Increased Tumor Cell Invasion Research Articles

∆Np63α inhibits Rac1 activation and cancer cell invasion through suppression of PREX1

ΔNp63α, a member of the p53 family of transcription factors, plays a critical role in maintaining the proliferative potential of stem cells in the stratified epithelium. Although ΔNp63α is considered an oncogene and is frequently overexpressed in squamous cell carcinoma, loss of ΔNp63α expression is associated with increased tumor cell invasion and metastasis. We recently identified a ΔNp63α/miR-320a/PKCγ signaling axis that regulates cancer cell invasion by inhibiting phosphorylation of the small GTPase Rac1, a master switch of cell motility that positively regulates cell invasion in multiple human cancers. In this study, we identified a novel mechanism by which ΔNp63α negatively regulates Rac1 activity, by inhibiting the expression of the Rac-specific Guanine Exchange Factor PREX1. ΔNp63α knockdown in multiple squamous cell carcinoma cell lines leads to increased Rac1 activation, which is abrogated by treatment with the Rac1 inhibitor NSC23766. Furthermore, ΔNp63α negatively regulates PREX1 transcript and protein levels. Using a Rac-GEF activation assay, we also showed that ΔNp63α reduces the levels of active PREX1. The inhibition of the PREX1-Rac1 signaling axis by ΔNp63α leads to impaired cell invasion, thus establishing the functional relevance of this link. Our results elucidated a novel molecular mechanism by which ΔNp63α negatively affects cancer cell invasion and identifies the ΔNp63α/Rac1 axis as a potential target for metastasis.

GNAS mutations suppress cell invasion by activating MEG3 in growth hormone-secreting pituitary adenoma.

Approximately 30%-40% of growth hormone-secreting pituitary adenomas (GHPAs) harbor somatic activating mutations in GNAS (α subunit of stimulatory G protein). Mutations in GNAS are associated with clinical features of smaller and less invasive tumors. However, the role of GNAS mutations in the invasiveness of GHPAs is unclear. GNAS mutations were detected in GHPAs using a standard polymerase chain reaction (PCR) sequencing procedure. The expression of mutation-associated maternally expressed gene 3 (MEG3) was evaluated with RT-qPCR. MEG3 was manipulated in GH3 cells using a lentiviral expression system. Cell invasion ability was measured using a Transwell assay, and epithelial-mesenchymal transition (EMT)-associated proteins were quantified by immunofluorescence and western blotting. Finally, a tumor cell xenograft mouse model was used to verify the effect of MEG3 on tumor growth and invasiveness. The invasiveness of GHPAs was significantly decreased in mice with mutated GNAS compared with that in mice with wild-type GNAS. Consistently, the invasiveness of mutant GNAS-expressing GH3 cells decreased. MEG3 is uniquely expressed at high levels in GHPAs harboring mutated GNAS. Accordingly, MEG3 upregulation inhibited tumor cell invasion, and conversely, MEG3 downregulation increased tumor cell invasion. Mechanistically, GNAS mutations inhibit EMT in GHPAs. MEG3 in mutated GNAS cells prevented cell invasion through the inactivation of the Wnt/β-catenin signaling pathway, which was further validated in vivo. Our data suggest that GNAS mutations may suppress cell invasion in GHPAs by regulating EMT through the activation of the MEG3/Wnt/β-catenin signaling pathway.

DDR2-regulated arginase activity in ovarian cancer-associated fibroblasts promotes collagen production and tumor progression

Ovarian cancer has poor survival outcomes particularly for advanced stage, metastatic disease. Metastasis is promoted by interactions of stromal cells, such as cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME), with tumor cells. CAFs play a key role in tumor progression by remodeling the TME and extracellular matrix (ECM) to result in a more permissive environment for tumor progression. It has been shown that fibroblasts, in particular myofibroblasts, utilize metabolism to support ECM remodeling. However, the intricate mechanisms by which CAFs support collagen production and tumor progression are poorly understood. In this study, we show that the fibrillar collagen receptor, Discoidin Domain Receptor 2 (DDR2), promotes collagen production in human and mouse omental CAFs through arginase activity. CAFs with high DDR2 or arginase promote tumor colonization in the omentum. In addition, DDR2-depleted CAFs had decreased ornithine levels leading to decreased collagen production and polyamine levels compared to WT control CAFs. Tumor cell invasion was decreased in the presence CAF conditioned media (CM) depleted of DDR2 or arginase-1, and this invasion defect was rescued in the presence of CM from DDR2-depleted CAFs that constitutively overexpressed arginase-1. Similarly, the addition of exogenous polyamines to CM from DDR2-depleted CAFs led to increased tumor cell invasion. We detected SNAI1 protein at the promoter region of the arginase-1 gene, and DDR2-depleted CAFs had decreased levels of SNAI1 protein at the arginase-1 promoter region. Furthermore, high stromal arginase-1 expression correlated with poor survival in ovarian cancer patients. These findings highlight how DDR2 regulates collagen production by CAFs in the tumor microenvironment by controlling the transcription of arginase-1, and CAFs are a major source of arginase activity and L-arginine metabolites in ovarian cancer models.

Irradiated Mammary Spheroids Elucidate Mechanisms of Macrophage-Mediated Breast Cancer Recurrence.

While most patients with triple negative breast cancer receive radiation therapy to improve outcomes, a significant subset of patients continue to experience recurrence. Macrophage infiltration into radiation-damaged sites has been shown to promote breast cancer recurrence in pre-clinical models. However, the mechanisms that drive recurrence are unknown. Here, we developed a novel spheroid model to evaluate macrophage-mediated tumor cell recruitment. We characterized infiltrating macrophage phenotypes into irradiated mouse mammary tissue via flow cytometry. We then engineered a spheroid model of radiation damage with primary fibroblasts, macrophages, and 4T1 mouse mammary carcinoma cells using in vivo macrophage infiltration results to inform our model. We analyzed 4T1 infiltration into spheroids when co-cultured with biologically relevant ratios of pro-healing M2:pro-inflammatory M1 macrophages. Finally, we quantified interleukin 6 (IL-6) secretion associated with conditions favorable to tumor cell infiltration, and we directly evaluated the impact of IL-6 on tumor cell invasiveness in vitro and in vivo. In our in vivo model, we observed a significant increase in M2 macrophages in mouse mammary glands 10days post-irradiation. We determined that tumor cell motility toward irradiated spheroids was enhanced in the presence of a 2:1 ratio of M2:M1 macrophages. We also measured a significant increase in IL-6 secretion after irradiation both in vivo and in our model. This secretion increased tumor cell invasiveness, and tumor cell invasion and recruitment were mitigated by neutralizing IL-6. Our work suggests that interactions between infiltrating macrophages and damaged stromal cells facilitate breast cancer recurrence through IL-6 signaling. The online version contains supplementary material available at 10.1007/s12195-023-00775-x.

Overexpression of RAB31 in gastric cancer is associated with released exosomes and increased tumor cell invasion and metastasis.

Gastric cancer (GC) is one of most common cancers worldwide. Several studies have suggested that Rab31 functions as a membrane vesicle transport regulator; however, the mechanism by which RAB31 regulates exosome secretion and promotes metastasis remains to be clarified. We examined the expression of RAB31 protein and mRNA in GC tissue samples via immunohistochemistry and reverse transcription-polymerase chain reaction assays, respectively. We elucidated the function of RAB31 in GC cells by constructing a cell model and a pulmonary metastatic model of GC with overexpression of RAB31. Protein mass spectrometry was used to identify the exosomal protein. RAB31 expression increased at both the protein and mRNA levels with the development of GC. Cells overexpressing RAB31 showed an enhanced ability to migrate in both the in vitro cell model and the pulmonary metastatic model of GC. Exosome nanoparticle tracking analysis and electron microscopy revealed that the both the number and size of the exosomes secreted by GC cells were reduced when RAB31 expression was depleted. Injection of exosomes derived from RAB31 overexpressing cells promoted pulmonary metastasis in vivo. Analysis of the exosomal proteins revealed that PSMA1 was overexpressed in GC tissue in accordance with RAB31 expression. PSMA1 overexpression was highly associated with poor prognosis of GC patients. Our findings revealed a key role for RAB31 in GC metastasis through regulation of exosome secretion.

Advanced Cell Culture Models Illuminate the Interplay between Mammary Tumor Cells and Activated Fibroblasts.

The interaction between tumor cells and activated fibroblasts determines malignant features of desmoplastic carcinomas such as rapid growth, progression towards a metastatic phenotype, and resistance to chemotherapy. On one hand, tumor cells can activate normal fibroblasts and even reprogram them into CAFs through complex mechanisms that also involve soluble factors. Among them, transforming growth factor beta (TGF-β) and Platelet-Derived Growth Factor (PDGF) have an established role in the acquisition of pro-tumorigenic phenotypes by fibroblasts. On the other hand, activated fibroblasts release Interleukin-6 (IL-6), which increases tumor-cell invasiveness and chemoresistance. However, the interplay between breast cancer cells and fibroblasts, as well as the modes of action of TGF-β, PDGF, and IL-6, are difficult to investigate in vivo. Here, we validated the usage of advanced cell culture models as tools to study the interplay between mammary tumor cells and fibroblasts, taking mouse and human triple-negative tumor cells and fibroblasts as a case study. We employed two different settings, one permitting only paracrine signaling, the other both paracrine and cell-contact-based signaling. These co-culture systems allowed us to unmask how TGF-β, PDGF and IL-6 mediate the interplay between mammary tumor cells and fibroblasts. We found that the fibroblasts underwent activation induced by the TGF-β and the PDGF produced by the tumor cells, which increased their proliferation and IL-6 secretion. The IL-6 secreted by activated fibroblasts enhanced tumor-cell proliferation and chemoresistance. These results show that these breast cancer avatars possess an unexpected high level of complexity, which resembles that observed in vivo. As such, advanced co-cultures provide a pathologically relevant tractable system to study the role of the TME in breast cancer progression with a reductionist approach.

Abstract P6-14-18: Effect of 3q oncogene SEC62 on migration and proliferation of triple-negative breast cancer cells

Abstract Background Chromosome 3q26 amplifications represent a frequent alteration in various cancer entities including breast cancer. SEC62 – a 3q26 encoded gene – was identified as a potential onco- and tumor-driver gene for the pathogenesis of breast cancer. Although the precise physiological function of the respective protein Sec62 is not completely understood, Sec62 seems to induce an increased stress tolerance, enhanced cell migration and invasive potential in SEC62 overexpressing cells. SEC62 overexpressing breast cancer patients have been shown to have a higher rate of lymph node metastasis and poorer overall prognosis. Hence, we aimed to further evaluate the effect of Sec62 for triple-negative breast cancer by targeting cell migration and proliferation of triple-negative cell lines altered in their SEC62 expression. Objectives The aim of this study was to investigate the role of Sec62 for triple-negative breast cancer in cell culture using functional analyzes comparing cell migration and cell proliferation in triple-negative cell lines using the effects of siRNA-mediated Sec62 depletion in vitro. Material& Methods In this study, three SEC62 gene silencing experiments each with two different siRNAs directed against the SEC62 mRNA were carried out in comparison to a control siRNA in combination with cell proliferation and cell migration tests plus Western blots for the triple-negative breast cancer cell line CAL120 in order to determine the suspected causal relationship between SEC62- overexpression and an increased cell migration and thus an enhanced invasion potential of the cancer cells. The cell proliferation was examined in real time in the 96 well xCELLigence system and the cell migration using Fluoroblock without matrigel using fluorescence microscopy. Results In cell migration assays, the median migrated cell number in the control siRNA group was 263 (241-279), the cell number in the groups of the two siRNAs directed against the Sec62 mRNA was 72 (70-149) and 178 (96-276) (p < 0.01). In cell proliferation assays, the median cell index in the control siRNA group was 7.5 (7.4-7.6), while it was 7.4 (7.3-7 .5) and 7.6 (7.5-7.7) (p=0.37). In the first analyzes of the medians of the three gene silencing experiments of the cell line CAL120, the expected negative effect of the SEC62-siRNA on cell migration is confirmed, while, as expected, the effect on cell proliferation remains unchanged with decreasing Sec62 content. Conclusion In this in vitro study using cell migration and cell proliferation assays we found a correlation between SEC62 overexpression and increased cell migration. This implies a potential association between Sec62 and an increased tumor cell invasion in triple-negative breast cancer. Citation Format: Julia SM Zimmermann, Annika Cullmann, Askin Kaya, Merle Doerk, Maximilian Linxweiler, Marc P Radosa, Sven Lang, Martin Jung, Erich F Solomayer, Julia C Radosa. Effect of 3q oncogene SEC62 on migration and proliferation of triple-negative breast cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-18.

Germline gain-of-function MMP11 variant results in an aggressive form of colorectal cancer.

Matrix metalloproteinase-11 (MMP11) is an enzyme with proteolytic activity against matrix and nonmatrix proteins. Although most MMPs are secreted as inactive proenzymes and are later activated extracellularly, MMP11 is activated intracellularly by furin within the constitutive secretory pathway. It is a key factor in physiological tissue remodeling and its alteration may play an important role in the progression of epithelial malignancies and other diseases. TCGA colon and colorectal adenocarcinoma data showed that upregulation of MMP11 expression correlates with tumorigenesis and malignancy. Here, we provide evidence that a germline variant in the MMP11 gene (NM_005940: c.232C>T; p.(Pro78Ser)), identified by whole exome sequencing, can increase the tumorigenic properties of colorectal cancer (CRC) cells. P78S is located in the prodomain region, which is responsible for blocking MMP11's protease activity. This variant was detected in the proband and all the cancer-affected family members analyzed, while it was not detected in healthy relatives. In silico analyses predict that P78S could have an impact on the activation of the enzyme. Furthermore, our in vitro analyses show that the expression of P78S in HCT116 cells increases tumor cell invasion and proliferation. In summary, our results show that this variant could modify the structure of the MMP11 prodomain, producing a premature or uncontrolled activation of the enzyme that may contribute to an early CRC onset in these patients. The study of this gene in other CRC cases will provide further information about its role in CRC development, which might improve patient treatment in the future.

Identification and Characterization of Tunneling Nanotubes Involved in Human Mast Cell FcεRI-Mediated Apoptosis of Cancer Cells.

Simple SummaryMast cells (MCs) are ubiquitously found in most tissues and in and around tumors. Their role in cancer pathogenesis remains an open area of investigation, and their interactions with tumor cells has not been explored. Here, a novel mechanism of communication between human MCs and tumor cells involving tunneling nanotubes (TnT) and other membrane structures is described. The formation of these communication structures is dependent on MC receptors interacting with tumor antigens through tumor-specific immunoglobulins and results in tumor-killing mediators from MC entering the tumor cells. This mechanism underlying the MC killing of tumor cells has important implications in understanding cancer pathogenesis.Mast cells (MCs) are found in practically all tissues where they participate in innate and adaptive immune responses. They are also found in and around tumors, yet their interactions with cancer cells and the resulting impact on cancer cell growth and metastasis are not well understood. In this study, we examined a novel mechanism of IgE-FcεRI-mediated, intercellular communication between human adipose-derived mast cells (ADMC) and cancer cells. The formation of heterotypic tunneling nanotubes (TnT) and membrane structures between MCs and tumor cells in vitro was examined using microscopy and a diverse array of molecule-specific indicator dyes. We show that several MC-specific structures are dependent on the specific interactions between human tumor IgE-sensitized MCs and antigens on the tumor cell surface. The formation of TnT, membrane blebs and other MC-specific structures paralleled FcεRI-degranulation occurring within 30 min and persisting for up to 24 h. The TnT-specific adhesion of FcεRI-activated MCs to tumor cells was characterized by the transport of the MC granule content into the tumor cells, including tryptase and TNF-α. This interaction led to apoptosis of the tumor cells, which differs from previous studies examining tissue cells within the cancer microenvironment. The formation of heterotypic TnT results in stimulation of an invasive tumor cell phenotype and increased tumor cell invasion and chemoresistance of the cancer cells. These studies describe a heretofore-unrecognized mechanism underlying IgE-mediated interactions and FcεRI-activated MC-mediated killing of tumor cells through the formation of TnT.

Tubulin Carboxypeptidase Activity Promotes Focal Gelatin Degradation in Breast Tumor Cells and Induces Apoptosis in Breast Epithelial Cells That Is Overcome by Oncogenic Signaling.

Simple SummaryThe recent discovery of the genetic identity of the tubulin carboxypeptidase (TCP) provides a unique opportunity to study the role of the detyrosination of α-tubulin (deTyr-Tub), as performed by the TCP, in breast epithelial cells and breast cancer cells. Previous research has shown that elevated deTyr-Tub conveys a poor prognosis in breast cancer and is upregulated in a coordinated manner at the invasive margin of patient tumor samples. Using TCP expression constructs, we have shown that increased deTyr-Tub promotes apoptosis in normal breast epithelial cells, that does not occur in the same cells with an oncogenic KRas mutation or Bcl-2/Bcl-xL overexpression. Furthermore, the addition of the TCP to the breast cancer cell lines MDA-MB-231 and Hs578t, also harboring Ras mutations, leads to increased focal gelatin degradation.Post-translational modifications (PTMs) of the microtubule network impart differential functions across normal cell types and their cancerous counterparts. The removal of the C-terminal tyrosine of α-tubulin (deTyr-Tub) as performed by the tubulin carboxypeptidase (TCP) is of particular interest in breast epithelial and breast cancer cells. The recent discovery of the genetic identity of the TCP to be a vasohibin (VASH1/2) coupled with a small vasohibin-binding protein (SVBP) allows for the functional effect of this tubulin PTM to be directly tested for the first time. Our studies revealed the immortalized breast epithelial cell line MCF10A undergoes apoptosis following transfection with TCP constructs, but the addition of oncogenic KRas or Bcl-2/Bcl-xL overexpression prevents subsequent apoptotic induction in the MCF10A background. Functionally, an increase in deTyr-Tub via TCP transfection in MDA-MB-231 and Hs578t breast cancer cells leads to enhanced focal gelatin degradation. Given the elevated deTyr-Tub at invasive tumor fronts and the correlation with poor breast cancer survival, these new discoveries help clarify how the TCP synergizes with oncogene activation, increases focal gelatin degradation, and may correspond to increased tumor cell invasion. These connections could inform more specific microtubule-directed therapies to target deTyr-tubulin.

Nuclear Envelope Disruptions Induce DNA Damage and Invasiveness.

Nuclear envelope (NE) disruptions induce DNA damage which increases tumor cell invasion.

Rab11-FIP1 mediates epithelial-mesenchymal transition and invasion in esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) is the most common subtype of esophageal cancer worldwide. The most commonly mutated gene in ESCC is TP53. Using a combinatorial genetic and carcinogenic approach, we generate a novel mouse model of ESCC expressing either mutant or null p53 and show that mutant p53 exhibits enhanced tumorigenic properties and displays a distinct genomic profile. Through RNA-seq analysis, we identify several endocytic recycling genes, including Rab Coupling Protein (Rab11-FIP1), which are significantly downregulated in mutant p53 tumor cells. In 3-dimensional (3D) organoid models, genetic knockdown of Rab11-FIP1 results in increased organoid size. Loss of Rab11-FIP1 increases tumor cell invasion in part through mutant p53 but also in an independent manner. Furthermore, loss of Rab11-FIP1 in human ESCC cell lines decreases E-cadherin expression and increases mesenchymal lineage-specific markers, suggesting induction of epithelial-mesenchymal transition (EMT). Rab11-FIP1 regulates EMT through direct inhibition of Zeb1, a key EMT transcriptional factor. Our novel findings reveal that Rab11-FIP1 regulates organoid formation, tumor cell invasion, and EMT.

A role for GLUT3 in glioblastoma cell invasion that is not recapitulated by GLUT1

ABSTRACT The multifaceted roles of metabolism in invasion have been investigated across many cancers. The brain tumor glioblastoma (GBM) is a highly invasive and metabolically plastic tumor with an inevitable recurrence. The neuronal glucose transporter 3 (GLUT3) was previously reported to correlate with poor glioma patient survival and be upregulated in GBM cells to promote therapeutic resistance and survival under restricted glucose conditions. It has been suggested that the increased glucose uptake mediated by GLUT3 elevation promotes survival of circulating tumor cells to facilitate metastasis. Here we suggest a more direct role for GLUT3 in promoting invasion that is not dependent upon changes in cell survival or metabolism. Analysis of glioma datasets demonstrated that GLUT3, but not GLUT1, expression was elevated in invasive disease. In human xenograft derived GBM cells, GLUT3, but not GLUT1, elevation significantly increased invasion in transwell assays, but not growth or migration. Further, there were no changes in glycolytic metabolism that correlated with invasive phenotypes. We identified the GLUT3 C-terminus as mediating invasion: substituting the C-terminus of GLUT1 for that of GLUT3 reduced invasion. RNA-seq analysis indicated changes in extracellular matrix organization in GLUT3 overexpressing cells, including upregulation of osteopontin. Together, our data suggest a role for GLUT3 in increasing tumor cell invasion that is not recapitulated by GLUT1, is separate from its role in metabolism and survival as a glucose transporter, and is likely broadly applicable since GLUT3 expression correlates with metastasis in many solid tumors.

Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion.

Cluster of differentiation (CD) 166 or activated leukocyte cell adhesion molecule (ALCAM) is a transmembrane molecule known to be an intercellular adhesion factor. The expression and function of ALCAM in medulloblastoma (MB), a pediatric brain tumor with highly advanced molecular genetics, remains unclear. Therefore, this study aimed to clarify the significance and functional role of ALCAM expression in MB. ALCAM expression in 45 patients with MB was evaluated by immunohistochemical analysis of formalin-fixed paraffin-embedded clinical specimens and the relationship between ALCAM expression and pathological type/molecular subgroup, such as WNT, SHH, Group 3, and Group 4, was examined. Eight ALCAM positive (18%), seven partially positive (16%), and 30 negative (67%) cases were detected. All seven cases of the WNT molecular subgroup were ALCAM positive and ALCAM expression strongly correlated with this subgroup (P < 0.0001). In addition, functional studies using MB cell lines revealed ALCAM expression affected proliferation and migration as a positive regulator in vitro. However, ALCAM silencing did not affect survival or the formation of leptomeningeal dissemination in an orthotopic mouse model, but did induce a malignant phenotype with increased tumor cell invasion at the dissemination sites (P = 0.0029). In conclusion, our results revealed that ALCAM exhibited highly specific expression in the WNT subgroup of MB. Furthermore, we demonstrated that the cell kinetics of MB cell lines can be altered by the expression of ALCAM.

A TRACER culture invasion assay to probe the impact of cancer associated fibroblasts on head and neck squamous cell carcinoma cell invasiveness.

Cancer associated fibroblasts (CAFs) are a major cellular component of the tumour stroma and have been shown to promote tumour cell invasion and disease progression. CAF-cancer cell interactions are bi-directional and occur via both soluble factor dependent and extracellular matrix (ECM) remodelling mechanisms, which are incompletely understood. Previously we developed the Tissue Roll for Analysis of Cellular Environment and Response (TRACER), a novel stacked paper tumour model in which cells embedded in a hydrogel are infiltrated into a porous cellulose scaffold that is then rolled around an aluminum core to generate a multi-layered 3D tissue. Here, we use the TRACER platform to explore the impact of CAFs derived from three different patients on the invasion of two head and neck squamous cell carcinoma (HNSCC) cell lines (CAL33 and FaDu). We find that co-culture with CAFs enhances HNSCC tumour cell invasion into an acellular collagen layer in TRACER and this enhanced migration occurs independently of proliferation. We show that CAF-enhanced invasion of CAL33 cells is driven by a soluble factor independent mechanism, likely involving CAF mediated ECM remodelling via matrix metalloprotenases (MMPs). Furthermore, we find that CAF-enhanced tumour cell invasion is dependent on the spatial pattern of collagen density within the culture. Our results highlight the utility of the co-culture TRACER platform to explore soluble factor independent interactions between CAFs and tumour cells that drive increased tumour cell invasion.

Functional significance of U2AF1 S34F mutations in lung adenocarcinomas

The functional role of U2AF1 mutations in lung adenocarcinomas (LUADs) remains incompletely understood. Here, we report a significant co-occurrence of U2AF1 S34F mutations with ROS1 translocations in LUADs. To characterize this interaction, we profiled effects of S34F on the transcriptome-wide distribution of RNA binding and alternative splicing in cells harboring the ROS1 translocation. Compared to its wild-type counterpart, U2AF1 S34F preferentially binds and modulates splicing of introns containing CAG trinucleotides at their 3′ splice junctions. The presence of S34F caused a shift in cross-linking at 3′ splice sites, which was significantly associated with alternative splicing of skipped exons. U2AF1 S34F induced expression of genes involved in the epithelial-mesenchymal transition (EMT) and increased tumor cell invasion. Finally, S34F increased splicing of the long over the short SLC34A2-ROS1 isoform, which was also associated with enhanced invasiveness. Taken together, our results suggest a mechanistic interaction between mutant U2AF1 and ROS1 in LUAD.

Overexpression of DCLK1-AL Increases Tumor Cell Invasion, Drug Resistance, and KRAS Activation and Can Be Targeted to Inhibit Tumorigenesis in Pancreatic Cancer.

Oncogenic KRAS mutation plays a key role in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis with nearly 95% of PDAC harboring mutation-activated KRAS, which has been considered an undruggable target. Doublecortin-like kinase 1 (DCLK1) is often overexpressed in pancreatic cancer, and recent studies indicate that DCLK1+ PDAC cells can initiate pancreatic tumorigenesis. In this study, we investigate whether overexpressing DCLK1 activates RAS and promotes tumorigenesis, metastasis, and drug resistance. Human pancreatic cancer cells (AsPC-1 and MiaPaCa-2) were infected with lentivirus and selected to create stable DCLK1 isoform 2 (alpha-long, AL) overexpressing lines. The invasive potential of these cells relative to vector control was compared using Matrigel coated transwell assay. KRAS activation and interaction were determined by a pull-down assay and coimmunoprecipitation. Gemcitabine, mTOR (Everolimus), PI3K (LY-294002), and BCL-2 (ABT-199) inhibitors were used to evaluate drug resistance downstream of KRAS activation. Immunostaining of a PDAC tissue microarray was performed to detect DCLK1 alpha- and beta-long expression. Analysis of gene expression in human PDAC was performed using the TCGA PAAD dataset. The effects of targeting DCLK1 were studied using xenograft and Pdx1CreKrasG12DTrp53R172H/+ (KPC) mouse models. Overexpression of DCLK1-AL drives a more than 2-fold increase in invasion and drug resistance and increased the activation of KRAS. Evidence from TCGA PAAD demonstrated that human PDACs expressing high levels of DCLK1 correlate with activated PI3K/AKT/MTOR-pathway signaling suggesting greater KRAS activity. High DCLK1 expression in normal adjacent tissue of PDAC correlated with poor survival and anti-DCLK1 mAb inhibited pancreatic tumor growth in vivo in mouse models.

Abstract 1951: Increased glioma collagen is associated with greater blood flow and peri-tumoral fluid flux, but less infiltration

Abstract Introduction: Composition of the glioma microenvironment (TME) determines tumor growth rate, invasion, metastasis and resistance to treatment. Compared to normal brain, tumor extracellular matrix (ECM) has a higher concentration of structural proteins including collagen, laminin, tenascin, and vinculin. We compared collagen expression in two rat models of glioma, human U251 grown in immune-compromised rats and 9L in syngeneic Fischer-344 rats, representing primary and recurrent glioma features, respectively. Experimental procedures: The U251 and 9L cells were implanted in the right brain hemisphere of female RNU rats (n=10) and Fischer-344 rats (n=7), respectively. Rats were imaged 2 to 3 weeks after implantation by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Tumor size, blood flow, apparent diffusion coefficient (ADC), blood-to-tumor forward volumetric transfer constant (Ktrans), peri-tumoral contrast flux (Flux), hydraulic conductivity (K), and extracellular volume fraction (VD) in the tumor (VD-tumor), tumor rim (VD-rim) and its periphery (VD-peri) along with tumor interstitial fluid pressure (TIFP) were measured. Immediately after imaging, brains were processed for paraffin embedding and histopathology. Brain sections (6 µm) containing the tumor were stained for collagen using Picrosirius red and adjacent brain sections with hematoxylin and eosin (H&amp;E). MRI data were compared by t-tests and significance inferred at p≤0.05. Digital images of Picrosirius red staining were analyzed using ImageJ for collagen expression expressed as % fraction of total tumor area. It was compared to the MRI biomarkers, and to the patterns of tumor cell dispersion into normal brain observed on H&amp;E stained images. Results: Tumor diameters averaged 4 mm for both models at the time of imaging. The U251 tumors showed greater Flux (p=0.03), higher blood flow (p=0.02), smaller VD_tumor (p=0.01) and VD_rim (p=0.02) values than the 9L tumors. TIFP also tended to be higher in the U251 model (p=0.06). Collagen expression in U251 tumors was significantly higher than in 9L tumors (p=0.002). On H&amp;E stained sections, the U251 tumors showed a well delineated tumor margin with very few cells invading the host tissue. In contrast, the 9L tumors showed cancer cells infiltrating singly and in clusters, along white matter tracts as well as in perivascular spaces. Conclusions: Increased collagen content was associated with elevated blood flow, Flux and TIFP, but less tumor cell infiltration in the U251 glioma. Observations of low collagen content in conjunction with increased tumor cell invasion in the 9L model suggest that treatment with collagenase, while increasing drug penetration may also make the tumors more infiltrative. A closer examination of TME to determine the composition of ECM proteins that regulate glioma aggressiveness and modulate its response to treatments is warranted. Note: This abstract was not presented at the meeting. Citation Format: Tavarekere N. Nagaraja, Rasha Elmghirbi, Stephen L. Brown, Ian Y. Lee, Glauber Cabral, Swayamprava Panda, Robert A. Knight, James R. Ewing. Increased glioma collagen is associated with greater blood flow and peri-tumoral fluid flux, but less infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1951.

Abstract LB-110: Proteasome addiction a new therapeutic opportunity to treat PTEN-deficient brain tumors

Abstract One of the most common genetic alterations in glioblastoma (30-40%) occurs in the tumor suppressor gene PTEN (phosphatase and tensin homolog), where loss of function has been mechanistically linked to increased tumor cell invasion, and to a lack of radio- and chemo-therapy response. To identify new drug compounds that target PTEN-deficient brain tumors we performed a high throughput drug screening using patient-derived GBM spheres and found that PTEN-deficient samples were highly sensitive to proteasome inhibitors. We confirmed PTEN dependency to proteasome inhibition by genetically overexpressing or deleting PTEN in GBM cells and established that the drug inhibition response relied on PTEN enzymatic activity. Additionally, proteasome inhibition specifically suppressed tumor growth and increased survival of mice orthotopically engrafted with human PTEN-null glioblastoma cancer stem cells. Mechanistically, we determined that PTEN-deficient cells were more sensitive to proteasome inhibition due to an increase in protein synthesis rate and loss of autophagy activity associated with activation of the PI3K/mTOR pathway. Finally, integrated in silico analysis of the Samsung Medical Center dataset corroborated that samples with PTEN/EGFR/PI3K alterations were significantly more sensitive to Carfilzomib, an FDA-approved proteasome inhibitor. This study reveals that PTEN-deficient cells are "proteasome addictive" and opens a new therapeutic opportunity to treat glioblastoma PTEN-deficient patients. Citation Format: Jorge A. Benitez, Darren Finlay, Alexandre Rosa-Campos, Jianhui Ma, Tomoyuki Koga, Kristiina Vuori, Frank Furnari. Proteasome addiction a new therapeutic opportunity to treat PTEN-deficient brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-110.

Periprostatic Adipose Tissue Favors Prostate Cancer Cell Invasion in an Obesity-Dependent Manner: Role of Oxidative Stress.

Prostate gland is surrounded by periprostatic adipose tissue (PPAT), which is increasingly believed to play a paracrine role in prostate cancer progression. Our previous work demonstrates that adipocytes promote homing of prostate cancer cells to PPAT and that this effect is upregulated by obesity. Here, we show that once tumor cells have invaded PPAT (mimicked by an in vitro model of coculture), they establish a bidirectional crosstalk with adipocytes, which promotes tumor cell invasion. Indeed, tumor cells induce adipocyte lipolysis and the free fatty acids (FFA) released are taken up and stored by tumor cells. Incubation with exogenous lipids also stimulates tumor cell invasion, underlining the importance of lipid transfer in prostate cancer aggressiveness. Transferred FFAs (after coculture or exogenous lipid treatment) stimulate the expression of one isoform of the pro-oxidant enzyme NADPH oxidase, NOX5. NOX5 increases intracellular reactive oxygen species (ROS) that, in turn, activate a HIF1/MMP14 pathway, which is responsible for the increased tumor cell invasion. In obesity, tumor-surrounding adipocytes are more prone to activate the depicted signaling pathway and to induce tumor invasion. Finally, the expression of NOX5 and MMP14 is upregulated at the invasive front of human tumors where cancer cells are in close proximity to adipocytes and this process is amplified in obese patients, underlining the clinical relevance of our results. IMPLICATIONS: Our work emphasizes the key role of adjacent PPAT in prostate cancer dissemination and proposes new molecular targets for the treatment of obese patients exhibiting aggressive diseases.