The Prolactin Inducible Protein Modulates Antitumor Immune Responses and Metastasis in a Mouse Model of Triple Negative Breast Cancer

The prolactin inducible protein (PIP) is expressed to varying degrees in more than 90% of breast cancers (BCs). Although high levels of PIP expression in BC has been shown to correlate with better prognosis and patient response to chemotherapy, some studies suggest that PIP may also play a role in metastasis. Here, we investigated the role of PIP in BC using the well-established 4T1 and E0771 mouse BC cell lines. Stable expression of PIP in both cell lines did not significantly alter their proliferation, migration, and response to anticancer drugs in vitro compared to empty vector control. To assess the effect of PIP expression on breast tumorigenesis in vivo, the 4T1 syngeneic transplantable mouse model was utilized. In immunocompetent syngeneic BALB/c mice, PIP-expressing 4T1 primary tumors displayed delayed tumor onset and reduced tumor growth, and this was associated with higher percentages of natural killer cells and reduced percentages of type 2 T-helper cells in the tumor environment. The delayed tumor onset and growth were abrogated in immunodeficient mice, suggesting that PIP-mediated modulation of primary tumor growth involves an intact immune system. Paradoxically, we also observed that PIP expression was associated with a higher number of 4T1 colonies in the lungs in both the immunocompetent and immunodeficient mice. Gene expression analysis of PIP-expressing 4T1 cells (4T1-PIP) revealed that genes associated with tumor metastasis such as CCL7, MMP3 and MMP13, were significantly upregulated in 4T1-PIP cells when compared to the empty vector control (4T1-EV) cells. Collectively, these studies strongly suggest that PIP may possess a double-edge sword effect in BC, enhancing both antitumor immunity as well as metastasis.

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3123 TGFbeta, Early Cytokine Dysregulation, and Airway Smooth Muscle Dysfunction in Cystic Fibrosis

Journal of Clinical and Translational Science ◽

10.1017/cts.2019.55 ◽

2019 ◽

Vol 3 (s1) ◽

pp. 22-23

Author(s):

Elizabeth L Kramer ◽

Rhonda Szczesniak ◽

Weiji Su ◽

Satish Madala ◽

Kristin Hudock ◽

...

Keyword(s):

Smooth Muscle ◽

Lung Function ◽

Mouse Model ◽

Lung Disease ◽

Airway Smooth Muscle ◽

Principal Components ◽

Muscle Dysfunction ◽

Empty Vector Control ◽

Empty Vector ◽

Smooth Muscle Dysfunction

OBJECTIVES/SPECIFIC AIMS: This study aims to first describe the unique cytokine profile and TGFbeta levels of young children with CF, then understand the pathologic effects of TGFbeta on lung function in a CF animal model. These powerful translational studies linking observations in clinical disease with a transgenic mouse model allow us a unique opportunity to investigate the role of TGFbeta in early CF lung disease. METHODS/STUDY POPULATION: Cytokine levels (TGFbeta, TNFalpha, IL-8, IL-6, HNE, and IL-1beta) in bronchoalveolar lavage fluid (BALF) from CF patients (n = 15) and non-CF control patients (n = 21) under 6 years old were determined by ELISA and Luminex assay. Tracheotomized patients without significant underlying lung disease were chosen as non-CF inflamed control patients, as they had similar levels of neutrophilic inflammation and infection as CF patients. The percentage of BAL neutrophils (% PMNs) in each sample was assessed. The relationships between cytokines were analyzed using linear regression and principal components analysis. In animal studies, CF and non-CF mice (n = 4-5 per group) were treated with intratracheal adenoviral TGFbeta1 vector, an empty vector control, or PBS. After one week, animals were collected; lung function, response to the bronchoconstrictor methacholine, and rescue with albuterol were measured utilizing a FlexiVent machine. Lungs were collected for histology. Immunohistochemistry for alpha-SMA was performed and pictures of all cross-sectional airways were obtained. Burden of ASM was assessed by dividing the square root of alpha-SMA stained airway smooth muscle by the basement membrane perimeter length of each airway. RESULTS/ANTICIPATED RESULTS: Patient characteristics of CF and non-CF inflamed control patients were similar in terms of age (3.6 yrs vs 3.3 yrs respectively, p = 0.49), positive BAL culture (13% vs 14%, p = 0.94), and % PMNs (65% vs 56%, p = 0.64). Despite these similarities, TGFbeta levels were 2-fold higher in CF versus non-CF BAL (p = 0.034). Analysis of BAL cytokines from both patient groups showed that three principal components describe 86% of total variance across the cytokine variables. These components represent different contributions from the cytokines, with TGFbeta, IL6, and % PMNs comprising one component of similarly regulated inflammatory markers. These components can distinguish CF versus non-CF patients with 77% accuracy (area under the curve: 0.77). TGFbeta concentrations were uniquely associated with increased IL-6 in CF samples (r = 0.74; p = 0.0015) but did not demonstrate association with other cytokines. After TGFbeta exposure, CF mice demonstrated greater abnormalities in airway resistance than non-CF mice, with heightened response to methacholine. Importantly, this increase in airway obstruction in CF mice was reversible with albuterol treatment, indicating airway smooth muscle dysfunction as a principal driver of lung function abnormalities. Furthermore, TGFbeta induced an increased ASM burden on lung histology in both CF and non-CF mice (p<0.05). IL-6 levels in the BAL of CF mice showed greater increases after TGFbeta treatment compared to non-CF mice (p<0.05). Empty vector control treatment did not cause lung pathology. DISCUSSION/SIGNIFICANCE OF IMPACT: Young children with CF have a unique pattern of pulmonary inflammation compared to inflamed non-CF control patients. In CF, TGFbeta pulmonary levels are uniquely associated with IL-6, a driver of ASM dysfunction in other pulmonary diseases. We followed up this clinical observation study by investigating the effect of TGFbeta on pulmonary disease in a mouse model. CF mice demonstrate increased pulmonary IL-6, airway obstruction, and ASM dysfunction after TGFbeta exposure. This study provides evidence that TGFbeta is associated with a distinct cytokine pattern that may promote ASM dysfunction in early CF lung disease. Understanding the mechanism of early CF pathophysiology will be critical in developing targeted therapeutics that can prevent early lung damage.

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Plant-mediated RNA interference of effector gene Mc16D10L confers resistance against Meloidogyne chitwoodi in diverse genetic backgrounds of potato and reduces pathogenicity of nematode offspring

Nematology ◽

10.1163/15685411-00002796 ◽

2014 ◽

Vol 16 (6) ◽

pp. 669-682 ◽

Cited By ~ 18

Author(s):

Phuong T.Y. Dinh ◽

Linhai Zhang ◽

Phuong T.Y. Dinh ◽

Linhai Zhang ◽

...

Keyword(s):

Rna Interference ◽

Vector Control ◽

Potato Production ◽

Russet Burbank ◽

Solanum Bulbocastanum ◽

Meloidogyne Chitwoodi ◽

Egg Masses ◽

Empty Vector Control ◽

Empty Vector ◽

Effector Gene

Meloidogyne chitwoodi is a major problem for potato production in the Pacific Northwest of the USA. In spite of long-term breeding efforts no commercial potato cultivars with resistance to M. chitwoodi exist to date. The resistance gene against M. chitwoodi has been introgressed from Solanum bulbocastanum into cultivated potato (S. tuberosum), but M. chitwoodi pathotypes are able to overcome this resistance. In this study, an RNA interference (RNAi) transgene targeting the M. chitwoodi effector gene Mc16D10L was introduced into potato cvs Russet Burbank and Désirée, and the advanced breeding line PA99N82-4, which carries the gene. Stable transgenic lines were generated for glasshouse infection assays. At 35 days after inoculation (DAI) with M. chitwoodi race 1 the number of egg masses (g root)−1 formed on RNAi lines of cvs Russet Burbank and Désirée was reduced significantly by up to 68% compared to empty vector control plants. At 55 DAI, the number of eggs was reduced significantly by up to 65%. In addition, RNAi of Mc16D10L significantly reduced the development of egg masses and eggs formed by the resistance-breaking M. chitwoodi pathotype Roza on PA99N82-4 by up to 47 and 44%, respectively. Importantly, the plant-mediated silencing effect of Mc16D10L was transmitted to M. chitwoodi offspring and significantly reduced pathogenicity in the absence of selection pressure on empty vector control plants. This finding suggests that the RNAi effect is stable and nematode infection decreases regardless of the genotype of the host once the RNAi process has been initiated in the nematode through a transgenic plant. In summary, plant-mediated down-regulation of effector gene Mc16D10L provides a promising new tool for molecular breeding against M. chitwoodi.

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Silencing and overexpression of the γ-subunit of Na-K-ATPase directly affect survival of IMCD3 cells in response to hypertonic stress

AJP Renal Physiology ◽

10.1152/ajprenal.00077.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. F1142-F1147 ◽

Cited By ~ 5

Author(s):

Juan M. Capasso ◽

Christopher J. Rivard ◽

Tomas Berl

Keyword(s):

Cell Survival ◽

Vector Control ◽

Collecting Duct ◽

Critical Role ◽

Hypertonic Stress ◽

Empty Vector Control ◽

Empty Vector ◽

Γ Subunit ◽

Subunit Expression ◽

The Impact

The γ-subunit of Na-K-ATPase is robustly expressed in inner medullary collecting duct (IMCD)3 cells either acutely challenged or adapted to hypertonicity but not under isotonic conditions. Circumstantial evidence suggests that this protein may be important for the survival of renal cells in a hypertonic environment. However, no direct proof for such a contention has been forthcoming. The complete mRNA sequences of either γ-subunit isoforms were spliced into an expression vector and transfected into IMCD3 cells. Multiple clones stably expressed γ-subunit protein under isotonic conditions. Clones expressing the γb isoform showed enhanced survival at lethal acute hypertonicity compared with either γa isoform or empty vector (control) expressing clones. We also evaluated the loss of γ-subunit expression on the survival of IMCD3 cells exposed to hypertonicity employing silencing RNA techniques. Multiple stable γ-subunit-specific siRNA clones were obtained and exposed to sublethal hypertonicity. Under these conditions, both the level of γ mRNA and protein was essentially undetectable. The impact of silencing γ-subunit expression resulted in a 70% reduction at 48 h ( P < 0.01) in cell survival compared with empty vector (control) clones. γ siRNA clones showed a 45% decrease in myo-inositol uptake compared with controls after an 18-h exposure to sublethal hypertonicity. Taken together, these data demonstrate a direct and critical role of the γ-subunit on IMCD3 cell survival and/or adaptation in response to ionic hypertonic stress.

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Cytochemical, cytogenetic, immunophenotypic and tumorigenic characterization of two hairy cell lines

Blood ◽

10.1182/blood.v71.2.422.bloodjournal712422 ◽

1988 ◽

Vol 71 (2) ◽

pp. 422-429

Author(s):

GB Faguet ◽

KL Satya-Prakash ◽

JF Agee

Keyword(s):

Cell Lines ◽

Nuclear Antigen ◽

Small Subset ◽

Tartrate Resistant Acid Phosphatase ◽

Hairy Cell ◽

Primary Tumors ◽

Immunodeficient Mice ◽

Barr Virus ◽

Epstein Barr ◽

B Lineage

Two cell lines (EH and HK) were derived from two patients with hairy cell leukemia (HCL). Both patients exhibited a clinical picture characteristic of HCL, including splenomegaly, cytopenias, and tartrate- resistant acid phosphatase (TRAP)-positive “hairy” lymphocytes in blood and marrow. EH and HK were demonstrably of B lineage, as judged by cytochemistry and immunophenotype, including expression of B1, B2, and LEU-12 antigens and of monoclonal surface immunoglobulins (slgs). Monoclonality was confirmed by clonal karyotype abnormality demonstrated in cell line HK. HCL parentage suggested by cytochemistry and electron microscopy was confirmed by the immunophenotypic observation that HCL lines expressed antigens alpha S-HCL1, alpha S- HCL3, and cCLLa. While alpha S-HCL1 and alpha S-HCL3 are nonspecific, their co-expression is characteristic of HCL cells. The cCLLa is a novel 69-kd membrane HCL-associated polypeptide antigen not shared by circulating normal T or B lymphocytes nor by malignant cells from unrelated lymphoid or nonlymphoid malignancies. The doubling time of EH and HK was 24 and 36 hours, respectively. While HK included a small subset of Epstein-Barr virus (EBV) nuclear antigen-positive cells, EH cells were homogeneously negative for the presence of this antigen. Both cell lines were consistently implantable in irradiation- preconditioned immunodeficient mice giving rise to primary tumors and widespread metastasis.

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Senescent Thyrocytes, Similarly to Thyroid Tumor Cells, Elicit M2-like Macrophage Polarization In Vivo

Biology ◽

10.3390/biology10100985 ◽

2021 ◽

Vol 10 (10) ◽

pp. 985

Author(s):

Mara Mazzoni ◽

Giuseppe Mauro ◽

Lucia Minoli ◽

Loredana Cleris ◽

Maria Chiara Anania ◽

...

Keyword(s):

Cell Lines ◽

Macrophage Polarization ◽

Critical Role ◽

Thyroid Tumor ◽

Thyroid Cells ◽

Immunodeficient Mice ◽

Tumor Onset ◽

Tumor Stages

Inflammation plays a critical role in thyroid cancer onset and progression. We previously characterized the in vitro interplay between macrophages and senescent human thyrocytes and thyroid tumor-derived cell lines, modeling the early and the late thyroid tumor phases, respectively. We reported that both models are able to induce pro-tumoral M2-like macrophage polarization, through the activation of the COX2-PGE2 axis. Here, we investigated the presence of macrophage infiltrating cells in mouse xenografts derived from the above described cells models. We showed that subcutaneous injection in immunodeficient mice of both senescent human thyrocytes and thyroid tumor-derived cell lines elicits macrophage recruitment. Furthermore, considering the type of macrophage infiltrate, we observed a stronger infiltration of Arginase I positive cells (M2-like). Overall, these results demonstrate the in vivo capability of senescent and tumor thyroid cells to recruit and polarize macrophages, suggesting that the promotion of a pro-tumoral microenvironment through tumor associated macrophages may occurs in late as well as in early thyroid tumor stages, favoring tumor onset and progression.

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Cytochemical, cytogenetic, immunophenotypic and tumorigenic characterization of two hairy cell lines

Blood ◽

10.1182/blood.v71.2.422.422 ◽

1988 ◽

Vol 71 (2) ◽

pp. 422-429 ◽

Cited By ~ 13

Author(s):

GB Faguet ◽

KL Satya-Prakash ◽

JF Agee

Keyword(s):

Cell Lines ◽

Nuclear Antigen ◽

Small Subset ◽

Tartrate Resistant Acid Phosphatase ◽

Hairy Cell ◽

Primary Tumors ◽

Immunodeficient Mice ◽

Barr Virus ◽

Epstein Barr ◽

B Lineage

Abstract Two cell lines (EH and HK) were derived from two patients with hairy cell leukemia (HCL). Both patients exhibited a clinical picture characteristic of HCL, including splenomegaly, cytopenias, and tartrate- resistant acid phosphatase (TRAP)-positive “hairy” lymphocytes in blood and marrow. EH and HK were demonstrably of B lineage, as judged by cytochemistry and immunophenotype, including expression of B1, B2, and LEU-12 antigens and of monoclonal surface immunoglobulins (slgs). Monoclonality was confirmed by clonal karyotype abnormality demonstrated in cell line HK. HCL parentage suggested by cytochemistry and electron microscopy was confirmed by the immunophenotypic observation that HCL lines expressed antigens alpha S-HCL1, alpha S- HCL3, and cCLLa. While alpha S-HCL1 and alpha S-HCL3 are nonspecific, their co-expression is characteristic of HCL cells. The cCLLa is a novel 69-kd membrane HCL-associated polypeptide antigen not shared by circulating normal T or B lymphocytes nor by malignant cells from unrelated lymphoid or nonlymphoid malignancies. The doubling time of EH and HK was 24 and 36 hours, respectively. While HK included a small subset of Epstein-Barr virus (EBV) nuclear antigen-positive cells, EH cells were homogeneously negative for the presence of this antigen. Both cell lines were consistently implantable in irradiation- preconditioned immunodeficient mice giving rise to primary tumors and widespread metastasis.

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Protein Tyrosine Phosphatase Type-1 (PTPN1) Is Frequently Mutated In Primary Mediastinal B Cell Lymphoma and Hodgkin Lymphoma

Blood ◽

10.1182/blood.v122.21.242.242 ◽

2013 ◽

Vol 122 (21) ◽

pp. 242-242

Author(s):

Jay Gunawardana ◽

Fong Chun Chan ◽

Adele Telenius ◽

Bruce W. Woolcock ◽

Robert Kridel ◽

...

Keyword(s):

Cell Lines ◽

Protein Tyrosine Phosphatase ◽

Cell Lymphoma ◽

Tyrosine Phosphatase ◽

B Cell Lymphoma ◽

Clinical Samples ◽

Hek 293 ◽

Empty Vector Control ◽

Empty Vector ◽

293 Cells

Abstract Introduction Hodgkin Lymphoma (HL) accounts for 11% of all lymphomas and despite being one of the most curable lymphomas, 20% of HL patients still ultimately die of their disease. Similarly, a proportion of cases of primary mediastinal B cell lymphoma (PMBCL) have refractory disease or early relapse and frequently fail second-line therapy. Development of more targeted therapeutic approaches is impeded by the lack of knowledge about the mutational landscape in the cancer genomes of these lymphomas. PTPN1 is a protein tyrosine phosphatase gene that encodes the protein, PTP1B. PTP1B dephosphorylates tyrosine residues on many activated kinases to maintain cellular homeostasis. As overactive receptor kinases are critical oncogenic events in cancer, we hypothesized that constitutively active Janus kinase-Signal transducer and activation of transcription (JAK-STAT) observed in HL and PMBCL are in part due to a mutated PTPN1 gene with an impaired functional ability to dephosphorylate this constitutive signaling pathway. Methods and samples Biopsies at the time of primary diagnosis were obtained for 49 PMBCL and 30 HL patients from the British Columbia Cancer Agency, Arizona Lymphoma Repository and the Hôpital Henri Mondor Pathology Department. DNA from PMBCL samples, microdissected Hodgkin Reed Sternberg (HRS) cells and 12 lymphoma-derived cell lines were extracted for PTPN1 exonic PCR amplification (nested PCR was used for HRS cell DNA) and Sanger sequencing. PTPN1 was silenced in a HL cell line (KMH2) by lentiviral transduction of a vector expressing shRNA and confirmed by quantitative real time (qRT) PCR. Wild type and mutant PTPN1 cDNA were cloned into the mammalian expression vector pcDNA 3.1 and expressed in HEK-293 cells. Protein expression of clinical samples, silenced and expressed cells were analyzed by immunohistochemistry and western blotting. Comparisons between groups were performed using two-sample student t tests. Results After exclusion of reported single nucleotide polymorphisms (SNPs) and silent mutations, 16 PTPN1 coding sequence mutations were found in our PMBCL cohort, corresponding to 14 mutations (29%) in clinical samples and 2 in PMBCL-dervied cell lines. Twelve additional mutations were discovered in our HL cohort, corresponding to 6 mutations (20%) in HRS cell samples and another 6 in HL-derived cell lines. In total, 14 (54%) missense, 4 (15%) frameshift, 3 (12%) single amino acid deletions, 4 (15%) nonsense mutations, and 1 (4%) promoter mutation were observed. Eight of these mutations were confirmed as somatic by sequencing of matched constitutional DNA. Silencing of PTPN1 resulted in hyperphosphorylation of JAK1, JAK2, STAT3, STAT5, STAT6 and up-regulation of the oncogenes, MYC and BCL6. Ectopic expression of nonsense and missense PTPN1 mutants in HEK-293 cells led to sustained phosphorylation of STAT6 in comparison to the empty vector control (densitometric values Q9* 0.5 vs. 1.0, R156* 0.7 vs. 1.0, M74L 0.4 vs. 1.0 and M282L 0.8 vs. 1.0). Furthermore, no phosphatase activity was observed for the nonsense mutants and moderate phosphatase activity for the missense mutants using a tyrosine phosphatase-specific substrate (fold change Q9* 2.0, R156* 1.9, M74L 46.7, M282L 46.0 and WT 58.3, compared to empty vector control). Immunohistochemical analysis showed that PTPN1 mutations correspond to decreased protein expression in PMBCL (p=0.03). Discussion PTPN1 is recurrently mutated in PMBCL and HL contributing to constitutive JAK-STAT signaling and oncogene dysregulation. These data suggest PTPN1 mutations as novel driver alterations in these lymphomas and might provide a novel, rational therapeutic target for treating HL and PMBCL patients. Disclosures: Savage: Eli-Lilly: Consultancy. Connors:F Hoffmann-La Roche: Research Funding; Roche Canada: Research Funding.

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The Microanatomy of the Leukemic Stem Cell Niche in Murine Chronic Myelogenous Leukemia

Blood ◽

10.1182/blood.v124.21.351.351 ◽

2014 ◽

Vol 124 (21) ◽

pp. 351-351 ◽

Cited By ~ 1

Author(s):

Melanie Meister ◽

Joel A Spencer ◽

Juwell Wu ◽

Cher Zhao ◽

Lymperi Stefania ◽

...

Keyword(s):

Bone Marrow ◽

Vector Control ◽

Chronic Myelogenous Leukemia ◽

Leukemic Cell ◽

Myelogenous Leukemia ◽

Empty Vector Control ◽

Empty Vector ◽

Cml Model ◽

Number Of Cells

Abstract Objectives and background: Constituents of the bone marrow microenvironment (BMM) influence the proliferation, differentiation and location of hematopoietic stem and progenitor cells (HSPC). Dependent on their maturation stage, different subsets of HSPC are localized at distinct sites in the BMM. This location depends on HSPC-intrinsic, as well as HSPC-extrinsic factors. The BMM protects leukemic stem cells (LSC) from treatment with tyrosine kinase inhibitors or chemotherapy. We, therefore, investigated the microanantomy of the LSC niche hypothesizing that it may differ from the normal HSPC niche. Methods: We used a combination of confocal and 2-photon intravital microscopy (IVM) of the murine calvarium and well-described retroviral models of BCR-ABL1+chronic myelogenous leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). Results: We show here that BCR-ABL1+Lin–c-Kit+Sca-1+ (LKS) CD150+CD48– (LKS SLAM) cells, which harbor the LSC fraction in the CML model, homed to locations further away from the endosteum than their normal counterparts. Prior in-vitro treatment of BCR-ABL1+ LKS with imatinib mesylate, considered standard of care in CML, reversed this phenotype and the cells were found closer to the endosteum. Native BCR-ABL1, as well as the imatinib-resistant BCR-ABL1 point mutants BCR-ABL1Y253F, BCR-ABL1E255K, BCR-ABL1T315I and BCR-ABL1M351T had similar intrinsic catalytic activity, but the BCR-ABL1Y253F, BCR-ABL1E255K, and BCR-ABL1T315I mutants increased the IL-3-independent proliferative capacity of 32D cells relative to native BCR-ABL1. BCR-ABL1Y253F and BCR-ABL1M351T caused increased transformation of primary BM B-lymphoid progenitors in vitro and led to accelerated induction of B-ALL in mice. In the CML model, BCR-ABL1Y253F and BCR-ABL1T315Iinduced myeloproliferative neoplasia with shortened survival and features of accelerated phase disease compared to native BCR-ABL1, whereas BCR-ABL1T315I LKS cells homed closer to osteoblastic cells than LKS cells expressing native BCR-ABL1. Sequential in vivo tracking of leukemic progenitor growth by IVM showed a similar nadir in the number of cells per leukemic cell ‘nest’ 11 days after irradiation and IV transplantation in recipients of DsRed+BCR-ABL1+ or empty vector control-transduced bone marrow. However, between days 18-25 after transplantation there was a significant increase in the number of cells per leukemic cell ‘nest’ compared to the empty vector control group. Sequential immunohistochemistry and TUNEL assays of leukemic bone sections in imatinib- or vehicle-treated recipient mice with CML showed that initial BCR-ABL1+ growth tends to occur at locations further away from the endosteum, whereas erythroid islands were found closer to the endosteum and trabeculae. Apoptosis in response to imatinib appeared most prominent in the metaphysis. Lastly, we could demonstrate by IVM in the CML model that treatment of mice with a combination of imatinib plus granulocyte colony-stimulating factor led to ‘emptying’ of the LSC niche and superior eradication of BCR-ABL1+ leukemic cells compared to treatment with imatinib alone. Conclusions: In summary, these data suggest that the microanatomy of the LSC niche in CML differs from the normal hematopoietic niche. BCR-ABL1 mutation status may affect the positioning of CML LSC in the microenvironment, and location in the niche may be altered pharmacologically, suggesting that niche location may influence clinical outcome. Disclosures Krause: Glycomimetics. Inc.: Research Funding.

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Whole-Exome Analysis Of DLBCL Tumors Reveals a Unique Genetic Signature Associated With Aggressive Disease

Blood ◽

10.1182/blood.v122.21.499.499 ◽

2013 ◽

Vol 122 (21) ◽

pp. 499-499

Author(s):

Anne J Novak ◽

Yan W Asmann ◽

Matthew J Maurer ◽

Chen Wang ◽

Susan L Slager ◽

...

Keyword(s):

Patient Outcome ◽

Vector Control ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Response To Therapy ◽

Empty Vector Control ◽

Aggressive Disease ◽

Empty Vector ◽

Aggressive Form ◽

Whole Exome

Abstract While extensive, the genetic studies of DLBCL tumors to date have primarily focused on somatic mutations that are associated with development of lymphoma, i.e., driver mutations. Identification of key genetic events that impact and/or predict response to therapy and clinical outcome of lymphoma patients has not been accomplished. In an exploratory study, we used whole-exome sequencing to identify novel biomarkers that can predict which patients have an aggressive form of DLBCL, and therefore would likely benefit from a different and more aggressive treatment plan. Using whole-exome sequencing data from 54 newly diagnosed DLBCL patients, we evaluated the association of somatic coding single nucleotide (cSNV) and copy number (CNV) variants with aggressive DLBCL. All patients were treated with R-CHOP or immunochemotherapy, and disease aggressiveness was based on relapse, with patients classified as having aggressive disease (AD) (n=13, defined as treatment failure, relapse, or progression within 24 months of diagnosis) versus non-aggressive disease (n=41, defined as achieving at least 24 months of relapse-free survival). An in-house algorithm, patternCNV was used to detect somatic CNVs. Logistic regression was used to assess statistical significance. In the cSNV analysis, 24 genes were found to be significantly (p ≤ 0.05) associated with AD. Of these genes, CIITA (p=0.01) was previously identified as a mutational target in DLBCL and is a recurrent gene fusion partner in lymphoid cancers. Metacore analyses showed that seven of these genes, including CIITA, can be placed in the same regulatory network centered around CREB1. Interestingly, the CREB binding protein (CREBBP) is a known target of pathogenic mutations in DLBCL. In the CNV analyses, we identified 245 gene amplifications and 209 gene deletions associated with AD (p ≤ 0.05). Deleted genes were localized in three major chromosomal regions, 6p22.3-6p22.1 (26 genes), 6q13-6q16.3 (31 genes), and 6q21-6q24.2 (86 genes). In the CNV amplification analysis, the genes were localized in two major chromosomal regions, 3q12.3-3q29 (90 genes) and 19q13.12-19q13.43 (122 genes). We also quantified the association of each regional deletion/amplification with patient outcome. As expected, each deleted region was univariately associated with time to relapse: 6p22.3-6p22.1 (HR = 7.19, 95% CI: 2.66-19.46, p=1.33x10-5), 6q13-6q16.3 (HR = 4.44, 95% CI: 1.74-11.32, p=6.84x10-4), and 6q21-6q24.2 (HR = 5.42, 95% CI: 2.12-13.87, p=9.23x10-5) as well as the two amplification regions: 3q12.3-3q29 (HR = 4.60, 95% CI: 1.49-14.24, p=4.27x10-3) and 19q13.12 -19q13 (HR = 13.79, 95% CI: 4.39-43.34, p=2.81x10-8). Deletions at 6q21-6q24.2 have been previously reported in DLBCL and two tumor suppressor genes associated with lymphoma pathogenesis are localized in the region, TNFAIP3 (p=0.14) and PRDM1 (p=0.02), the latter of which was significantly associated with AD. However, the genes in the 6p21 locus that were most significant (p=0.002) were SLC22A16, GPR6, SOBP, MATTL24, DDO, and REV3L. SLC22A16, which also had cSNVs in two tumors, is an organic cation transporter that has been shown to transport chemotherapeutic drugs including doxorubicin. Successful drug response has been correlated with the level of activity and expression of this transporter in tumor cells. To determine if expression of SLC22A16 would have an impact on doxorubicin transport, and may therefore impact response to R-CHOP we overexpressed either an empty vector control or SLC22A16 in OCI-Ly7 DLBCL cells, which do not express endogenous SLC22A16 mRNA. We found that OCI-Ly7 cells expressing SLC22A16 have increased 14C-doxorubicin uptake and are more sensitive to doxorubicin-induced cell death when compared to the empty vector control cells. These data suggest that loss of SLC22A16 expression through gene deletion could impact the ability of DLBCL cells to respond to therapy and indicates an aggressive form of the disease. In summary, our data are the first to use whole exome sequencing to identify genetic variants associated with aggressive DLBCL, which will require replication in additional samples. Nevertheless, our data highlight the role for somatic CNVs in patient outcome and we biologically validate the potential impact of deletions in SLC22A16 at 6p21. Disclosures: No relevant conflicts of interest to declare.

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Integrin alpha-V is an important driver in pancreatic adenocarcinoma progression

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01946-2 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Marius Kemper ◽

Alina Schiecke ◽

Hanna Maar ◽

Sergey Nikulin ◽

Andrey Poloznikov ◽

...

Keyword(s):

Tumor Growth ◽

Cell Lines ◽

Epithelial Mesenchymal Transition ◽

Xenograft Model ◽

Ductal Adenocarcinoma ◽

Integrin Subunit ◽

Mesenchymal Transition ◽

Immunodeficient Mice ◽

Model Cell

Abstract Background Mesothelial E- and P-selectins substantially mediate the intraperitoneal spread of Pancreatic ductal adenocarcinoma (PDA) cells in xenograft models. In the absence of selectins in the host, the integrin subunit alpha-V (ITGAV, CD51) was upregulated in the remaining metastatic deposits. Here we present the first experimental study to investigate if ITGAV plays a functional role in PDA tumor growth and progression with a particular focus on intraperitoneal carcinomatosis. Methods Knockdown of ITGAV was generated using an RNA interference-mediated approach in two PDA cell lines. Tumor growth, intraperitoneal and distant metastasis were analyzed in a xenograft model. Cell lines were characterized in vitro. Gene expression of the xenograft tumors was analyzed. Patient samples were histologically classified and associations to survival were evaluated. Results The knockdown of ITGAV in PDA cells strongly reduces primary tumor growth, peritoneal carcinomatosis and spontaneous pulmonary metastasis. ITGAV activates latent TGF-β and thereby drives epithelial-mesenchymal transition. Combined depletion of ITGAV on the tumor cells and E- and P-selectins in the tumor-host synergistically almost abolishes intraperitoneal spread. Accordingly, high expression of ITGAV in PDA cells was associated with reduced survival in patients. Conclusion Combined depletion of ITGAV in PDA cells and E- and P-selectins in host mice massively suppresses intraperitoneal carcinomatosis of PDA cells xenografted into immunodeficient mice, confirming the hypothesis of a partly redundant adhesion cascade of metastasizing cancer cells. Our data strongly encourage developing novel therapeutic approaches for the combined targeting of E- and P-selectins and ITGAV in PDA.

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