (EMT). In Trp53R172H tumor cells, p53 occupies the Csf-1 promoter. The Csf-1 locus is enriched with histone 3 lysine 27 acetylation (H3K27ac), which is likely permissive for fostering an interaction between bromodomain-containing domain 4 (BRD4) and p53-R172H to regulate Csf-1 transcription. Inhibition of BRD4 not only reduces tumor invasion and lung metastasis but also reduces circulating CSF-1 levels. Overall, our results establish a novel p53-R172H-dependent BRD4-CSF-1 axis that promotes ESCC lung metastasis and suggest avenues for therapeutic strategies for this difficult-to-treat disease., Significance: The invasion-metastasis cascade is a recalcitrant barrier to effective cancer therapy. We establish that the p53-R172H-dependent BRD4-CSF-1 axis is a mediator of prometastatic properties, correlates with patient survival and tumor stages, and its inhibition significantly reduces tumor cell invasion and lung metastasis. This axis can be exploited for therapeutic advantage. This article is featured in Selected Articles from This Issue, p. 2489., (©2023 American Association for Cancer Research.)

Cancer-associated fibroblasts (CAF) can promote tumor growth, metastasis, and therapeutic resistance in esophageal squamous cell carcinoma (ESCC), but the mechanisms of action remain elusive. Our objective was to identify secreted factor(s) that mediate the communication between CAFs and ESCC tumor cells with the aim of identifying potential druggable targets. Through unbiased cytokine arrays, we have identified CC motif chemokine ligand 5 (CCL5) as a secreted factor that is increased upon co-culture of ESCC cells and CAFs, which we replicated in esophageal adenocarcinoma (EAC) with CAFs. Loss of tumor-cell-derived CCL5 reduces ESCC cell proliferation in vitro and in vivo and we propose this is mediated, in part, by a reduction in ERK1/2 signaling. Loss of tumor-derived CCL5 reduces the percentage of CAFs recruited to xenograft tumors in vivo. CCL5 is a ligand for the CC motif receptor 5 (CCR5), for which a clinically approved inhibitor exists, namely Maraviroc. Maraviroc treatment reduced tumor volume, CAF recruitment, and ERK1/2 signaling in vivo, thus, mimicking the effects observed with genetic loss of CCL5. High CCL5 or CCR5 expression is associated with worse prognosis in low-grade esophageal carcinomas., Implications: These data highlight the role of CCL5 in tumorigenesis and the therapeutic potential of targeting the CCL5-CCR5 axis in ESCC., (©2023 American Association for Cancer Research.)

Cholesterol dependence is an essential characteristic of pancreatic ductal adenocarcinoma (PDAC). Cholesterol 25-hydroxylase (CH25H) catalyzes monooxygenation of cholesterol into 25-hydroxycholesterol, which is implicated in inhibiting cholesterol biosynthesis and in cholesterol depletion. Here, we show that, within PDAC cells, accumulation of cholesterol was facilitated by the loss of CH25H. Methylation of the CH25H gene and decreased levels of CH25H expression occurred in human pancreatic cancers and was associated with poor prognosis. Knockout of Ch25h in mice accelerated progression of Kras-driven pancreatic intraepithelial neoplasia. Conversely, restoration of CH25H expression in human and mouse PDAC cells decreased their viability under conditions of cholesterol deficit, and decelerated tumor growth in immune competent hosts. Mechanistically, the loss of CH25H promoted autophagy resulting in downregulation of MHC-I and decreased CD8+ T-cell tumor infiltration. Re-expression of CH25H in PDAC cells combined with immune checkpoint inhibitors notably inhibited tumor growth. We discuss additional benefits that PDAC cells might gain from inactivation of CH25H and the potential translational importance of these findings for therapeutic approaches to PDAC., Implications: Loss of CH25H by pancreatic cancer cells may stimulate tumor progression and interfere with immunotherapies., (©2022 American Association for Cancer Research.)

Carriers of a pathogenic/likely pathogenic (P/LP) BRCA1/BRCA2/ATM/PALB2 variant are at increased risk of pancreatic ductal adenocarcinoma (PDAC), yet current guidelines recommend surveillance only for those with a family history of PDAC. We aimed to investigate outcomes of endoscopic ultrasound (EUS)-based PDAC surveillance in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC. We performed a retrospective analysis of all P/LP BRCA1/BRCA2/ATM/PALB2 carriers who underwent EUS at a tertiary care center. Of 194 P/LP BRCA1/BRCA2/ATM/PALB2 carriers who underwent EUS, 64 (33%) had no family history of PDAC and had at least 1 EUS for PDAC surveillance. These individuals underwent 143 total EUSs, were predominantly female (72%), and BRCA2 carriers (73%), with the majority having a personal history of cancer other than PDAC (67%). The median age at time of first EUS was 62 years [interquartile range (IQR), 53-67 years] and a median of 2 EUSs (IQR 1-3) were performed per patient, with a median of 3 years (IQR 2-4.5 years) between the first and last EUS for those with more than 1 EUS. Pancreatic abnormalities were detected in 44%, including cysts in 27%, and incidental luminal abnormalities in 41%. Eight percent developed a new pancreatic mass or cyst during surveillance, 2 individuals developed PDAC, and no serious complications resulted from surveillance. After discussion of the risks, limitations, and potential benefits, PDAC surveillance can be considered in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC; however, the effectiveness of PDAC surveillance in this population requires further study. PREVENTION RELEVANCE: BRCA1/BRCA2/ATM/PALB2 carriers have increased pancreatic ductal adenocarcinoma (PDAC) risk, yet are typically not eligible for PDAC surveillance in the absence of PDAC family history. Herein we describe outcomes of PDAC surveillance in BRCA1/BRCA2/ATM/PALB2 carriers without a family history of PDAC, showing that PDAC surveillance can be considered in this high-risk group., (©2021 American Association for Cancer Research.)

Pancreatic cancer metastasis is a leading cause of cancer-related deaths, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Here, we report that the parathyroid hormone-related protein (PTHrP encoded by PTHLH ) is frequently amplified as part of the KRAS amplicon in patients with pancreatic cancer. PTHrP upregulation drives the growth of both primary and metastatic tumors in mice and is highly enriched in pancreatic ductal adenocarcinoma metastases. Loss of PTHrP-either genetically or pharmacologically-dramatically reduces tumor burden, eliminates metastasis, and enhances overall survival. These effects are mediated in part through a reduction in epithelial-to-mesenchymal transition, which reduces the ability of tumor cells to initiate metastatic cascade. Spp1 , which encodes osteopontin, is revealed to be a downstream effector of PTHrP. Our results establish a new paradigm in pancreatic cancer whereby PTHrP is a driver of disease progression and emerges as a novel therapeutic vulnerability. SIGNIFICANCE: Pancreatic cancer often presents with metastases, yet no strategies exist to pharmacologically inhibit this process. Herein, we establish the oncogenic and prometastatic roles of PTHLH , a novel amplified gene in pancreatic ductal adenocarcinoma. We demonstrate that blocking PTHrP activity reduces primary tumor growth, prevents metastasis, and prolongs survival in mice. This article is highlighted in the In This Issue feature, p. 1601 ., (©2021 American Association for Cancer Research.)

KRAS mutation is present in approximately 30% of human lung adenocarcinomas. Although recent advances in targeted therapy have shown great promise, effective targeting of KRAS remains elusive, and concurrent alterations in tumor suppressors render KRAS- mutant tumors even more resistant to existing therapies. Contributing to the refractoriness of KRAS -mutant tumors are immunosuppressive mechanisms, such as increased presence of suppressive regulatory T cells (Treg) in tumors and elevated expression of the inhibitory receptor PD-1 on tumor-infiltrating T cells. Treatment with BET bromodomain inhibitors is beneficial for hematologic malignancies, and they have Treg-disruptive effects in a non-small cell lung cancer (NSCLC) model. Targeting PD-1-inhibitory signals through PD-1 antibody blockade also has substantial therapeutic impact in lung cancer, although these outcomes are limited to a minority of patients. We hypothesized that the BET bromodomain inhibitor JQ1 would synergize with PD-1 blockade to promote a robust antitumor response in lung cancer. In the present study, using Kras +/LSL-G12D ; Trp53 L/L (KP) mouse models of NSCLC, we identified cooperative effects between JQ1 and PD-1 antibody. The numbers of tumor-infiltrating Tregs were reduced and activation of tumor-infiltrating T cells, which had a T-helper type 1 (Th1) cytokine profile, was enhanced, underlying their improved effector function. Furthermore, lung tumor-bearing mice treated with this combination showed robust and long-lasting antitumor responses compared with either agent alone, culminating in substantial improvement in the overall survival of treated mice. Thus, combining BET bromodomain inhibition with immune checkpoint blockade offers a promising therapeutic approach for solid malignancies such as lung adenocarcinoma. Cancer Immunol Res; 6(10); 1234-45. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated fibroblasts (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting growth factors, modifying the extracellular matrix, supporting angiogenesis, and suppressing antitumor immune responses. Through an unbiased approach, we have established that IL-6 mediates cross-talk between tumor cells and CAF not only by supporting tumor cell growth, but also by promoting fibroblast activation. As a result, IL-6 receptor (IL6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant three-dimensional (3D) organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL6Rα antibody, suppressed tumor growth in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head and neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor growth and altered STAT3 and ERK1/2 gene signatures. We used small-molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL6 is a major contributor to the dynamic cross-talk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL6Rα and downstream signaling pathways as a novel targeted therapy in oral-upper-GI cancers. Significance: These findings demonstrate the interaction of esophageal cancer and cancer-associated fibroblasts through IL-6 signaling, providing rationale for a novel therapeutic approach to target these cancers. Cancer Res; 78(17); 4957-70. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The RNA-binding protein LIN28B plays an important role in development, stem cell biology, and tumorigenesis. LIN28B has two isoforms: the LIN28B-long and -short isoforms. Although studies have revealed the functions of the LIN28B-long isoform in tumorigenesis, the role of the LIN28B-short isoform remains unclear and represents a major gap in the field. The LIN28B-long and -short isoforms are expressed in a subset of human colorectal cancers and adjacent normal colonic mucosa, respectively. To elucidate the functional and mechanistic aspects of these isoforms, colorectal cancer cells (Caco-2 and LoVo) were generated to either express no LIN28B or the -short or -long isoform. Interestingly, the long isoform suppressed LET-7 expression and activated canonical RAS/ERK signaling, whereas the short isoform did not. The LIN28B-long isoform-expressing cells demonstrated increased drug resistance to 5-fluorouracil and cisplatin through the upregulation of ERCC1, a DNA repair gene, in a LET-7 -dependent manner. The LIN28B-short isoform preserved its ability to bind pre- let-7 , without inhibiting the maturation of LET-7 , and competed with the LIN28B-long isoform for binding to pre- let-7 Coexpression of the short isoform in the LIN28B-long isoform-expressing cells rescued the phenotypes induced by the LIN28B-long isoform. Implications: This study demonstrates the differential antagonistic functions of the LIN28B-short isoform against the LIN28B-long isoform through an inability to degrade LET-7 , which leads to the novel premise that the short isoform may serve to counterbalance the long isoform during normal colonic epithelial homeostasis, but its downregulation during colonic carcinogenesis may reveal the protumorigenic effects of the long isoform. Mol Cancer Res; 16(3); 403-16. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The degree of heterogeneity among cancer stem cells (CSC) remains ill-defined and may hinder effective anti-CSC therapy. Evaluation of oral cancers for such heterogeneity identified two compartments within the CSC pool. One compartment was detected using a reporter for expression of the H3K4me3 demethylase JARID1B to isolate a JARID1B(high) fraction of cells with stem cell-like function. JARID1B(high) cells expressed oral CSC markers including CD44 and ALDH1 and showed increased PI3K pathway activation. They were distinguished from a fraction in a G0-like cell-cycle state characterized by low reactive oxygen species and suppressed PI3K/AKT signaling. G0-like cells lacked conventional CSC markers but were primed to acquire stem cell-like function by upregulating JARID1B, which directly mediated transition to a state expressing known oral CSC markers. The transition was regulated by PI3K signals acting upstream of JARID1B expression, resulting in PI3K inhibition depleting JARID1B(high) cells but expanding the G0-like subset. These findings define a novel developmental relationship between two cell phenotypes that may jointly contribute to CSC maintenance. Expansion of the G0-like subset during targeted depletion of JARID1B(high) cells implicates it as a candidate therapeutic target within the oral CSC pool. Cancer Res; 76(18); 5538-49. ©2016 AACR., Competing Interests: All authors declare no conflict of interest related to this work., (©2016 American Association for Cancer Research.)

Background: Better methods are needed to predict risk of progression for Barrett's esophagus. We aimed to determine whether a tissue systems pathology approach could predict progression in patients with nondysplastic Barrett's esophagus, indefinite for dysplasia, or low-grade dysplasia., Methods: We performed a nested case-control study to develop and validate a test that predicts progression of Barrett's esophagus to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), based upon quantification of epithelial and stromal variables in baseline biopsies. Data were collected from Barrett's esophagus patients at four institutions. Patients who progressed to HGD or EAC in ≥1 year (n = 79) were matched with patients who did not progress (n = 287). Biopsies were assigned randomly to training or validation sets. Immunofluorescence analyses were performed for 14 biomarkers and quantitative biomarker and morphometric features were analyzed. Prognostic features were selected in the training set and combined into classifiers. The top-performing classifier was assessed in the validation set., Results: A 3-tier, 15-feature classifier was selected in the training set and tested in the validation set. The classifier stratified patients into low-, intermediate-, and high-risk classes [HR, 9.42; 95% confidence interval, 4.6-19.24 (high-risk vs. low-risk); P < 0.0001]. It also provided independent prognostic information that outperformed predictions based on pathology analysis, segment length, age, sex, or p53 overexpression., Conclusion: We developed a tissue systems pathology test that better predicts risk of progression in Barrett's esophagus than clinicopathologic variables., Impact: The test has the potential to improve upon histologic analysis as an objective method to risk stratify Barrett's esophagus patients. Cancer Epidemiol Biomarkers Prev; 25(6); 958-68. ©2016 AACR., (©2016 American Association for Cancer Research.)

Unlabelled: Pancreatic cancer is projected to become the second leading cause of cancer-related death in the United States by 2020. A familial aggregation of pancreatic cancer has been established, but the cause of this aggregation in most families is unknown. To determine the genetic basis of susceptibility in these families, we sequenced the germline genomes of 638 patients with familial pancreatic cancer and the tumor exomes of 39 familial pancreatic adenocarcinomas. Our analyses support the role of previously identified familial pancreatic cancer susceptibility genes such as BRCA2, CDKN2A, and ATM, and identify novel candidate genes harboring rare, deleterious germline variants for further characterization. We also show how somatic point mutations that occur during hematopoiesis can affect the interpretation of genome-wide studies of hereditary traits. Our observations have important implications for the etiology of pancreatic cancer and for the identification of susceptibility genes in other common cancer types., Significance: The genetic basis of disease susceptibility in the majority of patients with familial pancreatic cancer is unknown. We whole genome sequenced 638 patients with familial pancreatic cancer and demonstrate that the genetic underpinning of inherited pancreatic cancer is highly heterogeneous. This has significant implications for the management of patients with familial pancreatic cancer., (©2015 American Association for Cancer Research.)

Unlabelled: The colon tumor microenvironment is becoming increasingly recognized as a complex but central player in the development of many cancers. Previously, we identified an oncogenic role for the mRNA-binding protein IMP1 (IGF2BP1) in the epithelium during colon tumorigenesis. In the current study, we reveal the contribution of stromal IMP1 in the context of colitis-associated colon tumorigenesis. Interestingly, stromal deletion of Imp1 (Dermo1Cre;Imp1(LoxP/LoxP), or Imp1(ΔMes)) in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer resulted in increased tumor numbers of larger size and more advanced histologic grade than controls. In addition, Imp1(ΔMes) mice exhibited a global increase in protumorigenic microenvironment factors, including enhanced inflammation and stromal components. Evaluation of purified mesenchyme from AOM/DSS-treated Imp1(ΔMes) mice demonstrated an increase in hepatocyte growth factor (HGF), which has not been associated with regulation via IMP1. Genetic knockdown of Imp1 in human primary fibroblasts confirmed an increase in HGF with Imp1 loss, demonstrating a specific, cell-autonomous role for Imp1 loss to increase HGF expression. Taken together, these data demonstrate a novel tumor-suppressive role for IMP1 in colon stromal cells and underscore an exquisite, context-specific function for mRNA-binding proteins, such as IMP1, in disease states., Implications: The tumor-suppressive role of stromal IMP1 and its ability to modulate protumorigenic factors suggest that IMP1 status is important for the initiation and growth of epithelial tumors., (©2015 American Association for Cancer Research.)

Myeloid-derived suppressor cells (MDSC) are an immunosuppressive population of immature myeloid cells found in advanced-stage cancer patients and mouse tumor models. Production of inducible nitric oxide synthase (iNOS) and arginase, as well as other suppressive mechanisms, allows MDSCs to suppress T-cell-mediated tumor clearance and foster tumor progression. Using an unbiased global gene expression approach in conditional p120-catenin knockout mice (L2-cre;p120ctn(f/f)), a model of oral-esophageal cancer, we have identified CD38 as playing a vital role in MDSC biology, previously unknown. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation, and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in other mouse tumor models of esophageal carcinogenesis, and CD38(high) MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38(high) MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38(low) MDSCs, likely as a result of increased iNOS production. In addition, we have identified novel tumor-derived factors, specifically IL6, IGFBP3, and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38(+) MDSCs in peripheral blood of advanced-stage cancer patients and validated targeting CD38 in vivo as a novel approach to cancer therapy., (©2015 American Association for Cancer Research.)

Unlabelled: Pancreatic cancer is one of the deadliest cancers with poor survival rates and limited therapeutic options. To improve the understanding of this disease's biology, a prerequisite for the generation of novel therapeutics, new platforms for rapid and efficient genetic and therapeutic screening are needed. Therefore, a combined in vitro/in vivo hybrid shRNA assay was developed using isolated murine primary pancreatic ductal cells (PDCs), in which oncogenic Kras(G12D) could be activated in vitro by genomic recombination through 4OH-tamoxifen-induced nuclear translocation of Cre-ERT2 expressed under control of the ROSA26 promoter. Further genetic manipulation was achieved through selective and stable RNAi against the tumor suppressors p16(Ink4a) (CDKN2A) or Trp53 (TP53) using lentiviral gene delivery. Treatment of PDCs with 4OH-tamoxifen increased phosphorylation of ERK downstream of KRAS, and subsequent lentiviral transduction resulted in sustained target gene repression. Double-mutant PDCs were then reintroduced into the pancreata of NOD-SCID-gamma (NSG) mice and monitored for tumor growth. Orthotopic implantation of PDCs carrying the activated Kras(G12D)-allele and shRNA against p16(Ink4a) or Trp53 resulted in tumor growth, metastasis, and reduced survival of NSG mice. In contrast, Kras(G12D) alone was not sufficient to induce tumor growth., Implications: The combinatory in vitro/in vivo approach described in this study allows for rapid and efficient identification of genes involved in carcinogenesis and opens new avenues for the development of therapeutic strategies to improve cancer treatment., (©2015 American Association for Cancer Research.)

Unlabelled: Protein arginine methyltransferase 5 (PRMT5) has been implicated as a key modulator of lymphomagenesis. Whether PRMT5 has overt oncogenic function in the context of leukemia/lymphoma and whether it represents a therapeutic target remains to be established. We demonstrate that inactivation of PRMT5 inhibits colony-forming activity by multiple oncogenic drivers, including cyclin D1, c-MYC, NOTCH1, and MLL-AF9. Furthermore, we demonstrate that PRMT5 overexpression specifically cooperates with cyclin D1 to drive lymphomagenesis in a mouse model, revealing inherent neoplastic activity. Molecular analysis of lymphomas revealed that arginine methylation of p53 selectively suppresses expression of crucial proapoptotic and antiproliferative target genes, thereby sustaining tumor cell self-renewal and proliferation and bypassing the need for the acquisition of inactivating p53 mutations. Critically, analysis of human tumor specimens reveals a strong correlation between cyclin D1 overexpression and p53 methylation, supporting the biomedical relevance of this pathway., Significance: We have identified and functionally validated a crucial role for PRMT5 for the inhibition of p53-dependent tumor suppression in response to oncogenic insults. The requisite role for PRMT5 in the context of multiple lymphoma/leukemia oncogenic drivers suggests a molecular rationale for therapeutic development., (©2015 American Association for Cancer Research.)

Pancreatic ductal adenocarcinomas (PDAC) are highly invasive and metastatic neoplasms commonly unresponsive to current drug therapy. Overwhelmingly, PDAC harbors early constitutive, oncogenic mutations in K-Ras(G12D) that exist prior to invasion. Histologic and genetic analyses of human PDAC biopsies also exhibit increased expression of extracellular signal-regulated kinase (ERK) 1/2 and proinvasive matrix metalloproteinases (MMP), indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K-Ras/ERK1/2 signaling and its influence on MMP-directed stromal invasion in primary human pancreatic ductal epithelial cells (PDEC) have yet to be elucidated in three-dimensions. Expression of oncogenic K-Ras(G12D) alone in genetically defined PDECs reveals increased invadopodia and epithelial-to-mesenchymal transition markers, but only when cultured in a three-dimensional model incorporating a basement membrane analog. Activation of ERK2, but not ERK1, also occurs only in K-Ras(G12D)-mutated PDECs cultured in three-dimensions and is a necessary intracellular signaling event for invasion based upon pharmacologic and short hairpin RNA (shRNA) inhibition. Increased active invasion of K-Ras(G12D) PDECs through the basement membrane model is associated with a specific microarray gene expression signature and induction of MMP endopeptidases. Specifically, MMP-1 RNA, its secreted protein, and its proteolytic cleavage activity are amplified in K-Ras(G12D) PDECs when assayed by real-time quantitative PCR, ELISA, and fluorescence resonance energy transfer (FRET). Importantly, shRNA silencing of MMP-1 mimics ERK2 inhibition and disrupts active, vertical PDEC invasion. ERK2 isoform and MMP-1 targeting are shown to be viable strategies to attenuate invasion of K-Ras(G12D)-mutated human pancreatic cancer cells in a three-dimensional tumor microenvironment., (©2011 AACR.)

LIN28B is a homologue of LIN28 that induces pluripotency when expressed in conjunction with OCT4, SOX2, and KLF4 in somatic fibroblasts. LIN28B represses biogenesis of let-7 microRNAs and is implicated in both development and tumorigenesis. Recently, we have determined that LIN28B overexpression occurs in colon tumors. We conducted a comprehensive analysis of LIN28B protein expression in human colon adenocarcinomas. We found that LIN28B overexpression correlates with reduced patient survival and increased probability of tumor recurrence. To elucidate tumorigenic functions of LIN28B, we constitutively expressed LIN28B in colon cancer cells and evaluated tumor formation in vivo. Tumors with constitutive LIN28B expression exhibit increased expression of colonic stem cell markers LGR5 and PROM1, mucinous differentiation, and metastasis. Together, our findings point to a function for LIN28B in promoting colon tumor pathogenesis, especially metastasis.

Insulin-like growth factor 2 mRNA-binding protein-1 (IMP-1) is an oncofetal protein that binds directly to and stabilizes oncogenic c-Myc and regulates, in turn, its posttranscriptional expression and translation. In contrast to normal adult tissue, IMP-1 is reexpressed and/or overexpressed in human cancers. We show that knockdown of c-Myc in human colon cancer cell lines increases the expression of mature let-7 miRNA family members and downregulates several of its mRNA targets: IMP-1, Cdc34, and K-Ras. We further show that loss of IMP-1 inhibits Cdc34, Lin-28B, and K-Ras, suppresses SW-480 cell proliferation and anchorage-independent growth, and promotes caspase- and lamin-mediated cell death. We also found that IMP-1 binds to the coding region and 3'UTR of K-Ras mRNA. RNA microarray profiling and validation by reverse transcription PCR reveals that the p53-inducible proapoptotic protein CYFIP2 is upregulated in IMP-1 knockdown SW480 cells, a novel finding. We also show that overexpression of IMP-1 increases c-Myc and K-Ras expression and LIM2405 cell proliferation. Furthermore, we show that loss of IMP-1 induces Caspase-3- and PARP-mediated apoptosis, and inhibits K-Ras expression in SW480 cells, which is rescued by CYFIP2 knockdown. Importantly, analysis of 228 patients with colon cancers reveals that IMP-1 is significantly upregulated in differentiated colon tumors (P ≤ 0.0001) and correlates with K-Ras expression (r = 0.35, P ≤ 0.0001) relative to adjacent normal mucosa. These findings indicate that IMP-1, interrelated with c-Myc, acts upstream of K-Ras to promote survival through a novel mechanism that may be important in colon cancer pathogenesis., (©2011 AACR.)

Chronic infectious diseases, such as Helicobacter pylori infection, can promote cancer in a large part through induction of chronic inflammation. Oncogenic K-ras mutation in epithelial cells activates inflammatory pathways, which could compensate for a lack of infectious stimulus. Gastric histopathology and putative progenitor markers [doublecortin and calcium/calmodulin-dependent protein kinase-like 1 (Dcamkl1) and keratin 19 (K19)] in K19-K-ras-V12 (K19-kras) transgenic mice were assessed at 3, 6, 12, and 18 months of age, in comparison with Helicobacter felis-infected wild-type littermates. Inflammation was evaluated by reverse transcription-PCR of proinflammatory cytokines, and K19-kras mice were transplanted with green fluorescent protein (GFP)-labeled bone marrow. Both H. felis infection and K-ras mutation induced upregulation of proinflammatory cytokines, expansion of Dcamkl1(+) cells, and progression to oxyntic atrophy, metaplasia, hyperplasia, and high-grade dysplasia. K19-kras transgenic mice uniquely displayed mucous metaplasia as early as 3 months and progressed to high-grade dysplasia and invasive intramucosal carcinoma by 20 months. In bone marrow-transplanted K19-kras mice that progressed to dysplasia, a large proportion of stromal cells were GFP(+) and bone marrow-derived, but only rare GFP(+) epithelial cells were observed. GFP(+) bone marrow-derived cells included leukocytes and CD45(-) stromal cells that expressed vimentin or α smooth muscle actin and were often found surrounding clusters of Dcamkl1(+) cells at the base of gastric glands. In conclusion, the expression of mutant K-ras in K19(+) gastric epithelial cells can induce chronic inflammation and promote the development of dysplasia., (©2010 AACR.)

Human squamous cell cancers are the most common epithelially derived malignancies. One example is esophageal squamous cell carcinoma (ESCC), which is associated with a high mortality rate that is related to a propensity for invasion and metastasis. Here, we report that periostin, a highly expressed cell adhesion molecule, is a key component of a novel tumor-invasive signature obtained from an organotypic culture model of engineered ESCC. This tumor-invasive signature classifies with human ESCC microarrays, underscoring its utility in human cancer. Genetic modulation of periostin promotes tumor cell migration and invasion as revealed in gain-of-loss and loss-of-function experiments. Inhibition of epidermal growth factor receptor signaling and restoration of wild-type p53 function were each found to attenuate periostin, suggesting the interdependence of two common genetic alterations with periostin function. Collectively, our studies reveal periostin as an important mediator of ESCC tumor invasion and they indicate that organotypic (three-dimensional) culture can offer an important tool to discover novel biological effectors in cancer., (Copyright 2010 AACR.)

K-ras is the most commonly mutated oncogene in pancreatic cancer and its activation in murine models is sufficient to recapitulate the spectrum of lesions seen in human pancreatic ductal adenocarcinoma (PDAC). Recent studies suggest that Notch receptor signaling becomes reactivated in a subset of PDACs, leading to the hypothesis that Notch1 functions as an oncogene in this setting. To determine whether Notch1 is required for K-ras-induced tumorigenesis, we used a mouse model in which an oncogenic allele of K-ras is activated and Notch1 is deleted simultaneously in the pancreas. Unexpectedly, the loss of Notch1 in this model resulted in increased tumor incidence and progression, implying that Notch1 can function as a tumor suppressor gene in PDAC., (Copyright 2010 AACR.)

Transforming growth factor-beta (TGF-beta) is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive about which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-beta. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to the enrichment of an EMT-competent cellular subpopulation among telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by the induction of cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT on TGF-beta stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in the induction of p15(INK4B) and p16(INK4A), reactivating the EGFR-dependent senescence program. Importantly, TGF-beta-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or the activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing the expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis., ((c)2010 AACR.)

Pancreatic ductal adenocarcinoma is the overwhelmingly predominant form of pancreatic cancer and the second most common type of gastrointestinal cancer (behind colorectal cancer) in the United States. Recent exciting advances in two areas of pancreatic ductal adenocarcinoma (i.e., the development and characterization of genetically engineered mouse models and the dissection of the genetic basis of hereditary forms in families) have been illuminating. These preclinical models and clinical syndromes provide the first tangible basis for progress in screening and prevention in high-risk populations and in the development of molecular diagnostics and experimental therapeutics.

The antineoplastic drug sorafenib (BAY 43-9006) is a multikinase inhibitor that targets the serine-threonine kinase B-Raf as well as several tyrosine kinases. Given the numerous molecular targets of sorafenib, there are several potential anticancer mechanisms of action, including induction of apoptosis, cytostasis, and antiangiogenesis. We observed that sorafenib has broad activity in viability assays in several human tumor cell lines but selectively induces apoptosis in only some lines. Sorafenib was found to decrease Mcl-1 levels in most cell lines tested, but this decrease did not correlate with apoptotic sensitivity. Sorafenib slows cell cycle progression and prevents irradiated cells from reaching and accumulating at G2-M. In synchronized cells, sorafenib causes a reversible G1 delay, which is associated with decreased levels of cyclin D1, Rb, and phosphorylation of Rb. Although sorafenib does not affect intrinsic radiosensitivity using in vitro colony formation assays, it significantly reduces colony size. In HCT116 xenograft tumor growth delay experiments in mice, sorafenib alters radiation response in a schedule-dependent manner. Radiation treatment followed sequentially by sorafenib was found to be associated with the greatest tumor growth delay. This study establishes a foundation for clinical testing of sequential fractionated radiation followed by sorafenib in gastrointestinal and other malignancies.

To investigate pathways of human esophageal squamous cell transformation, we generated esophageal tumor cells using human telomerase- and SV40-immortalized primary esophageal epithelial cells (EPC2) by overexpression of selected combinations of oncogenes. H-Ras, c-Myc, or Akt, but not epidermal growth factor receptor (EGFR), induced transformed colonies in soft agar. By contrast, bioluminescence imaging of genetically altered immortalized esophageal cells revealed that Akt, EGFR, or H-Ras, but not c-Myc, resulted in tumor formation in immunodeficient mice. H-Ras-driven tumors showed highly tumorigenic phenotypes with 2.6 +/- 0.6 days for doubling, whereas Akt and EGFR tumors doubled every 9.5 +/- 1.6 and 6.1 +/- 1.2 days, respectively. H-Ras-driven tumors expressed the hypoxia-inducible factor target Glut1, whereas Akt- or EGFR-driven tumors had evidence of angiogenesis and no detectable Glut1 expression. Proliferation rates among these tumors were similar, but there was reduced apoptosis in the more aggressive H-Ras-driven tumors that also developed aneuploidy and multiple centrosomes. c-Myc overexpression did not result in tumorigenic conversion but introduction of Bcl-XL into c-Myc-expressing cells generated tumors. Although cytokeratin expression was typical of squamous carcinoma, gene expression profiling was done to compare the four different types of engineered tumors with human esophageal squamous cell carcinomas and adenocarcinomas. Interestingly, c-Myc plus Bcl-XL transformants mimicked squamous carcinomas, whereas H-Ras-, EGFR-, and Akt-driven tumors were similar to adenocarcinomas in their molecular profiles. These genetically engineered models may provide new platforms for understanding human esophagus cancer and may assist in the evaluation of new therapies.

In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and loss of E-cadherin is a hallmark of tumor progression fostering cancer cell invasion and metastasis. To examine E-cadherin loss in squamous cell cancers, we used primary human esophageal epithelial cells (keratinocytes) as a platform and retrovirally transduced wild-type and dominant-negative forms of E-cadherin into these cells. We found decreased cell adhesion in the cells expressing dominant-negative E-cadherin, thereby resulting in enhanced migration and invasion. To analyze which molecular pathway(s) may modulate these changes, we conducted microarray analysis and found up-regulation of transforming growth factor beta receptor II (TbetaRII) in the wild-type E-cadherin-overexpressing cells, which was confirmed by real-time PCR and Western blot analyses. To investigate the in vivo relevance of this finding, we analyzed tissue microarrays of paired esophageal squamous cell carcinomas and adjacent normal esophagus, and we could show a coordinated loss of E-cadherin and TbetaRII in approximately 80% of tumors. To determine if there may be an E-cadherin-dependent regulation of TbetaRII, we show the physical interaction of E-cadherin with TbetaRII and that this is mediated through the extracellular domains of E-cadherin and TbetaRII, respectively. In addition, TbetaRI is recruited to this complex. When placed in the context of three-dimensional cell culture, which reflects the physiologic microenvironment, TbetaRII-mediated cell signaling is dependent upon intact E-cadherin function. Our results, which suggest that E-cadherin regulates TbetaRII function, have important implications for epithelial carcinogenesis characterized through the frequent occurrence of E-cadherin and TbetaRII loss.

Environmental and occupational exposure to arsenite is associated with an increased risk of human cancers, including skin, urinary bladder, and respiratory tract cancers. Although much evidence suggests that alterations in cell cycle machinery are implicated in the carcinogenic effect of arsenite, the molecular mechanisms underlying the cell cycle alterations are largely unknown. In the present study, we observed that exposure of human keratinocyte HaCat cells to arsenite resulted in the promotion of cell cycle progression, especially G(1)-S transition. Further studies found that arsenite exposure was able to induce cyclin D1 expression. The induction of cyclin D1 by arsenite required nuclear factor-kappaB (NF-kappaB) activation, because the inhibition of IkappaB phosphorylation by overexpression of the dominant-negative mutant, IKKbeta-KM, impaired arsenite-induced cyclin D1 expression and G1-S transition. The requirement of IkappaB kinase beta (IKKbeta) for cyclin D1 induction was further confirmed by the findings that arsenite-induced cyclin D1 expression was totally blocked in IKKbeta knockout (IKKbeta(-/-)) mouse embryo fibroblasts. In addition, knockdown of cyclin D1 expression using cyclin D1-specific small interference RNA significantly blocked arsenite-induced cell cycle progression in HaCat cells. Taken together, our results show that arsenite-induced cell cycle from G(1) to S phase transition is through IKKbeta/NF-kappaB/cyclin D1-dependent pathway.

Normal human somatic cells have a finite life span and undergo replicative senescence after a limited number of cell divisions. Erosion of telomeric DNA has emerged as a key factor in senescence, which is antagonized during cell immortalization and transformation. To clarify the involvement of telomerase in the immortalization of keratinocytes, catalytic subunit of telomerase (hTERT) expression was restored in normal human esophageal epithelial cells (EPC2). EPC2-hTERT cells overcame senescence and were immortalized without p16INK4a genetic or epigenetic alterations. p16INK4a was expressed at moderate levels and remained functional as evidenced by induction with UV treatment and binding to cyclin-dependent kinase 4 and 6. There were no mutations in the p53 gene, and p53 was functionally intact. Importantly, senescence could be activated in the immortalized EPC2-hTERT cells by overexpression of oncogenic H-ras or p16INK4a. Furthermore, the EPC2-hTERT cells yielded basal cell hyperplasia in an innovative organotypic culture system in contrast to a normal epithelium from parental cells. These comprehensive results indicate that the expression of telomerase induces immortalization of normal human esophageal keratinocytes without inactivation of p16INK4a/pRb pathway or abrogation of the p53 pathway.

Overexpression of cyclin D1 and disruption of cell cycle control in G(1) occur frequently in human esophageal cancer. Transgenic (TG) mice with cyclin D1 overexpression targeted to the oral-esophageal tissue by the EBV ED-L2 promoter showed increased severity in esophageal dysplasia without cancer development, after multiple doses of N-nitrosomethylbenzylamine (NMBA). Dietary zinc deficiency (ZD) in mice enhances cellular proliferation in esophagus/forestomach and susceptibility to NMBA-induced carcinogenesis. We investigated whether cyclin D1 overexpression in TG mice, together with ZD, might lead to unchecked cell proliferation and accelerated NMBA-induced tumorigenesis. Five-week-old TG and wild-type (WT) mice were fed a ZD- or -sufficient (ZS) diet, forming four groups: ZD:TG; ZS:TG; ZD:WT; and ZS:WT. After 4 weeks, animals were given a single intragastric NMBA dose and were sacrificed 25 and 77 days later. Without NMBA, cell proliferation was greatest in ZD:TG esophagus/forestomach, followed by ZD:WT, and then ZS:TG>/=ZS:WT. The high rate of cell proliferation was accompanied by overexpression of cell cycle progression and tumorigenesis biomarkers, including proliferating cell nuclear antigen, cyclin D1, cyclin-dependent kinase 4, p53, cytokeratin 14, epidermal growth factor receptor, and by a reduced rate of apoptosis. ZD substantially increased forestomach tumor incidence in TG mice: 85% of ZD:TG versus 14% of ZS:TG mice had forestomach tumors (P < 0.001), with progression to malignancy occurring only in ZD:TG tumors. Additionally, 14% of ZD:TG mice developed esophageal tumors and esophageal intestinal metaplasia at 77 days. Thus, cyclin D1 overexpression, in cooperation with ZD, decontrols cell proliferation, ensuring cell expansion, a prerequisite for cancer development.

A frequent genetic alteration found in premalignant stages of pancreatic adenocarcinoma is K-ras oncogene point mutation. The mechanistic basis for the inability of K-ras mutation to transform pancreatic ductal cells is unclear, although cooperating events with p16 inactivation, p53 mutation, and SMAD 4 mutation are recognized to be necessary. We have generated a novel mouse model in which the cytokeratin 19 promoter, specifically active in pancreatic ductal cells but not other cell types of the pancreas, is fused to mutant K-ras. This is of direct relevance to human pancreatic cancer because premalignant lesions are found specifically in ductal cells. There is dramatic periductal lymphocytic infiltration in the pancreata of transgenic mice, predominantly CD4+ T lymphocytes, which may act as an adaptive immune response to activated ras-mediated signaling. In addition, gene array analysis reveals an induction of N-cadherin in transgenic mice pancreatic ductal cells, the significance of which relates to promotion of cell adhesion and deterrence of cell migration. Apart from these important biological considerations, there is parallel activity of the cytokeratin 19 promoter in the stem cell region of the gastric epithelium, namely in mucous neck cells. Activated K-ras in this context causes mucous neck cell hyperplasia, a precursor to gastric adenocarcinoma. There is concomitant parietal cell decrease, which is a key step toward gastric adenocarcinoma. Taken together, we have defined how mutant K-ras signaling modulates important molecular events in the initiating events of pancreatic and gastric carcinogenesis.

beta-Catenin plays an important role in signal transduction pathways that regulate cellular differentiation and proliferation. The increased concentration of this protein in the cytoplasm favors its binding to the T-cell factor (TCF) family of DNA-binding proteins, and it subsequently translocates to the nucleus, where it induces transcription of specific genes. We explored mechanisms that lead to activation of beta-catenin/TCF-dependent transcription in esophageal squamous cell carcinoma (ESCC) independent of adenomatous polyposis coli and beta-catenin mutation. Electrophoresis mobility shift assay demonstrated that TCF4 and beta-catenin form a complex and have DNA binding activity. However, there was no constitutive activation of beta-catenin/TCF-dependent transcription. Coculture experiments demonstrated that Wnt-1, but not Wnt-5A and Wnt-7A, activated the TCF reporter gene. Additionally, when cultured with Wnt-1-conditioned media, ESCC cell lines showed an accumulation of beta-catenin in the cytoplasm. Although both Wnt and epidermal growth factor inactivate glycogen synthase kinase 3beta, activation of epidermal growth factor receptor did not stabilize beta-catenin. A comparison of extracellular stimuli suggests that specific Wnt family members stabilize beta-catenin with resulting activation of TCF-dependent transcription in ESCC.

The p53 gene family, comprising p53, p63, and p73, has overlapping and distinctive functional roles. These members share structural similarities allowing for dynamic interplay in the activation of genes that are important in development and key cellular functions, such as the induction of apoptosis. Whereas p53 is a classical tumor suppressor gene, p63 and p73 do not share this feature in cancer formation and progression. The compensation in the expression level of these members in a background that is deficient for one of them has not been examined previously. Given the importance of p63 in the development and differentiation of oral-esophageal stratified squamous epithelia and the absence of oral-esophageal tumors in p53-null mice, we postulated and describe herein that p63 expression is associated with the loss of p53 in a p53-deficient background. Both full-length and amino-truncated forms of p63 are expressed and increased in oral-esophageal epithelia of p53-null mice when compared with wild-type mice, and the induction of p21 may potentially be preserved through the increase of p63.

The stratified squamous epithelium is a model system in which to define molecular mechanisms underlying the switch from proliferation to differentiation. This can be achieved through the functional dissection of keratin gene promoters. Having previously established the importance of keratin 4 in maintaining the differentiated phenotype in corneal epithelial cells, we investigated the role of Sp1-mediated transactivation of the keratin 4 promoter given the role of Sp1 in differentiation and cell cycle progression. Sp1 transactivation of the keratin 4 promoter was diminished in cyclin D1-overexpressing cells, which may be mediated through a newly described direct interaction between Sp1 and cyclin D1 and opposed by a complex between Sp1 and pRB.

Chromosomal allelic losses have varying frequency in breast cancer, with key regions including chromosomes 1, 3p, 7q, 9p, 16q, 17, and 22q. Recently, we have been able to map a new target region of allelic loss on chromosome 22q involved in colorectal cancer. The aim of the current investigation was to determine whether this target region may also be involved in human breast carcinogenesis. Thirty-six pairs of matched normal and tumor specimens from breast cancer patients, as well as eight breast cancer-derived cell lines, were genotyped using 17 microsatellite markers spanning chromosome 22q. Allelic deletion was found in 19 of 36 tumors (53%), and the pattern observed in those cases with partial losses was consistent with a region flanked by D22S1171 and D22S928. This interval overlaps that identified in colorectal cancer and comprises nearly 1.1 Mb. This study provides evidence of a common region of deletion on chromosome 22q13 involved in both breast and colorectal cancers and underscores the existence of putative tumor suppressor gene(s) at this location.

The cyclin D1 oncogene is critical in the progression of the cell cycle through the G1 phase. It is frequently overexpressed in squamous cell carcinomas originating from the head/neck and esophagus. Yet, the functional consequences of aberrant cyclin D1 overexpression are not entirely understood apart from increased cell proliferation. To address this question, we have developed a transgenic mouse model in which the EBV ED-L2 promoter targets cyclin D1 to the stratified squamous epithelium in a tissue-specific fashion to the tongue and esophagus, thereby resulting in a dysplastic phenotype. We now demonstrate that the dysplastic phenotype is associated with increased cell proliferation based on proliferating cell nuclear antigen overexpression and abnormalities in cyclin-dependent kinase 4, epidermal growth factor receptor, and p53. In aggregate, these studies suggest that alterations in certain oncogenes and tumor suppressor genes occur early during head/neck and esophageal carcinogenesis.

CD44 is the transmembrane adhesion molecule which binds hyaluronate. The gene encoding CD44 is found on chromosome 11p and comprises 20 exons. Differential splicing of the 10 extracellular juxtamembranous exons (v1-10) generates the major isoforms of CD44. The major CD44 isoform found on hematopoetic cells (CD44s) contains none of the variably expressed exons, while the major isoform expressed on epithelial cells [CD44(v8-10)] contains exons v8-10. Metastasis-specific isoforms of CD44 were first documented in a model of rat pancreatic adenocarcinoma [CD44(v4-7), CD44(v6-7)] and subsequently in other cancers. This study is the first characterization of CD44 isoforms in primary and metastatic human pancreatic adenocarcinomas. CD44 isoforms were analyzed in specimens of 15 primary and 6 metastatic pancreatic adenocarcinomas as well as in 6 specimens of control pancreata by two different methods. Radiolabeled reverse transcriptase-PCR coupled with 8% PAGE allowed analysis of the major isoforms of CD44, while Southern blot hybridization with [alpha-32P]dCTP-labeled probes permitted analysis for metastasis-specific CD44 isoforms containing CD44(v6) or CD44(v8-10). No differences in the expression of CD44(v8-10) and CD44s were found among the primary and metastatic pancreatic adenocarcinomas, and control specimens of pancreata. However, a novel CD44(v6) isoform was found in metastatic lesions and may represent the human homologue of the rat pancreatic adenocarcinoma metastasis-associated CD44 isoform.