Development of 3D-iNET ORION: a novel, pre-clinical, three-dimensional in vitro cell model for modeling human metastatic neuroendocrine tumor of the pancreas.

Neuroendocrine tumors (NETs) of the pancreas are rare neoplasms that present complex challenges to diagnosis and treatment due to their indolent course. The incidence of pancreatic neuroendocrine tumors has increased significantly over the past two decades. A limited number of pancreatic neuroendocrine cell lines are currently available for the research. Here, we present 3D-iNET ORION, a novel 3-dimensional (spheroid) cell line, isolated from human pancreatic neuroendocrine tumor liver metastasis. Three-dimensionally grown (3D) cancer cell lines have gained interest over the past years as 3D cancer cell lines better recapitulate the in vivo structure of tumors, and are more suitable for in vitro and in vivo experiments. 3D-iNET ORION cancer cell line showed high potential to form tumorspheres when embedded in Matrigel matrix and expresses synaptophysin and EpCAM. Electron microscopy analysis of cancer cell line proved the presence of dense neurosecretory granules. When xenografted into athymic mice, 3D-iNET ORION cells produce slow-growing tumors, positive for chromogranin and synaptophysin. Human Core Exome Panel Analysis has shown that 3DiNET ORION cell line retains the genetic aberration profile detected in the original tumor. In conclusion, our newly developed neuroendocrine cancer cell line can be considered as a new research tool for in vitro and in vivo experiments.

Targeting SMAD-Dependent Signaling: Considerations in Epithelial and Mesenchymal Solid Tumors.

SMADs are the canonical intracellular effector proteins of the TGF-ß (transforming growth factor-ß). SMADs translocate from plasma membrane receptors to the nucleus regulated by many SMAD-interacting proteins through phosphorylation and other post-translational modifications that govern their nucleocytoplasmic shuttling and subsequent transcriptional activity. The signaling pathway of TGF-ß/SMAD exhibits both tumor-suppressing and tumor-promoting phenotypes in epithelial-derived solid tumors. Collectively, the pleiotropic nature of TGF-ß/SMAD signaling presents significant challenges for the development of effective cancer therapies. Here, we review preclinical studies that evaluate the efficacy of inhibitors targeting major SMAD-regulating and/or -interacting proteins, particularly enzymes that may play important roles in epithelial or mesenchymal compartments within solid tumors.

Multidimensional screening of pancreatic cancer spheroids reveals vulnerabilities in mitotic and cell-matrix adhesion signaling that associate with metastatic progression and decreased patient survival.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, with a median survival of less than 12 months and a 5-year survival of less than 10 %. Here, we have established an image-based screening pipeline for quantifying single PDAC spheroid dynamics in genetically and phenotypically diverse PDAC cell models. Wild-type KRas PDAC cells formed tight/compact spheroids - compaction of these structures was completely blocked by cytoplasmic dynein and focal adhesion kinase (FAK) inhibitors. In contrast, PDAC cells containing mutant KRas formed loosely aggregated spheroids that grew significantly slower following inhibition of polo-like kinase 1 (PLK1) or focal adhesion kinase (FAK). Independent of genetic background, multicellular PDAC-mesenchymal stromal cell (MSC) spheroids self-organized into structures with an MSC-dominant core. The inclusion of MSCs into wild-type KRas PDAC spheroids modestly affected their compaction; however, MSCs significantly increased the compaction and growth of mutant KRas PDAC spheroids. Notably, exogenous collagen 1 potentiated PANC1 spheroid compaction while ITGA1 knockdown in PANC1 cells blocked MSC-induced PANC1 spheroid compaction. In agreement with a role for collagen-based integrin adhesion complexes in stromal cell-induced PDAC phenotypes, we also discovered that MSC-induced PANC1 spheroid growth was completely blocked by the ITGB1 immunoneutralizing antibody mAb13. Finally, multiplexed single-cell immunohistochemical analysis of a 25 patient PDAC tissue microarray revealed a relationship between decreased variance in Spearman r correlation for ITGA1 and PLK1 expression within the tumor cell compartment of PDAC in patients with advanced disease stage, and elevated expression of both ITGA1 and PLK1 in PDAC was found to be associated with decreased patient survival. Taken together, this work uncovers new therapeutic vulnerabilities in PDAC that are relevant to the progression of this stromal cell-rich malignancy and which may reveal strategies for improving patient outcomes.

Patient-Derived Organoids as Therapy Screening Platforms in Cancer Patients.

Patient-derived organoids (PDOs) developed ex vivo and in vitro are increasingly used for therapeutic screening. They provide a more physiologically relevant model for drug discovery and development compared to traditional cell lines. However, several challenges remain to be addressed to fully realize the potential of PDOs in therapeutic screening. This paper summarizes recent advancements in PDO development and the enhancement of PDO culture models. This is achieved by leveraging materials engineering and microfabrication technologies, including organs-on-a-chip and droplet microfluidics. Additionally, this work discusses the application of PDOs in therapy screening to meet diverse requirements and overcome bottlenecks in cancer treatment. Furthermore, this work introduces tools for data processing and analysis of organoids, along with their microenvironment. These tools aim to achieve enhanced readouts. Finally, this work explores the challenges and future perspectives of using PDOs in drug development and personalized screening for cancer patients.

Aqueous humor TGFß and fibrillin-1 in Tsk mice reveal clues to POAG pathogenesis.

Aqueous humor (AH) and blood levels of transforming growth factor ß (TGFß) are elevated in idiopathic primary open angle glaucoma (POAG) representing a disease biomarker of unclear status and function. Tsk mice display a POAG phenotype and harbor a mutation of fibrillin-1, an important regulator of TGFß bioavailability. AH TGFß2 was higher in Tsk than wild-type (WT) mice (by 34%; p = 0.002; ELISA); similarly, AH TGFß2 was higher in human POAG than controls (2.7-fold; p = 0.00005). As in POAG, TGFß1 was elevated in Tsk serum (p = 0.01). Fibrillin-1 was detected in AH from POAG subjects and Tsk mice where both had similar levels relative to controls (p = 0.45). 350 kDa immunoblot bands representing WT full-length fibrillin-1 were present in human and mouse AH. A 418 kDa band representing mutant full-length fibrillin-1 was present only in Tsk mice. Lower molecular weight fibrillin-1 antibody-reactive bands were present in similar patterns in humans and mice. Certain bands (130 and 32 kDa) were elevated only in human POAG and Tsk mice (p ≤ 0.04 relative to controls) indicating discrete isoforms relevant to disease. In addition to sharing a phenotype, Tsk mice and human POAG subjects had common TGFß and fibrillin-1 features in AH and also blood that are pertinent to understanding glaucoma pathogenesis.

Fibronectin, DHPS and SLC3A2 Signaling Cooperate to Control Tumor Spheroid Growth, Subcellular eIF5A1/2 Distribution and CDK4/6 Inhibitor Resistance.

Extracellular matrix (ECM) protein expression/deposition within and stiffening of the breast cancer microenvironment facilitates disease progression and correlates with poor patient survival. However, the mechanisms by which ECM components control tumorigenic behaviors and responses to therapeutic intervention remain poorly understood. Fibronectin (FN) is a major ECM protein controlling multiple processes. In this regard, we previously reported that DHPS-dependent hypusination of eIF5A1/2 is necessary for fibronectin-mediated breast cancer metastasis and epithelial to mesenchymal transition (EMT). Here, we explored the clinical significance of an interactome generated using hypusination pathway components and markers of intratumoral heterogeneity. Solute carrier 3A2 (SLC3A2 or CD98hc) stood out as an indicator of poor overall survival among patients with basal-like breast cancers that express elevated levels of DHPS. We subsequently discovered that blockade of DHPS or SLC3A2 reduced triple negative breast cancer (TNBC) spheroid growth. Interestingly, spheroids stimulated with exogenous fibronectin were less sensitive to inhibition of either DHPS or SLC3A2 - an effect that could be abrogated by dual DHPS/SLC3A2 blockade. We further discovered that a subset of TNBC cells responded to fibronectin by increasing cytoplasmic localization of eIF5A1/2. Notably, these fibronectin-induced subcellular localization phenotypes correlated with a G0/G1 cell cycle arrest. Fibronectin-treated TNBC cells responded to dual DHPS/SLC3A2 blockade by shifting eIF5A1/2 localization back to a nucleus-dominant state, suppressing proliferation and further arresting cells in the G2/M phase of the cell cycle. Finally, we observed that dual DHPS/SLC3A2 inhibition increased the sensitivity of both Rb-negative and -positive TNBC cells to the CDK4/6 inhibitor palbociclib. Taken together, these data identify a previously unrecognized mechanism through which extracellular fibronectin controls cancer cell tumorigenicity by modulating subcellular eIF5A1/2 localization and provides prognostic/therapeutic utility for targeting the cooperative DHPS/SLC3A2 signaling axis to improve breast cancer treatment responses.

Fibrillin-1 mutant mouse captures defining features of human primary open glaucoma including anomalous aqueous humor TGF beta-2.

Primary open angle glaucoma (POAG) features an optic neuropathy, elevated aqueous humor (AH) TGFß2, and major risk factors of central corneal thickness (CCT), increasing age and intraocular pressure (IOP). We examined Tight skin (Tsk) mice to see if mutation of fibrillin-1, a repository for latent TGFß, is associated with characteristics of human POAG. We measured: CCT by ocular coherence tomography (OCT); IOP; retinal ganglion cell (RGC) and optic nerve axon counts by microscopic techniques; visual electrophysiologic scotopic threshold responses (STR) and pattern electroretinogram (PERG); and AH TGFß2 levels and activity by ELISA and MINK epithelial cell-based assays respectively. Tsk mice had open anterior chamber angles and compared with age-matched wild type (WT) mice: 23% thinner CCT (p < 0.003); IOP that was higher (p < 0.0001), more asymmetric (p = 0.047), rose with age (p = 0.04) and had a POAG-like frequency distribution. Tsk mice also had RGCs that were fewer (p < 0.04), declined with age (p = 0.0003) and showed increased apoptosis and glial activity; fewer optic nerve axons (p = 0.02); abnormal axons and glia; reduced STR (p < 0.002) and PERG (p < 0.007) visual responses; and higher AH TGFß2 levels (p = 0.0002) and activity (p = 1E-11) especially with age. Tsk mice showed defining features of POAG, implicating aberrant fibrillin-1 homeostasis as a pathogenic contributor to emergence of a POAG phenotype.

A SNAI2-PEAK1-INHBA stromal axis drives progression and lapatinib resistance in HER2-positive breast cancer by supporting subpopulations of tumor cells positive for antiapoptotic and stress signaling markers.

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Specifically, we identified that the fibroblastic stroma in HER2-positive breast cancer patient tissue stains positive for both nuclear SNAI2 and cytoplasmic PEAK1. Furthermore, mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells in a PEAK1-dependent manner. Analysis of PEAK1-dependent secreted factors from MSCs revealed INHBA/activin-A as a necessary factor in the conditioned media of PEAK1-expressing MSCs that promotes lapatinib resistance. Single-cell CycIF analysis of MSC-breast cancer cell co-cultures identified enrichment of p-Akthigh/p-gH2AXlow, MCL1high/p-gH2AXlow and GRP78high/VIMhigh breast cancer cell subpopulations by the presence of PEAK1-expressing MSCs and lapatinib treatment. Bioinformatic analyses on a PEAK1-centric stroma-tumor cell gene set and follow-up immunostaining of co-cultures predict targeting antiapoptotic and stress pathways as a means to improve targeted therapy responses and patient outcomes in HER2-positive breast cancer and other stroma-rich malignancies. These data provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA stromal cell axis.

Cell surface GRP78 promotes stemness in normal and neoplastic cells.

Reliable approaches to identify stem cell mechanisms that mediate aggressive cancer could have great therapeutic value, based on the growing evidence of embryonic signatures in metastatic cancers. However, how to best identify and target stem-like mechanisms aberrantly acquired by cancer cells has been challenging. We harnessed the power of reprogramming to examine GRP78, a chaperone protein generally restricted to the endoplasmic reticulum in normal tissues, but which is expressed on the cell surface of human embryonic stem cells and many cancer types. We have discovered that (1) cell surface GRP78 (sGRP78) is expressed on iPSCs and is important in reprogramming, (2) sGRP78 promotes cellular functions in both pluripotent and breast cancer cells (3) overexpression of GRP78 in breast cancer cells leads to an induction of a CD24-/CD44+ tumor initiating cell (TIC) population (4) sGRP78+ breast cancer cells are enriched for stemness genes and appear to be a subset of TICs (5) sGRP78+ breast cancer cells show an enhanced ability to seed metastatic organ sites in vivo. These collective findings show that GRP78 has important functions in regulating both pluripotency and oncogenesis, and suggest that sGRP78 marks a stem-like population in breast cancer cells that has increased metastatic potential in vivo.

Secretomes from metastatic breast cancer cells, enriched for a prognostically unfavorable LCN2 axis, induce anti-inflammatory MSC actions and a tumor-supportive premetastatic lung.

Cancer metastasis is responsible for the clear majority of cancer-related deaths. Survival and expansion of cancer cells at secondary sites requires that these premetastatic microenvironments be primed by primary tumor cells and their secreted factors. Efforts to date have been limited by immune-deficient in vivo models and/or the need for finely-tuned analysis time points that reduce contributions from early-disseminating cancer cells. In this regard, we developed a tumor cell-free syngeneic breast cancer model for characterizing tumor cell secretome-mediated reprogramming of premetastatic tissues. We demonstrate that secretomes from metastatic breast cancer cells differentially regulate the lung and brain, promoting a tumor-supportive lung microenvironment with both elevated CD73 expression and decreased TNFα expression. Using in vitro models of CD73-positive mesenchymal stem cells (MSCs) and macrophages/monocytes, we tested whether MSCs can mediate anti-inflammatory effects of metastatic breast cancer cells. Notably, conditioned media from metastatic Py230 cells reprogrammed the secretomes of MSCs toward an anti-inflammatory state. Mining transcriptome data from Py8119 and Py230 cells revealed a lipocalin 2 (LCN2) axis that is selectively expressed in the metastatic Py230 cells, predicts poor breast cancer patient survival and is elevated in circulating serum of mice chronically treated with conditioned media from Py230 cells. Taken together, these results establish the utility of an immune-competent tumor cell-free model for characterizing the mechanisms of breast cancer cell priming of the premetastatic niche, demonstrate that MSCs can mediate the anti-inflammatory effects of metastatic breast cancer cells and substantiate LCN2 as a promising therapeutic target for blocking breast cancer progression.

Identification of myosin II as a cripto binding protein and regulator of cripto function in stem cells and tissue regeneration.

Cripto regulates stem cell function in normal and disease contexts via TGFbeta/activin/nodal, PI3K/Akt, MAPK and Wnt signaling. Still, the molecular mechanisms that govern these pleiotropic functions of Cripto remain poorly understood. We performed an unbiased screen for novel Cripto binding proteins using proteomics-based methods, and identified novel proteins including members of myosin II complexes, the actin cytoskeleton, the cellular stress response, and extracellular exosomes. We report that myosin II, and upstream ROCK1/2 activities are required for localization of Cripto to cytoplasm/membrane domains and its subsequent release into the conditioned media fraction of cultured cells. Functionally, we demonstrate that soluble Cripto (one-eyed pinhead in zebrafish) promotes proliferation in mesenchymal stem cells (MSCs) and stem cell-mediated wound healing in the zebrafish caudal fin model of regeneration. Notably, we demonstrate that both Cripto and myosin II inhibitors attenuated regeneration to a similar degree and in a non-additive manner. Taken together, our data present a novel role for myosin II function in regulating subcellular Cripto localization and function in stem cells and an important regulatory mechanism of tissue regeneration. Importantly, these insights may further the development of context-dependent Cripto agonists and antagonists for therapeutic benefit.

ITGA1 is a pre-malignant biomarker that promotes therapy resistance and metastatic potential in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has single-digit 5-year survival rates at <7%. There is a dire need to improve pre-malignant detection methods and identify new therapeutic targets for abrogating PDAC progression. To this end, we mined our previously published pseudopodium-enriched (PDE) protein/phosphoprotein datasets to identify novel PDAC-specific biomarkers and/or therapeutic targets. We discovered that integrin alpha 1 (ITGA1) is frequently upregulated in pancreatic cancers and associated precursor lesions. Expression of ITGA1-specific collagens within the pancreatic cancer microenvironment significantly correlates with indicators of poor patient prognosis, and depleting ITGA1 from PDAC cells revealed that it is required for collagen-induced tumorigenic potential. Notably, collagen/ITGA1 signaling promotes the survival of ALDH1-positive stem-like cells and cooperates with TGFß to drive gemcitabine resistance. Finally, we report that ITGA1 is required for TGFß/collagen-induced EMT and metastasis. Our data suggest that ITGA1 is a new diagnostic biomarker and target that can be leveraged to improve patient outcomes.

A novel method for RNA extraction from FFPE samples reveals significant differences in biomarker expression between orthotopic and subcutaneous pancreatic cancer patient-derived xenografts.

Next-generation sequencing (NGS) can identify and validate new biomarkers of cancer onset, progression and therapy resistance. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) cancer samples from patients represent a rich resource for linking molecular signatures to clinical data. However, performing NGS on FFPE samples is limited by poor RNA purification methods. To address this hurdle, we developed an improved methodology for extracting high-quality RNA from FFPE samples. By briefly integrating a newly-designed micro-homogenizing (mH) tool with commercially available FFPE RNA extraction protocols, RNA recovery is increased by approximately 3-fold while maintaining standard A260/A280 ratios and RNA quality index (RQI) values. Furthermore, we demonstrate that the mH-purified FFPE RNAs are longer and of higher integrity. Previous studies have suggested that pancreatic ductal adenocarcinoma (PDAC) gene expression signatures vary significantly under in vitro versus in vivo and in vivo subcutaneous versus orthotopic conditions. By using our improved mH-based method, we were able to preserve established expression patterns of KRas-dependency genes within these three unique microenvironments. Finally, expression analysis of novel biomarkers in KRas mutant PDAC samples revealed that PEAK1 decreases and MST1R increases by over 100-fold in orthotopic versus subcutaneous microenvironments. Interestingly, however, only PEAK1 levels remain elevated in orthotopically grown KRas wild-type PDAC cells. These results demonstrate the critical nature of the orthotopic tumor microenvironment when evaluating the clinical relevance of new biomarkers in cells or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to enhance and expand future FFPE-RNA-NGS cancer biomarker studies.

Ascending the PEAK1 toward targeting TGFß during cancer progression: Recent advances and future perspectives.

Cancer is the second leading cause of death in the United States. Mortality in patients with solid, epithelial-derived tumors strongly correlates with disease stage and the systemic metastatic load. In such cancers, notable morphological and molecular changes have been attributed to cells as they pass through a continuum of epithelial-mesenchymal transition (EMT) states and many of these changes are essential for metastasis. While cancer metastasis is a complex cascade that is regulated by cell-autonomous and microenvironmental influences, it is well-accepted that understanding and controlling metastatic disease is a viable method for increasing patient survival. In the past 5 years, the novel non-receptor tyrosine kinase PEAK1 has surfaced as a central regulator of tumor progression and metastasis in the context of solid, epithelial cancers. Here, we review this literature with a special focus on our recent work demonstrating that PEAK1 mediates non-canonical pro-tumorigenic TGFß signaling and is an intracellular control point between tumor cells and their extracellular microenvironment. We conclude with a brief discussion of potential applications derived from our current understanding of PEAK1 biology.

Identification of a PEAK1/ZEB1 signaling axis during TGFß/fibronectin-induced EMT in breast cancer.

Transforming Growth Factor beta (TGFß) is the archetypal member of the TGFß superfamily of ligands and has pleiotropic functions during normal development, adult tissue homeostasis and pathophysiological processes such as cancer. In epithelial cancers TGFß signaling can either suppress tumor growth or promote metastasis via the induction of a well-characterized epithelial-mesenchymal transition (EMT) program. We recently reported that PEAK1 kinase mediates signaling cross talk between TGFß receptors and integrin/Src/MAPK pathways and functions as a critical molecular regulator of TGFß-induced breast cancer cell proliferation, migration, EMT and metastasis. Here, we examined the breast cancer cell contexts in which TGFß induces both EMT and PEAK1, and discovered this event to be unique to oncogene-transformed mammary epithelial cells and triple-negative breast cancer cells. Using the Cancer BioPortal database, we identified PEAK1 co-expressors across multiple malignancies that are also common to the TGFß response gene signature (TBRS). We then used the ScanSite database to identify predicted protein-protein binding partners of PEAK1 and the PEAK1-TBRS co-expressors. Analysis of the Cytoscape interactome and Babelomics-derived gene ontologies for a novel gene set including PEAK1, CRK, ZEB1, IL11 and COL4A1 enabled us to hypothesize that PEAK1 may be regulating TGFß-induced EMT via its interaction with or regulation of these other genes. In this regard, we have demonstrated that PEAK1 is necessary for TGFß to induce ZEB1-mediated EMT in the context of fibronectin/ITGB3 activation. These studies and future mechanistic studies will pave the way toward identifying the context in which TGFß blockade may significantly improve breast cancer patient outcomes.

PEAK1 Acts as a Molecular Switch to Regulate Context-Dependent TGFß Responses in Breast Cancer.

Transforming Growth Factor ß (TGFß) has dual functions as both a tumor suppressor and a promoter of cancer progression within the tumor microenvironment, but the molecular mechanisms by which TGFß signaling switches between these outcomes and the contexts in which this switch occurs remain to be fully elucidated. We previously identified PEAK1 as a new non-receptor tyrosine kinase that associates with the cytoskeleton, and facilitates signaling of HER2/Src complexes. We also showed PEAK1 functions downstream of KRas to promote tumor growth, metastasis and therapy resistance using preclinical in vivo models of human tumor progression. In the current study, we analyzed PEAK1 expression in human breast cancer samples and found PEAK1 levels correlate with mesenchymal gene expression, poor cellular differentiation and disease relapse. At the cellular level, we also observed that PEAK1 expression was highest in mesenchymal breast cancer cells, correlated with migration potential and increased in response to TGFß-induced epithelial-mesenchymal transition (EMT). Thus, we sought to evaluate the role of PEAK1 in the switching of TGFß from a tumor suppressing to tumor promoting factor. Notably, we discovered that high PEAK1 expression causes TGFß to lose its anti-proliferative effects, and potentiates TGFß-induced proliferation, EMT, cell migration and tumor metastasis in a fibronectin-dependent fashion. In the presence of fibronectin, PEAK1 caused a switching of TGFß signaling from its canonical Smad2/3 pathway to non-canonical Src and MAPK signaling. This report is the first to provide evidence that PEAK1 mediates signaling cross talk between TGFß receptors and integrin/Src/MAPK pathways and that PEAK1 is an important molecular regulator of TGFß-induced tumor progression and metastasis in breast cancer. Finally, PEAK1 overexpression/upregulation cooperates with TGFß to reduce breast cancer sensitivity to Src kinase inhibition. These findings provide a rational basis to develop therapeutic agents to target PEAK1 expression/function or upstream/downstream pathways to abrogate breast cancer progression.

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.

CRIPTO/GRP78 signaling maintains fetal and adult mammary stem cells ex vivo.

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.

Proteomic and biochemical methods to study the cytoskeletome.

The cytoskeleton is fundamental to many cellular functions including cell proliferation, differentiation, adhesion, and migration. It is composed of actin, microtubules, intermediate filaments, and integrin cell surface receptors, which form focal adhesions with the extracellular matrix. These elements are highly integrated in the cell providing a rigid network of interconnected cables and protein scaffolds, which generate force and mechanical support to maintain cell shape and movement. However, the cytoskeleton is not just a simple compilation of static filaments that dictate cell adhesion and morphology-it is highly plastic with the inherent ability to assemble and disassemble in response to diverse and complex cellular cues. Thus, biochemical and proteomic methods are needed to better understand the cytoskeleton network and its dynamic signal transduction functions in health and disease. This chapter describes methods for the biochemical enrichment and mass spectrometry-based proteomic analyses of the cytoskeletome. We also detail how these methods can be used to investigate the cytoskeletome of migrating cells and their purified pseudopodia membrane projections.