XLSX file - 43K, Number of metastatic deposits for Ecd Scr and KD cell injected mice

Supplementary Table 1 from Overexpression of RhoA Induces Preneoplastic Transformation of Primary Mammary Epithelial Cells

Supplementary Figures 1-4 from Overexpression of RhoA Induces Preneoplastic Transformation of Primary Mammary Epithelial Cells

Supplementary Table 2 from Overexpression of RhoA Induces Preneoplastic Transformation of Primary Mammary Epithelial Cells

Supplementary Figures 1-2 from Cyclooxygenase-2 Expression during Immortalization and Breast Cancer Progression

The potency of polymeric micelle-based doxorubicin, SP1049C, against cancer stem cells (CSCs) in triple negative breast cancer (TNBC) is evaluated. CSCs with high epithelial specific antigen (ESA), high CD44 and low CD24 expression levels were derived from the TNBC cancer cells, MDA-MB-231 and MDA-MB-468. These CSCs were resistant to free doxorubicin (Dox) and displayed increased colony formation, migration, and invasion in vitro, along with higher tumorigenicity in vivo, compared to the parental and non-CSCs counterparts. SP1049C downregulated the expression and inhibited the functional activity of the breast cancer resistance protein (BCRP/ABCG2) in CSCs. The polymeric micelle drug had higher cytotoxicity and potency in reducing the colony formation of CSCs compared to the free drug. It was also more potent in inhibiting the tumor growth in the orthotopic animal tumor models derived from CSCs. These results indicate that SP1049C is active against CSCs and has potential in treating TNBC.

Prostate cancer remains one of the most common cancers diagnosed in men and one of the leading causes of cancer death in men. Tumor development and progression have been shown to be highly influenced not simply by the genetic makeup of a cell, but by its surrounding stroma, particularly fibroblasts. It has been demonstrated that prostate cancer-associated fibroblasts (CAFs, which are located marginal to the prostate tumor), differ from prostate normal-associated fibroblast (NAFs, which are located distal to the prostate tumor), on their contribution to tumor progression. However, human prostate cancer in-vitro model systems have focused largely on prostate cancer epithelial cells exclusively. A need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we utilized prostate cancer-associated fibroblasts (CAFs), prostate normal-associated fibroblasts (NAFs) and normal prostate epithelial (PrE) cells; all three lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and they were continuously passaged for at least 15 passages without any indications of a decrease in growth rate. All cell lines express appropriate specific cell lineage markers for either fibroblasts or epithelial cells. Fibroblasts expressed TE7 and alpha smooth muscle actin (a-SMA), while prostate epithelial cells expressed cytokeratin 5, low levels of prostate specific antigen (PSA) and high levels of p63 throughout their continuous passage; all characteristics in accord with their primary cell counterparts. Next, cell proliferation was measured for various prostate-derived epithelial cells under the influence of CAFs and NAFs cells. Normal prostate epithelial cell proliferation was inhibited and produced a visible morphological change in the cells in the presence of CAFs or CAF-conditioned medium. Meanwhile, the effects of stromal cells on prostate cancer cells was cell line dependent, demonstrating both promotion and inhibition of growth of selected cancer cells. Surprisingly, both CAFs and NAFs promoted cancer cell growth, but CAFs promoted a greater increase in cell proliferation than NAFs in some cancer cell lines. This study demonstrates that these three hTERT immortalized cells from human prostate are a valuable model system for the study of prostate cancer cell progression and tumor micro environment studies. Citation Format: Luis G. Rodriguez, Russell E. McDaniel, Xiangshan Zhao, Chaozhong Zou. An authenticated in vitro model for prostate microenvironment studies utilizing prostate epithelial cells and stromal-derived cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2115.

Abstract Tumor development begins with mutational changes to the genetic makeup of a cell, but its progression is not solely determined by the mutated cell, but also by the tumor’s microenvironment. Prostate cancer, a leading cancer diagnosed in men, has been shown to be highly influenced by its surrounding stroma, particularly fibroblasts. It has been demonstrated that cancer-associated prostate fibroblast (CAFs) differ from normal-associated prostate fibroblast (NAFs). However, human prostate cancer model systems have focused largely on prostate cancer epithelial cells. Currently, a need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we characterized three prostate-derived cells: prostate cancer-associated fibroblast (CAFs), prostate normal-associated fibroblast (NAFs) and prostate cancer epithelial (PrE) cells; all three lines were immortalized by hTERT (human telomerase reverse transcriptase) alone, and have been continuously passaged for more than 40 PDL in our hands. Our data shows that the hTERT immortalized CAFs proliferate faster than the NAFs; in addition, both CAFs and NAFs express fibroblast markers such as TE7 and alpha smooth muscle actin (α-SMA), while neither cell line expresses epithelial marker such as CK14. Both CAFs and NAFs also express elevated levels of α-SMA upon TGF-β stimulation. All three prostate-derived cells express the prostate specific marker AR, and show similar markers staining after long time passaging. Importantly, conditioned media collected from CAFs promotes tumor cell growth better than NAF conditioned media. In conclusion, CAFs, NAFs, and immortalized prostate cancer epithelium may provide a very valuable model system for the study of prostate cancer cell progression and tumor microenvironment studies. Citation Format: Luis G. Rodriguez, Russell E. McDaniel, Xiangshan Zhao, Elizabeth Turner, Christopher Annesi, Chaozhong Zou. Characterization of hTERT-immortalized prostate-derived stromal and epithelial cells: An authentic in vitro model for tumor microenvironment studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4948. doi:10.1158/1538-7445.AM2017-4948

Background: We have established the critical role of ADA3 as a coactivator of estrogen receptor (ER), as well as its role in cell cycle progression. Furthermore, we showed that ADA3 is predominantly nuclear in mammary epithelium, and in ER+, but is cytoplasmic in ER- breast cancers, the latter correlating with poor survival. However, the role of nuclear ADA3 in human mammary epithelial cells (hMECs), and in ER+ breast cancer cells, as well as the importance of ADA3 expression in relation to patient prognosis and survival in ER+ breast cancer have remained uncharacterized.Methods: We overexpressed ADA3 in hMECs or in ER+ breast cancer cells and assessed the effect on cell proliferation. The expression of ADA3 was analyzed then correlated with the expression of various prognostic markers, as well as survival of breast cancer patients.Results: Overexpression of ADA3 in ER-hMECs as well as in ER+ breast cancer cell lines enhanced cell proliferation. These cells showed increased cyclin B and c-MYC, decreased p27 and increased SKP2 levels. This was accompanied by increased mRNA levels of early response genes c-FOS, EGR1, and c-MYC. Analysis of breast cancer tissue specimens showed a significant correlation of ADA3 nuclear expression with c-MYC expression. Furthermore, nuclear ADA3 and c-MYC expression together showed significant correlation with tumor grade, mitosis, pleomorphism, NPI, ER/PR status, Ki67 and p27 expression. Importantly, within ER+ cases, expression of nuclear ADA3 and c-MYC also significantly correlated with Ki67 and p27 expression. Univariate Kaplan Meier analysis of four groups in the whole, as well as the ER+ patients showed that c-MYC and ADA3 combinatorial phenotypes showed significantly different breast cancer specific survival with c-MYC-high and ADA3-Low subgroup had the worst outcome. Using multivariate analyses within the whole cohort and the ER+ subgroups, the significant association of ADA3 and c-MYC expression with patients' outcome was independent of tumor grade, stage and size, and ER status.Conclusion: ADA3 overexpression enhances cell proliferation that is associated with increased expression of c-MYC. Expression patterns with respect to ADA3/c-MYC can divide patients into four significantly different subgroups, with c-MYC High and ADA3 Low status independently predicting poor survival in patients.

Breast cancer is characterized into different molecular subtypes, and each subtype is characterized by differential gene expression that are associated with distinct survival outcomes in patients. PIK3CA mutations are commonly associated with most breast cancer subtypes. More recently PIK3CA mutations have been shown to induce tumor heterogeneity and are associated with activation of EGFR-signaling and reduced relapse free survival in basal subtype of breast cancer. Thus, understanding what determines PIK3CA induced heterogeneity and oncogenesis, is an important area of investigation. In this study, we assessed the effect of mutant PIK3CA together with mutant Ras plus mutant p53 on oncogenic behavior of two distinct stem/progenitor breast cell lines, designated as K5+/K19- and K5+/K19+. Constructs were ectopically overexpressed in K5+/K19- and K5+/K19+ stem/progenitor cells, followed by various in-vitro and in-vivo analyses. Oncogene combination m-Ras/m-p53/m-PIK3CA efficiently transformed both K5+/K19- and K5+/K19+ cell lines in-vitro, as assessed by anchorage-independent soft agar colony formation assay. Significantly, while this oncogene combination induced a complete epithelial-to-mesenchymal transition (EMT) in K5+/K19- cell line, mostly epithelial phenotype with minor EMT component was seen in K5+/K19+ cell line. However, both K5+/K19- and K5+/K19+ transformed cells exhibited increased invasion and migration abilities. Analyses of CD44 and CD24 expression showed both cell lines had tumor-initiating CD44+/CD24low cell population, however transformed K5+/K19- cells had more proportion of these cells. Significantly, both cell types exhibited in-vivo tumorigenesis, and maintained their EMT and epithelial nature in-vivo in mice tumors. Notably, while both cell types exhibited increase in tumor-initiating cell population, differential EMT phenotype was observed in these cell lines. These results suggest that EMT is a cell type dependent phenomenon and does not dictate oncogenesis.

Abstract The RNA polymerase II mediated transcription requires the higher degree structure of chromatin to be relieved so that general transcription machinery can access the DNA. The acetylation of DNA bound histones at specific loci is the major epigenetic modification by which opening of chromatin is achieved. HATs (Histone Acetyl Transferase) are the enzymes that catalyze the acetylation of histones and require association with mediator proteins for their function. One such mediator protein is ADA3 (Alteration/Deficiency in Activation 3), which was initially discovered as a component of multi-protein complex that contains either GCN5 (General Control Non-repressed 5) or PCAF (p300/CBP Associated Factor) as HAT and subsequent studies showed that ADA3 associates with another HAT, p300. As a HAT interacting protein, ADA3 enhances the acetylation of histones as well as non-histone proteins, such as p53. We have recently shown that ADA3 is a cell cycle regulatory protein and is important for both G1 to S phase transition as well as mitosis. Conditional deletion of Ada3 from Ada3FL/FL MEFs (Mouse Embryonic Fibroblasts) causes cell cycle arrest and severe mitotic defects. To further explore the mechanism of ADA3 mediated mitosis, we performed ChIP-seq analyses and found that ADA3 bound to higher order repeat region of the centromere across most of the chromosomes. Further studies showed ADA3 interaction with centromere was mediated by a centromeric protein CENP-B. More importantly, siRNA mediated knockdown of ADA3 decreases the occupancy of CENP-B at centromere and causes chromosomal segregation defects during mitosis. These studies demonstrate a novel function of ADA3 in cell cycle regulation. Our current studies are focused on defining the exact mechanism of recruitment of ADA3 complex to CENP-B to regulate mitosis and its effect on genomic stability. Citation Format: Shashank Srivastava, Shakur Mohibi, June Wang-France, Sameer Mirza, Xiangshan Zhao, Hamid Band, Vimla Bnad. ADA3, a cell cycle regulator, regulates chromosome segregation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2719.

ADA3 (alteration/deficiency in activation 3) is a conserved component of several transcriptional co-activator and histone acetyltransferase (HAT) complexes. Recently, we generated Ada3 knock-out mice and demonstrated that deletion of Ada3 leads to early embryonic lethality. The use of Ada3(FL/FL) mouse embryonic fibroblasts with deletion of Ada3 using adenovirus Cre showed a critical role of ADA3 in cell cycle progression through mitosis. Here, we demonstrate an association of ADA3 with the higher order repeat region of the α-satellite region on human X chromosome centromeres that is consistent with its role in mitosis. Given the role of centromere proteins (CENPs) in mitosis, we next analyzed whether ADA3 associates with the centromere through CENPs. Both an in vivo proximity ligation assay and immunofluorescence studies confirmed the association of ADA3 with CENP-B protein, a highly conserved centromeric protein that binds to the 17-bp DNA sequences on α-satellite DNA. Deletional analysis showed that ADA3 directly associates with CENP-B through its N terminus, and a CENP-B binding-deficient mutant of ADA3 was incompetent in cell proliferation rescue. Notably, knockdown of ADA3 decreased binding of CENP-B onto the centromeres, suggesting that ADA3 is required for the loading of CENP-B onto the centromeres. Finally, we show that deletion of Ada3 from Ada3(FL/FL) mouse embryonic fibroblasts exhibited various chromosome segregation defects. Taken together, we demonstrate a novel ADA3 interaction with CENP-B-centromere that may account for its previously known function in mitosis. This study, together with its known function in maintaining genomic stability and its mislocalization in cancers, suggests an important role of ADA3 in mitosis.

Breast cancer is classified into different subtypes that are associated with different patient survival outcomes, underscoring the importance of understanding the role of precursor cell and genetic alterations in determining tumor subtypes. In this study, we evaluated the oncogenic phenotype of two distinct mammary stem/progenitor cell types designated as K5(+)/K19(-) or K5(+)/K19(+) upon introduction of identical combinations of oncogenes-mutant H-Ras (mRas) and mutant p53 (mp53), together with either wild-type ErbB2(wtErbB2) or wild-type EGFR (wtEGFR). We examined their tumor forming and metastasis potential, using both in-vitro and in-vivo assays. Both the combinations efficiently transformed K5(+)/K19(-) or K5(+)/K19(+) cells. Xenograft tumors formed by these cells were histologically heterogeneous, with variable proportions of luminal, basal-like and claudin-low type components depending on the cell types and oncogene combinations. Notably, K5(+)/K19(-) cells transformed with mRas/mp53/wtEGFR combination had a significantly longer latency for primary tumor development than other cell lines but more lung metastasis incidence than same cells expressing mRas/mp53/wtErbB2. K5(+)/K19(+) cells exhibit shorter overall tumor latency, and high metastatic potential than K5(+)/K19(-) cells, suggesting that these K19(+) progenitors are more susceptible to oncogenesis and metastasis. Our results suggest that both genetic alterations and cell type of origin contribute to oncogenic phenotype of breast tumors.

Abstract Breast cancer is classified in to different subtypes based on gene expression profiling. Significantly distinct subtypes present different outcome for patient survival, thus underscoring importance of understanding the role of precursor cell and genetic alterations in breast cancer subtypes. In the present study we are evaluating the role of defined combination of oncogenes in two distinct cell types in regulating the oncogenesis and metastatic behavior. We have earlier developed two hTERT-immortalized human stem/progenitor cell lines that are designated as K5+/K19- or K5+/K19+. We introduced different oncogenes in these cell lines and then examined their oncogenic potential, tumor formation and metastasis, using both in vitro and in vivo assays. We found that combinations of mutant H-Ras(mRas) and mutant p53(mp53), together with either wild type ErbB2(wtErbB2) or wild type EGFR(wtEGFR) efficiently transformed both cell types. Tumors formed by these cells were histologically heterogeneous, with variable proportions of luminal, basal-like and claudin-low type components dependent on cell types and oncogene combinations. Notably, K5+/K19- cells transformed with mRas/mp53/wtEGFR combination had significantly longer latency for primary tumor development than those expressing mRas/mp53/wtErbB2. Despite the longer latency, overall oncogene combination mRas/mp53/wtEGFR give early lung metastasis onset in both K5+/K19- and K5+/K19+ cells as compared to mRas/mp53/wtErbB2 indicating higher metastatic potential of this combination. Besides the effect of different oncogene combinations, we also found that K5+/K19+ cells overall had shorter primary tumor latency, and high metastatic potential than K5+/K19- cells. Histological and biochemical characterization of metastatic tumors is underway and will be discussed at the time of presentation. Our results suggest that both the genetic alteration and cell type contribute to oncogenicity of breast tumors. Citation Format: Divya Bhagirath, Xiangshan Zhao, William W. West, Hamid Band, Vimla Band. Contribution of cell types and genetic alterations in breast cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3233. doi:10.1158/1538-7445.AM2015-3233

BACKGROUND:Identification and characterization of molecular controls that regulate mammary stem and progenitor cell homeostasis are critical to our understanding of normal mammary gland development and its pathology.RESULTS:We demonstrate that conditional knockout of Sox9 in the mouse mammary gland results in impaired postnatal development. In short-term lineage tracing in the postnatal mouse mammary gland using Sox9-CreER driven reporters, Sox9 marked primarily the luminal progenitors and bipotent stem/progenitor cells within the basal mammary epithelial compartment. In contrast, long-term lineage tracing studies demonstrate that Sox9+ precursors gave rise to both luminal and myoepithelial cell lineages. Finally, fate mapping of Sox9 deleted cells demonstrates that Sox9 is essential for luminal, but not myoepithelial, lineage commitment and proliferation.CONCLUSIONS:These studies identify Sox9 as a key regulator of mammary gland development and stem/progenitor maintenance.

Transcriptional activation by estrogen receptor (ER) is a key step to breast oncogenesis. Given previous findings that ADA3 is a critical component of HAT complexes that regulate ER function and evidence that overexpression of other ER coactivators such as SRC-3 is associated with clinical outcomes in breast cancer, the current study was designed to assess the potential significance of ADA3 expression/localization in human breast cancer patients. In this study, we analyzed ADA3 expression in breast cancer tissue specimens and assessed the correlation of ADA3 staining with cancer progression and patient outcome. Tissue microarrays prepared from large series of breast cancer patients with long-term follow-ups were stained with anti-ADA3 monoclonal antibody using immunohistochemistry. Samples were analyzed for ADA3 expression followed by correlation with various clinicopathological parameters and patients' outcomes. We report that breast cancer specimens show predominant nuclear, cytoplasmic, or mixed nuclear + cytoplasmic ADA3 staining patterns. Predominant nuclear ADA3 staining correlated with ER+ status. While predominant cytoplasmic ADA3 staining negatively correlated with ER+ status, but positively correlated with ErbB2, EGFR, and Ki67. Furthermore, a positive correlation of cytoplasmic ADA3 was observed with higher histological grade, mitotic counts, Nottingham Prognostic Index, and positive vascular invasion. Patients with nuclear ADA3 and ER positivity have better breast cancer specific survival and distant metastasis free survival. Significantly, cytoplasmic expression of ADA3 showed a strong positive association with reduced BCSS and DMFS in ErbB2+/EGFR+ patients. Although in multivariate analyses ADA3 expression was not an independent marker of survival, predominant nuclear ADA3 staining in breast cancer tissues correlates with ER+ expression and together serves as a marker of good prognosis, whereas predominant cytoplasmic ADA3 expression correlates with ErbB2+/EGFR+ expression and together is a marker of poor prognosis. Thus, ADA3 cytoplasmic localization together with ErbB2+/EGFR+ status may serve as better prognostic marker than individual proteins to predict survival of patients.

Based on gene expression patterns, breast cancers can be divided into subtypes that closely resemble various developmental stages of normal mammary epithelial cells (MECs). Thus, understanding molecular mechanisms of MEC development is expected to provide critical insights into initiation and progression of breast cancer. Epidermal growth factor receptor (EGFR) and its ligands play essential roles in normal and pathological mammary gland. Signals through EGFR is required for normal mammary gland development. Ligands for EGFR are over-expressed in a significant proportion of breast cancers, and elevated expression of EGFR is associated with poorer clinical outcome. In the present study, we examined the effect of signals through EGFR on MEC differentiation using the human telomerase reverse transcriptase (hTERT)-immortalized human stem/progenitor MECs which express cytokeratin 5 but lack cytokeratin 19 (K5(+)K19(-) hMECs). As reported previously, these cells can be induced to differentiate into luminal and myoepithelial cells under appropriate culture conditions. K5(+)K19(-) hMECs acquired distinct cell fates in response to EGFR ligands epidermal growth factor (EGF), amphiregulin (AREG) and transforming growth factor alpha (TGFα) in differentiation-promoting MEGM medium. Specifically, presence of EGF during in vitro differentiation supported development into both luminal and myoepithelial lineages, whereas cells differentiated only towards luminal lineage when EGF was replaced with AREG. In contrast, substitution with TGFα led to differentiation only into myoepithelial lineage. Chemical inhibition of the MEK-Erk pathway, but not the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, interfered with K5(+)K19(-) hMEC differentiation. The present data validate the utility of the K5(+)K19(-) hMEC cells for modeling key features of human MEC differentiation. This system should be useful in studying molecular/biochemical mechanisms of human MEC differentiation.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Precisely regulated cell proliferation is essential for embryonic development as well as homeostasis in adult organs and tissues, whereas uncontrolled cell proliferation is a hallmark of cancer. Thus, elucidating how the cell cycle machinery is controlled is an important area of research in cancer cell biology. A large body of evidence has established a basic paradigm of the control of cell cycle progression involving the Retinoblastoma (Rb) protein family in conjunction with the E2F family of transcription factors. During G0/G1, interaction of hypo-phosphorylated Rb proteins with E2Fs prevents the transcription of E2F target genes. Cyclin-CDK complexes generated during cell cycle progression hyper-phosphorylate Rb, leading to release of Rb from E2Fs; this allows E2F target gene transcription and cell cycle progression. We previously identified the mammalian ortholog of Drosophila ecdysoneless (Ecd) protein as a novel and essential regulator of Rb-E2F-dependent cell cycle progression. Loss of Ecd retards the separation of Rb from E2F, arrests cells at G1/S boundary and prevents cell cycle progression. Consistent with role of Ecd in cell cycle, our recent studies show Ecd is overexpressed in breast cancer cell lines as well as in ductal carcinoma in situ and infiltrating ductal carcinomas of the breast. To understand the mechanism of Ecd overexpression and its correlation with oncogenesis, we overexpressed either Ecd alone, 61L-HRas alone or both genes together in hTERT-immortalized hMECs. These transfectants were then examined for their oncogenic properties. We observed while vector infected cells do not exhibit anchorage independence, all three transfectants showed anchorage independent proliferation, particularly Ecd+Ras showed a dramatic increase in anchorage independence in comparison with either Ecd or H-Ras. Similarly, other oncogenic traits such as proliferation on matrigel, invasion and migration were dramatically increased when both genes were expressed in comparison with individual genes. Considering the known oncogenic role of Ras in human cancer and the known role of Ecd in cell cycle, these data are highly significant and present a novel synergistic pathway for mammary cell transformation. We are currently defining the molecular mechanism of this synergistic oncogenic effect. In addition, we are generating Ecd overexpressed transgenic mice to examine the role of Ecd in oncogenesis in in vivo models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4177. doi:1538-7445.AM2012-4177

Abstract Purpose: To study the expression and function of a novel cell-cycle regulatory protein, human ecdysoneless (Ecd), during pancreatic cancer pathogenesis. Experimental Design: Immunohistochemical expression profiling of Ecd was done in nonneoplastic normal pancreatic tissues and pancreatic ductal adenocarcinoma lesions (from tissue microarray and Rapid Autopsy program) as well as precancerous PanIN lesions and metastatic organs. To analyze the biological significance of Ecd in pancreatic cancer progression, Ecd was stably knocked down in pancreatic cancer cell line followed by in vitro and in vivo functional assays. Results: Normal pancreatic ducts showed very weak to no Ecd expression compared to significant positive expression in pancreatic cancer tissues (mean ± SE composite score: 0.3 ± 0.2 and 3.8 ± 0.2 respectively, P < 0.0001) as well as in PanIN precursor lesions with a progressive increase in Ecd expression with increasing dysplasia (PanIN-1–PanIN-3). Analysis of matched primary tumors and metastases from patients with pancreatic cancer revealed that Ecd is highly expressed in both primary pancreatic tumor and in distant metastatic sites. Furthermore, knockdown of Ecd suppressed cell proliferation in vitro and tumorigenicity of pancreatic cancer cells in mice orthotopic tumors. Microarray study revealed that Ecd regulates expression of glucose transporter GLUT4 in pancreatic cancer cells and was subsequently shown to modulate glucose uptake, lactate production, and ATP generation by pancreatic cancer cells. Finally, knockdown of Ecd also reduced level of pAkt, key signaling molecule known to regulate aerobic glycolysis in cancer cells. Conclusion: Ecd is a novel tumor-promoting factor that is differentially expressed in pancreatic cancer and potentially regulates glucose metabolism within cancer cells. Clin Cancer Res; 18(22); 6188–98. ©2012 AACR.