Tracy Wright

KRas Induces a Src/PEAK1/ErbB2 Kinase Amplification Loop That Drives Metastatic Growth and Therapy Resistance in Pancreatic Cancer

Early biomarkers and effective therapeutic strategies are desperately needed to treat pancreatic ductal adenocarcinoma (PDAC), which has a dismal 5-year patient survival rate. Here, we report that the novel tyrosine kinase PEAK1 is upregulated in human malignancies, including human PDACs and pancreatic intraepithelial neoplasia (PanIN). Oncogenic KRas induced a PEAK1-dependent kinase amplification loop between Src, PEAK1, and ErbB2 to drive PDAC tumor growth and metastasis in vivo. Surprisingly, blockade of ErbB2 expression increased Src-dependent PEAK1 expression, PEAK1-dependent Src activation, and tumor growth in vivo, suggesting a mechanism for the observed resistance of patients with PDACs to therapeutic intervention. Importantly, PEAK1 inactivation sensitized PDAC cells to trastuzumab and gemcitabine therapy. Our findings, therefore, suggest that PEAK1 is a novel biomarker, critical signaling hub, and new therapeutic target in PDACs.

Cancer cell migration within 3D layer-by-layer microfabricated photocrosslinked PEG scaffolds with tunable stiffness.

Our current understanding of 3-dimensional (3D) cell migration is primarily based on results from fibrous scaffolds with randomly organized internal architecture. Manipulations that change the stiffness of these 3D scaffolds often alter other matrix parameters that can modulate cell motility independently or synergistically, making observations less predictive of how cells behave when migrating in 3D. In order to decouple microstructural influences and stiffness effects, we have designed and fabricated 3D polyethylene glycol (PEG) scaffolds that permit orthogonal tuning of both elastic moduli and microstructure. Scaffolds with log-pile architectures were used to compare the 3D migration properties of normal breast epithelial cells (HMLE) and Twist-transformed cells (HMLET). Our results indicate that the nature of cell migration is significantly impacted by the ability of cells to migrate in the third dimension. 2D ECM-coated PEG substrates revealed no statistically significant difference in cell migration between HMLE and HMLET cells among substrates of different stiffness. However, when cells were allowed to move along the third dimension, substantial differences were observed for cell displacement, velocity and path straightness parameters. Furthermore, these differences were sensitive to both substrate stiffness and the presence of the Twist oncogene. Importantly, these 3D modes of migration provide insight into the potential for oncogene-transformed cells to migrate within and colonize tissues of varying stiffness.

CRIPTO/GRP78 Signaling Maintains Fetal and Adult Mammary Stem Cells Ex Vivo

Summary Little is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell-surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers.

A Hypusine–eIF5A–PEAK1 Switch Regulates the Pathogenesis of Pancreatic Cancer

Deregulation of protein synthesis is a hallmark of cancer cell proliferation, survival, and metastatic progression. eIF5A1 and its highly related isoform eIF5A2 are translation initiation factors that have been implicated in a range of human malignancies, but how they control cancer development and disease progression is still poorly understood. Here, we investigated how eIF5A proteins regulate pancreatic ductal adenocarcinoma (PDAC) pathogenesis. eIF5A proteins are the only known proteins regulated by a distinct posttranslational modification termed hypusination, which is catalyzed by two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). The highly selective nature of the hypusine modification and its amenability to pharmacologic inhibition make eIF5A proteins attractive therapeutic targets. We found that the expression and hypusination of eIF5A proteins are upregulated in human PDAC tissues and in premalignant pancreatic intraepithelial neoplasia tissues isolated from Pdx-1-Cre: LSL-KRAS(G12D) mice. Knockdown of eIF5A proteins in PDAC cells inhibited their growth in vitro and orthotopic tumor growth in vivo, whereas amplification of eIF5A proteins increased PDAC cell growth and tumor formation in mice. Small-molecule inhibitors of DHPS and DOHH both suppressed eIF5A hypusination, preventing PDAC cell growth. Interestingly, we found that eIF5A proteins regulate PDAC cell growth by modulating the expression of PEAK1, a nonreceptor tyrosine kinase essential for PDAC cell growth and therapy resistance. Our findings suggest that eIF5A proteins utilize PEAK1 as a downstream effector to drive PDAC pathogenesis and that pharmacologic inhibition of the eIF5A-hypusine-PEAK1 axis may provide a novel therapeutic strategy to combat this deadly disease.

Eukaryotic Translation Initiation Factor 5A (EIF5A) Regulates Pancreatic Cancer Metastasis by Modulating RhoA and Rho-associated Kinase (ROCK) Protein Expression Levels

Background: Eukaryotic translation initiation factor 5A (eIF5A) regulates pancreatic cancer pathogenesis. Results: eIF5A was shown to control the expression of a set of key signaling molecules including RhoA and ROCK2, and to promote the invasive potential of pancreatic cancer cells. Conclusion: Hypusine/eIF5A/RhoA/ROCK cascade promotes pancreatic cancer cell metastasis. Significance: eIF5A may be a novel druggable target to treat metastatic pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with an overall survival rate of less than 5%. The poor patient outcome in PDAC is largely due to the high prevalence of systemic metastasis at the time of diagnosis and lack of effective therapeutics that target disseminated cells. The fact that the underlying mechanisms driving PDAC cell migration and dissemination are poorly understood have hindered drug development and compounded the lack of clinical success in this disease. Recent evidence indicates that mutational activation of K-Ras up-regulates eIF5A, a component of the cellular translational machinery that is critical for PDAC progression. However, the role of eIF5A in PDAC cell migration and metastasis has not been investigated. We report here that pharmacological inhibition or genetic knockdown of eIF5A reduces PDAC cell migration, invasion, and metastasis in vitro and in vivo. Proteomic profiling and bioinformatic analyses revealed that eIF5A controls an integrated network of cytoskeleton-regulatory proteins involved in cell migration. Functional interrogation of this network uncovered a critical RhoA/ROCK signaling node that operates downstream of eIF5A in invasive PDAC cells. Importantly, eIF5A mediates PDAC cell migration and invasion by modulating RhoA/ROCK protein expression levels. Together our findings implicate eIF5A as a cytoskeletal rheostat controlling RhoA/ROCK protein expression during PDAC cell migration and metastasis. Our findings also implicate the eIF5A/RhoA/ROCK module as a potential new therapeutic target to treat metastatic PDAC cells.

eIF5A-PEAK1 Signaling Regulates YAP1/TAZ Protein Expression and Pancreatic Cancer Cell Growth

In pancreatic ductal adenocarcinoma (PDAC), mutant KRAS stimulates the translation initiation factor eIF5A and upregulates the focal adhesion kinase PEAK1, which transmits integrin and growth factor signals mediated by the tumor microenvironment. Although eIF5A-PEAK1 signaling contributes to multiple aggressive cancer cell phenotypes, the downstream signaling processes that mediate these responses are uncharacterized. Through proteomics and informatic analyses of PEAK1-depleted PDAC cells, we defined protein translation, cytoskeleton organization, and cell-cycle regulatory pathways as major pathways controlled by PEAK1. Biochemical and functional studies revealed that the transcription factors YAP1 and TAZ are key targets of eIF5A-PEAK1 signaling. YAP1/TAZ coimmunoprecipitated with PEAK1. Interfering with eIF5A-PEAK1 signaling in PDAC cells inhibited YAP/TAZ protein expression, decreasing expression of stem cell-associated transcription factors (STF) including Oct4, Nanog, c-Myc, and TEAD, thereby decreasing three-dimensional (3D) tumor sphere growth. Conversely, amplified eIF5A-PEAK1 signaling increased YAP1/TAZ expression, increasing expression of STF and enhancing 3D tumor sphere growth. Informatic interrogation of mRNA sequence databases revealed upregulation of the eIF5A-PEAK1-YAP1-TEAD signaling module in PDAC patients. Taken together, our findings indicate that eIF5A-PEAK1-YAP signaling contributes to PDAC development by regulating an STF program associated with increased tumorigenicity. Cancer Res; 77(8); 1997-2007. ©2017 AACR.

Abstract 602: Silencing BRG1 in human cancers

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL BRG1 is a novel tumor suppressor that serves as the major catalytic subunit of the SWI/SNF complex. Because this complex is central to the function of a number of transcription factors and key cellular proteins and it is required for a plethora of signal transduction pathways, its loss can have devastating effects on cellular function. We know that BRG1 is not only lost in lung cancer but also in a variety of tumor types: between 10-40%. Hence, the loss of this protein plays an important role in cancer development and progression. In cell lines, it has been well document that BRG1 is mutated or altered in the vast majority of cell lines, but in primary lung cancer mutations are not found. Our analysis has led us to believe that BRG1 is epigenetically silenced like its homolog BRM. Hence, it may be possible to restore its expression in cancer cells. The loss of BRG1 impacts the sensitivity to Cisplatin by making cells more sensitive to the DNA damaging effects of this agent. Similarly, this loss also impacts sensitivity to EGFR inhibitors by making cells more resistant. Hence, the loss of BRG1 can help to explain the reciprocal sensitivity to these drugs seen in cancers. Hence, the BRG1 protein can play the role of a biomarker for chemotherapy sensitivity as well as being a potential target for therapy. Further research will reveal how this gene can best be integrated into clinical practice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 602. doi:10.1158/1538-7445.AM2011-602

Abstract LB-303: A hypusine-eIF5A-PEAK1 switch regulates the pathogenesis of pancreatic ductal adenocarcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Deregulation of mRNA translation and protein synthesis is a hallmark of cancer cell proliferation, survival, and metastatic progression. eIF5A1, and its highly related isoform eIF5A2, are critical translation initiation factors implicated in several human malignancies. Interestingly, eIF5A proteins are the only known proteins in nature that are regulated by a distinct posttranslational modification termed hypusination. This unique lysine modification ([N-(4-amino-2-hydroxybutyl)lysine]) involves polyamine biosynthesis and is catalyzed by two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). The highly selective nature of the hypusine modification and its amenability to pharmacological and genetic inhibition make the eIF5A-hypusine pathway an attractive therapeutic target to combat human cancers. Here, we investigated the role of eIF5A1/A2-hypusine pathway in pancreatic ductal adenocarcinoma (PDAC) pathogenesis. Hypusinated eIF5A1/A2 are upregulated in human PDAC tissues and in early stage PanIN tissues isolated from genetically engineered Pdx-Cre:LSL-KRASG12D mice. RNAi-mediated knockdown of eIF5A1/A2 in human PDAC cell lines inhibited cancer cell growth in vitro and inhibited tumor growth orthotopically in mice. In contrast, amplification of eIF5A1/A2 in PDAC cells increased orthotopic growth and tumor formation in mice. The deoxyhypusine hydroxylase inhibitor, CPX, and the deoxyhypusine synthase inhibitor, GC7, inhibited eIF5A1/A2 hypusination, preventing PDAC cell growth. Interestingly, we find that eIF5A1/A2 regulates cancer cell expansion by modulating the expression level of PEAK1, a non-receptor tyrosine kinase essential for PDAC cell growth. Our findings suggest that eIF5A1/A2 utilizes PEAK1 tyrosine kinase as a downstream effector to drive PDAC pathogenesis, and that pharmacological inhibition of the eIF5A1/A2 hypusination-PEAK1 axis may provide a novel therapeutic strategy to combat this deadly disease. Citation Format: Ken Fujimura, Tracy Wright, Jan Strnadel, Jonathan Kelber, Sharmeela Kaushal, Cristina Metildi, Andrew Lowy, Michael Bouvet, Richard Klemke. A hypusine-eIF5A-PEAK1 switch regulates the pathogenesis of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-303. doi:10.1158/1538-7445.AM2014-LB-303

Abstract 3035: Cripto/GRP78 signaling promotes the stem cell phenotype in normal and neoplastic mammary epithelial cells

Microenvironmental factors are critical regulators of stem cell and tumor cell behavior. However, few stem cell factors have been identified and targeted in breast and other cancers. Using a highly enriched stem cell pool isolated from fetal mammary rudiments and single cell analyses, we have identified the onco-fetal protein Cripto as a potent soluble factor that regulates the mammary stem cell state. Cripto is a GPI-anchored/secreted signaling protein and a known regulator of PI3K/AKT and TGF-beta pathways. Here, we develop a novel Cripto antagonist, ALK4L75A-Fc, which selectively blocks the growth factor-like effects of soluble Cripto. We show that this antagonist promotes the differentiation of mammary stem cells cultured ex vivo while Cripto treatment maintains the stem cell phenotype and yields colonies with enhanced mammary gland reconstitution capacity. We previously discovered that Cripto signaling requires binding to cell surface Glucose Response Protein 78kDa (GRP78). GRP78 is an HSP70 family member that is induced by stresses including hypoxia and glucose deprivation and it is highly expressed in tumors where these conditions prevail. We show here that cell surface GRP78 marks fetal mouse mammary stem cells and a population of bipotent adult mammary epithelial cells that are selectively responsive to soluble Cripto in vitro. GRP78high mammary epithelial cells also harbor substantially higher stem cell activity upon transplantation than GRP78low cells. Consistently, we further show that deletion of GRP78 from adult mammary epithelial cells ex vivo blocks mammary gland reconstitution. Finally, we find that Cripto antagonism with ALK4L75A-Fc inhibits the proliferation of triple negative breast cancer cell lines in vitro and that this antagonist can also inhibit tumor growth in vivo. Since Cripto and GRP78 are both induced by hypoxia and other stressful conditions found in tumors, our data raise the possibility that Cripto/GRP78 signaling may exacerbate breast and other cancers by increasing the number of stem cell-like tumor cells in tumor microenvironments where these conditions predominate. Citation Format: Benjamin T. Spike, Jonathan A. Kelber, Evan Booker, Madhuri Kalathur, Rose Rodewald, Julia Lipianskaya, Justin La, Marielle He, Tracy Wright, Richard Klemke, Geoffrey Wahl, Peter C. Gray. Cripto/GRP78 signaling promotes the stem cell phenotype in normal and neoplastic mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3035. doi:10.1158/1538-7445.AM2014-3035

Abstract 4911: Aggressive variants isolated from human pancreatic cancer passaging in mice are enriched in stem cell markers and PEAK1 expression.

A more aggressive variants of the human pancreatic cancer cell line BxPC-3 were generated by serially passaging primary pancreatic tumors in nude mice. Changes in tumorigenicity, proliferation, stem cell markers, PEAK1 expression and survival were determined. Orthotopic mouse models of human pancreatic cancer were established by injecting 1x10 6 BxPC-3-RFP pancreatic cancer cells into the pancreas of nude mice. Primary pancreatic tumors were harvested from premorbid mice to establish cell lines. The established cell line (10 6 ) was orthotopically injected into another set of mice. This cycle was repeated five times. Cells (10 6 ) of the sixth serially passaged cell line (P6) were orthotopically implanted into mice and monitored with weekly whole body fluorescence imaging. FACS and qPCR analysis were performed on primary and metastatic lesions for stem cell markers and PEAK1 expression. In a second group of mice, the parental (P0) and P6 cell lines were injected subcutaneously at different cell numbers (10 2 , 5 x 10 2 , 10 3 , 10 4 , 10 5 cells) to determine the minimum cell number that would produce a tumor. At 6 weeks, the tumors were harvested and weighed. The BxPC-3-RFP P6 variant had more rapid primary tumor growth and shortened overall survival in mice compared to the parental cell line (52.2±SE3.1 days vs 115.4±SE10.2 days, respectively, p 2 cells had tumor growth at 6 weeks compared to 0/5 mice injected with 10 2 cells of the P0 cell line (p=0.025). The P6 cell line also resulted in larger tumors at 6 weeks compared to the P0 cell line (102.2±28.5 mg vs 11.0±4.1 mg, respectively, p=0.002). qPCR evaluation demonstrated enrichment of stem cell markers (CD24, CD44, EpCAM) and PEAK1 expression in the P6 cell line compared to P0 cell line, by 4.6-, 15-, 4.2- and 16.3-fold, respectively. FACS demonstrated an increase in CD24+/CD44+/EpCAM+ cells from 69% in the parental cell line to 97.1% in the P6 cell line. In conclusion, serial passaging of tumor allows for in vivo selection of a more aggressive variant of the human pancreatic cancer cell line BxPC-3 associated with an enrichment in stem cell markers and an increase in PEAK1 expression. Citation Format: Cristina A. Metildi, Sharmeela Kaushal, Jan Strnadel, Tracy Wright, Jonathan A. Kelber, Richard L. Klemke, Robert M. Hoffman, Michael Bouvet. Aggressive variants isolated from human pancreatic cancer passaging in mice are enriched in stem cell markers and PEAK1 expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4911. doi:10.1158/1538-7445.AM2013-4911