Inducible Transgenic Model Research Articles

Lenvatinib Plus Anti-PD-1 Combination Therapy for Advanced Cancers: Defining Mechanisms of Resistance in an Inducible Transgenic Model of Thyroid Cancer.

Background: Combination therapy with lenvatinib plus programmed death-1 (PD-1) immune checkpoint blockades (ICBs) is under investigation in many solid tumors, including thyroid cancer. Lenvatinib is known to reduce angiogenesis and may overturn the immunosuppressive effects of vascular endothelial growth factor in the tumor microenvironment. Previous studies investigating the effects of VEGF receptor inhibition on the immune response were performed in rapidly growing tumor models where immune equilibrium is not established before treatment. We hypothesize that physiologically relevant preclinical models are necessary to define mechanisms of resistance to immune-targeted combination therapies. Methods: We utilized the TPO-CreER/BrafV600E/wt/Trp53Δex2-10/Δex2-10 inducible transgenic model of advanced thyroid cancer to investigate lenvatinib treatment in the context of an anti-PD-1 ICB. Following tumor establishment, 3.5 months postinduction, mice were treated with high- (10 mg/kg) or low-dose (2 mg/kg) lenvatinib, anti-PD-1, or combination of lenvatinib with anti-PD-1. Tumor volume and lung metastases were assessed in each group. Immune infiltrate was characterized by flow cytometry and immunohistochemistry, and TCRß sequencing was performed to further investigate the T cell response. Results: Both low- and high-dose lenvatinib reduced tumor volume, while anti-PD-1 had no effect, alone or in combination. Although both low- and high-dose lenvatinib reduced vascular density, low-dose lenvatinib was superior in controlling tumor size. Lung metastases and survival were not improved with therapy despite the effects of lenvatinib on primary tumor size. Low-dose lenvatinib treatment led to a subtle reduction in the dominant Ly6G+CD11b+ myeloid cell population and was associated with increased CD4+ T cell infiltrate and enrichment in 4-1BB+ and granzyme B+ CD4+ T cells and FoxP3+ regulatory T cells. Polyclonal T cell expansion was evident in the majority of mice, suggesting that a tumor-specific T cell response was generated. Conclusions: The effects of lenvatinib on the immune response were most pronounced in mice treated with low-dose lenvatinib, suggesting that dose should be considered in clinical application. While the immune-modulating potential of lenvatinib is encouraging, alterations in the immune milieu and T cell activation status were insufficient to sustain durable tumor regression, even with added anti-PD-1. Additional studies are necessary to develop more effective combination approaches in low-mutation burden tumors, such as thyroid cancer.

Sustained Club Cell Injury in Mice Induces Histopathologic Features of Deployment-Related Constrictive Bronchiolitis

Histopathologic evidence of deployment-related constrictive bronchiolitis (DRCB) has been identified in soldiers deployed to Southwest Asia. While inhalational injury to the airway epithelium is suspected, relatively little is known about the pathogenesis underlying this disabling disorder. Club cells are local progenitors critical for repairing the airway epithelium after exposure to various airborne toxins, and a prior study using an inducible transgenic murine model reported that 10 days of sustained targeted club cell injury causes constrictive bronchiolitis. To further understand the mechanisms leading to small airway fibrosis, a murine model was employed to show that sustained club cell injury elicited acute weight loss, caused increased local production of proinflammatory cytokines, and promoted accumulation of numerous myeloid cell subsets in the lung. Transition to a chronic phase was characterized by up-regulated expression of oxidative stress-associated genes, increased activation of transforming growth factor-β, accumulation of alternatively activated macrophages, and enhanced peribronchiolar collagen deposition. Comparative histopathologic analysis demonstrated that sustained club cell injury was sufficient to induce epithelial metaplasia, airway wall thickening, peribronchiolar infiltrates, and clusters of intraluminal airway macrophages that recapitulated key abnormalities observed in DRCB. Depletion of alveolar macrophages in mice decreased activation of transforming growth factor-β and ameliorated constrictive bronchiolitis. Collectively, these findings implicate sustained club cell injury in the development of DRCB and delineate pathways that may yield biomarkers and treatment targets for this disorder.

MicroRNA-21 Plays Multiple Oncometabolic Roles in the Process of NAFLD-Related Hepatocellular Carcinoma via PI3K/AKT, TGF-β, and STAT3 Signaling.

Simple SummaryThe PTS signaling (PI3K/AKT, TGF-β, and STAT3 Signaling) networks, regulated by microRNA-21, play roles in lipogenic factor regulation, tumor suppressor modulation and oncogenic activation. Our zebrafish model recreates the development of hepatocellular carcinoma (HCC) due to nonalcoholic fatty liver disease (NAFLD) concerning the physiological, metabolic, and histological aspects similar to that of human NAFLD-related HCC (NAHCC). Thus, microRNA-21 is critical in the pathogenesis of NAHCC, and serves as a novel therapeutic target in NAHCC progression.MicroRNA-21 (miR-21) is one of the most frequently upregulated miRNAs in liver diseases such as nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). However, mechanistic pathways that connect NAFLD and HCC remain elusive. We developed a doxycycline (Dox)-inducible transgenic zebrafish model (LmiR21) which exhibited an upregulation of miR-21 in the liver, which in turn induced the full spectrum of NAFLD, including steatosis, inflammation, fibrosis, and HCC, in the LmiR21 fish. Diethylnitrosamine (DEN) treatment led to accelerated liver tumor formation and exacerbated their aggressiveness. Moreover, prolonged miR-21 expression for up to ten months induced nonalcoholic steatohepatitis (NASH)-related HCC (NAHCC). Immunoblotting and immunostaining confirmed the presence of miR-21 regulatory proteins (i.e., PTEN, SMAD7, p-AKT, p-SMAD3, and p-STAT3) in human nonviral HCC tissues and LmiR21 models. Thus, we demonstrated that miR-21 can induce NAHCC via at least three mechanisms: First, the occurrence of hepatic steatosis increases with the decrease of ptenb, pparaa, and activation of the PI3K/AKT pathway; second, miR-21 induces hepatic inflammation (or NASH) through an increase in inflammatory gene expression via STAT3 signaling pathways, and induces liver fibrosis through hepatic stellate cell (HSC) activation and collagen deposition via TGF-β/Smad3/Smad7 signaling pathways; finally, oncogenic activation of Smad3/Stat3 signaling pathways induces HCC. Our LmiR21 models showed similar molecular pathology to the human cancer samples in terms of initiation of lipid metabolism disorder, inflammation, fibrosis and activation of the PI3K/AKT, TGF-β/SMADs and STAT3 (PTS) oncogenic signaling pathways. Our findings indicate that miR-21 plays critical roles in the mechanistic perspectives of NAHCC development via the PTS signaling networks.

Stimulation of hepatocarcinogenesis by activated cholangiocytes via Il17a/f1 pathway in kras transgenic zebrafish model

It has been well known that tumor progression is dependent on secreted factors not only from tumor cells but also from other surrounding non-tumor cells. In the current study, we investigated the role of cholangiocytes during hepatocarcinogenesis following induction of oncogenic krasV12 expression in hepatocytes using an inducible transgenic zebrafish model. Upon induction of carcinogenesis in hepatocytes, a progressive cell proliferation in cholangiocytes was observed. The proliferative response in cholangiocytes was induced by enhanced lipogenesis and bile acids secretion from hepatocytes through activation of Sphingosine 1 phosphate receptor 2 (S1pr2), a known cholangiocyte receptor involving in cholangiocyte proliferation. Enhancement and inhibition of S1pr2 could accelerate or inhibit cholangiocyte proliferation and hepatocarcinogenesis respectively. Gene expression analysis of hepatocytes and cholangiocytes showed that cholangiocytes stimulated carcinogenesis in hepatocytes via an inflammatory cytokine, Il17a/f1, which activated its receptor (Il17ra1a) on hepatocytes and enhanced hepatocarcinogenesis via an ERK dependent pathway. Thus, the enhancing effect of cholangiocytes on hepatocarcinogenesis is likely via an inflammatory loop.

Cell cycle-related kinase reprograms the liver immune microenvironment to promote cancer metastasis.

The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.

Treatment and Prevention of Lung Cancer Using a Virus-Infected Reprogrammed Somatic Cell-Derived Tumor Cell Vaccination (VIReST) Regime.

Lung cancer is one of the most commonly diagnosed cancer and despite therapeutic advances, mortality remains high. The long period of clinical latency associated with lung cancer provides an ideal window of opportunity to administer vaccines to at-risk individuals that can prevent tumor progression and initiate long-term anti-tumor immune surveillance. Here we describe a personalized vaccination regime that could be applied for both therapeutic and prophylactic prevention of lung cancer, based on the derivation of lung cancer cells from induced pluripotent stem cells. Stem cells from healthy mice were modified to express Cre-dependent KRASG12D and Trp53R172H prior to differentiation to lung progenitor cells. Subsequent viral delivery of Cre caused activation of exogenous driver mutations, resulting in transformation and development of lung cancer cells. iPSC-derived lung cancer cells were highly antigenically related to lung cancer cells induced in LSL-KRASG12D/+; Trp53R172H/+ transgenic mice and were antigenically unrelated to original pluripotent stem cells or pancreatic cancer cells derived using the same technological platform. For vaccination, induced lung cancer cells were infected with oncolytic Adenovirus or Vaccinia virus, to act as vaccine adjuvants, prior to delivery of vaccines sequentially to a murine inducible transgenic model of lung cancer. Application of this Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime primed tumor-specific T cell responses that significantly prolonged survival in both subcutaneous post-vaccine challenge models and induced transgenic models of lung cancer, demonstrating that stem cell-derived prophylactic vaccines may be a feasible intervention for treatment or prevention of lung cancer development in at-risk individuals.

Modulation of Mutant KrasG12D -Driven Lung Tumorigenesis In Vivo by Gain or Loss of PCDH7 Function.

PROTOCADHERIN 7 (PCDH7), a transmembrane receptor and member of the Cadherin superfamily, is frequently overexpressed in lung adenocarcinoma and is associated with poor clinical outcome. Although PCDH7 was recently shown to promote transformation and facilitate brain metastasis in lung and breast cancers, decreased PCDH7 expression has also been documented in colorectal, gastric, and invasive bladder cancers. These data suggest context-dependent functions for PCDH7 in distinct tumor types. Given that PCDH7 is a potentially targetable molecule on the surface of cancer cells, further investigation of its role in tumorigenesis in vivo is needed to evaluate the therapeutic potential of its inhibition. Here, we report the analysis of novel PCDH7 gain- and loss-of-function mouse models and provide compelling evidence that this cell-surface protein acts as a potent lung cancer driver. Employing a Cre-inducible transgenic allele, we demonstrated that enforced PCDH7 expression significantly accelerates KrasG12D -driven lung tumorigenesis and potentiates MAPK pathway activation. Furthermore, we performed in vivo somatic genome editing with CRISPR/Cas9 in KrasLSL-G12D ; Tp53fl/fl (KP) mice to assess the consequences of PCDH7 loss of function. Inactivation of PCDH7 in KP mice significantly reduced lung tumor development, prolonged survival, and diminished phospho-activation of ERK1/2. Together, these findings establish a critical oncogenic function for PCDH7 in vivo and highlight the therapeutic potential of PCDH7 inhibition for lung cancer. Moreover, given recent reports of elevated or reduced PCDH7 in distinct tumor types, the new inducible transgenic model described here provides a robust experimental system for broadly elucidating the effects of PCDH7 overexpression in vivo. IMPLICATIONS: In this study, we establish a critical oncogenic function for PCDH7 in vivo using novel mouse models and CRISPR/Cas9 genome editing, and we validate the therapeutic potential of PCDH7 inhibition for lung cancer.

Temporal Control of WNT Activity Regulates Tooth Number in Fish

Wnts determine cell polarity, cell proliferation, and cell differentiation during embryogenesis and play an essential role during tooth development initiation and morphogenesis. Wnt/β-catenin signaling has a time-dependent role in development because various signaling molecules that mutually interact are involved in the pathway, and tight regulation of the pathway is essential for normal development. Studies investigating how the Wnt/β-catenin signaling pathway controls the different stages of tooth development are rare. Specifically, the effects of Wnt/β-catenin signaling loss of function on different stages of tooth development are currently unknown. Here, we report the stage-dependent role of Wnt/β-catenin signaling in tooth development. In vivo loss and gain of function of Wnt/β-catenin signaling were implemented through the genetic overexpression of DKK1 with heat shock–inducible transgenic models and the pharmacologic inhibition of β-catenin destruction complex formation in zebrafish, respectively. We demonstrated that transient inhibition of Wnt/β-catenin signaling interrupted tooth development in a stage-dependent manner and conditional activation of Wnt/β-catenin signaling during 4V morphogenesis inhibited the development of 3V. These findings suggest that Wnt/β-catenin signaling plays an important role in the morphogenesis of teeth and the initiation of sequential tooth development in a stage-dependent manner.

Targeting Heat Shock Protein 70 to Ameliorate c-Jun Expression and Improve Demyelinating Neuropathy.

Increased expression of the c-jun transcription factor occurs in a variety of human neuropathies and is critical in promoting Schwann cell (SC) dedifferentiation and loss of the myelinated phenotype. Using cell culture models, we previously identified KU-32 as a novobiocin-based C-terminal heat shock protein 90 (Hsp90) inhibitor that decreased c-jun expression and the extent of demyelination. Additional chemical optimization has yielded KU-596 as a neuroprotective novologue whose mechanistic efficacy to improve a metabolic neuropathy requires the expression of Hsp70. The current study examined whether KU-596 therapy could decrease c-jun expression and improve motor function in an inducible transgenic model of a SC-specific demyelinating neuropathy (MPZ-Raf mice). Treating MPZ-Raf mice with tamoxifen activates the MAPK kinase pathway, increases c-jun expression and produces a profound demyelinating neuropathy characterized by a loss of motor function and paraparesis. KU-596 therapy did not interfere with MAPK activation but reduced c-jun expression, significantly improved motor performance, and ameliorated the extent of peripheral nerve demyelination in both prevention and intervention studies. Hsp70 was necessary for the drug's neuroprotective efficacy since MPZ-Raf × Hsp70 knockout mice did not respond to KU-596 therapy. Collectively, our data indicate that modulating Hsp70 may provide a novel therapeutic approach to attenuate SC c-jun expression and ameliorate the onset of certain demyelinating neuropathies in humans.

Activation of PPAR-α in the early stage of heart failure maintained myocardial function and energetics in pressure-overload heart failure.

Failing heart loses its metabolic flexibility, relying increasingly on glucose as its preferential substrate and decreasing fatty acid oxidation (FAO). Peroxisome proliferator-activated receptor α (PPAR-α) is a key regulator of this substrate shift. However, its role during heart failure is complex and remains unclear. Recent studies reported that heart failure develops in the heart of myosin heavy chain-PPAR-α transgenic mice in a manner similar to that of diabetic cardiomyopathy, whereas cardiac dysfunction is enhanced in PPAR-α knockout mice in response to chronic pressure overload. We created a pressure-overload heart failure model in mice through transverse aortic constriction (TAC) and activated PPAR-α during heart failure using an inducible transgenic model. After 8 wk of TAC, left ventricular (LV) function had decreased with the reduction of PPAR-α expression in wild-type mice. We examined the effect of PPAR-α induction during heart failure using the Tet-Off system. Eight weeks after the TAC operation, LV construction was preserved significantly by PPAR-α induction with an increase in PPAR-α-targeted genes related to fatty acid metabolism. The increase of expression of fibrosis-related genes was significantly attenuated by PPAR-α induction. Metabolic rates measured by isolated heart perfusions showed a reduction in FAO and glucose oxidation in TAC hearts, but the rate of FAO preserved significantly owing to the induction of PPAR-α. Myocardial high-energy phosphates were significantly preserved by PPAR-α induction. These results suggest that PPAR-α activation during pressure-overloaded heart failure improved myocardial function and energetics. Thus activating PPAR-α and modulation of FAO could be a promising therapeutic strategy for heart failure.NEW & NOTEWORTHY The present study demonstrates the role of PPAR-α activation in the early stage of heart failure using an inducible transgenic mouse model. Induction of PPAR-α preserved heart function, and myocardial energetics. Activating PPAR-α and modulation of fatty acid oxidation could be a promising therapeutic strategy for heart failure.

Bone Marrow-Specific Loss of ABI1 Induces Myelofibrosis through a Mechanism Involving Activation of NFκB

Primary myelofibrosis (PMF) is an age-related myeloproliferative neoplasm (MPN) characterized by progressive bone marrow fibrosis, increasingly ineffective hematopoiesis, vascular and inflammatory complications, and progression to acute myeloid leukemia (AML) in 20% of cases. PMF is the rarest and the most severe of MPNs, with unfavorable prognosis and challenging treatment. Constitutive activation of the JAK-STAT pathway is an essential contributor to the pathogenesis of PMF. Nonetheless, clinically available JAK inhibitors do not show consistent disease-modifying effects at the molecular and malignant stem cell levels, underscoring the need to identify other targetable pathogenic mechanisms in PMF and related MPNs.We recently described a new signaling cascade involved in the maintenance of the malignant phenotype of stem cells in chronic myeloid leukemia that affects their adhesiveness, interaction with the microenvironment and resistance to imatinib (Chorzalska et al., Leukemia 2014). These phenotypic changes were caused by loss of Abelson interactor-1 (Abi-1) which regulates integrin α4 and the Src family kinases (SFKs) signaling pathways. To further delineate how organ-specific absence of Abi-1 affects hematopoiesis and hematopoietic stem cells, we generated a conditional, inducible bone marrow-specific Abi-1 transgenic mouse knockout model. For this purpose, we crossed conditional Abi-1(fl/fl) knockout mice with transgenic Tg(Mx1-cre+/-) mice (B6.Cg-Tg(Mx1-cre+ 1Cgn/J) that express Cre recombinase under the control of the polyinosinic:polycytidylic acid (poly(I:C)) or interferon-inducible Mx1 promoter. Obtained Abi-1(fl/fl);Tg(Mx1- cre(+)) animals were subjected to bone marrow-specific Abi-1 deletion induced by intraperitoneal administration of poly(I:C), resulting in organ-specific Abi-1 knockout (Abi-1 KO). Abi-1(fl/fl);Tg(Mx1- cre(-)) animals subjected to the same poly(I:C) treatment (Abi-1 WT) were used as controls in all experiments (Figure 1A). We confirmed the absence of Abi-1 protein in the Abi-1 KO bone marrow by immunoblotting (Figure 1B). Abi-1 KO mice developed deranged hematopoiesis with progressive anemia, thrombocythemia, megakaryocytosis, and myeloid hyperplasia (Figure 1C). They also developed progressive splenomegaly with 1.4x, 2.4x, and 4.8x enlargement of the spleen at 4, 28, and 50 weeks after recombination, respectively, and progressive femur pallor (Figure 1D) with bone marrow fibrosis confirmed by trichrome and silver staining (Figure 1D). Using fluorescence-activated cell sorting, we determined that Abi-1 KO resulted in more than two-fold expansion of the most primitive long-term hematopoietic stem cells (Lin-CD34-Sca-1+c-Kit+ CD135-) in the bone marrow and 3.75-fold increase of LSK cells (Lin- Sca-1+c-Kit+) in the peripheral blood compared with controls (Figure 1E). Increased activities of the SFKs and NFκB were observed in Abi-1 KO bone marrow, and confirmed in human CD34+ cells isolated from the marrow of patients with PMF or AML (Figure 1F,G). Furthermore, CD34+ hematopoietic progenitors and granulocytes from PMF patients, as well as CD34+ hematopoietic progenitors from AML patients, showed decreased levels of ABI1 transcript (Figure 1H).Taken together, our results indicate that a targeted deactivation of Abi-1 in the bone marrow causes expansion of hematopoietic stem cells, myeloid hyperplasia with thrombocytosis and overproduction of collagen and reticulin fibers. The Abi-1 KO murine phenotype meets the Mouse Models of Human Cancers Consortium criteria for a myeloproliferative neoplasm with features resembling human PMF (Kogan et al., Blood 2002). These data indicate that loss of Abi-1 is sufficient for initiation of bone marrow fibrosis, and that Abi-1-SFKs-NFκB cross-talk may represent a new molecular target for the treatment of PMF.Bone marrow-specific, conditional Abi-1 KO animals represent a new model of PMF offering a unique opportunity to interrogate the molecular details of the Abi-1-driven mechanism that leads to the development of stem cell-derived MPNs, and to elucidate potential novel therapeutic approaches that directly address their pathogenesis. [Display omitted] DisclosuresOlszewski:Bristol-Myers Squibb: Consultancy; Genentech: Research Funding; TG Therapeutics: Research Funding. Reagan:Alexion: Honoraria; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

Intestinal-specific activatable Myb initiates colon tumorigenesis in mice.

Transcription factor Myb is overexpressed in most colorectal cancers (CRC). Patients with CRC expressing the highest Myb are more likely to relapse. We previously showed that mono-allelic loss of Myb in an Adenomatous polyposis coli (APC)-driven CRC mouse model (ApcMin/+) significantly improves survival. Here we directly investigated the association of Myb with poor prognosis and how Myb co-operates with tumor suppressor genes (TSGs) (Apc) and cell cycle regulator, p27. Here we generated the first intestinal-specific, inducible transgenic model; a MybER transgene encoding a tamoxifen-inducible fusion protein between Myb and the estrogen receptor-α ligand-binding domain driven by the intestinal-specific promoter, Gpa33. This was to mimic human CRC with constitutive Myb activity in a highly tractable mouse model. We confirmed that the transgene was faithfully expressed and inducible in intestinal stem cells (ISCs) before embarking on carcinogenesis studies. Activation of the MybER did not change colon homeostasis unless one p27 allele was lost. We then established that MybER activation during CRC initiation using a pro-carcinogen treatment, azoxymethane (AOM), augmented most measured aspects of ISC gene expression and function and accelerated tumorigenesis in mice. CRC-associated symptoms of patients including intestinal bleeding and anaemia were faithfully mimicked in AOM-treated MybER transgenic mice and implicated hypoxia and vessel leakage identifying an additional pathogenic role for Myb. Collectively, the results suggest that Myb expands the ISC pool within which CRC is initiated while co-operating with TSG loss. Myb further exacerbates CRC pathology partly explaining why high MYB is a predictor of worse patient outcome.

Controlled hypertension induces cerebrovascular and gene alterations in Cyp1a1-Ren2 transgenic rats

Hypertension is a major risk factor for small vessel disease and dementia, but the pathogenic mechanisms are not fully known. This study aimed to assess cerebrovascular alterations in response to different durations (4 or 6 months) of controlled hypertension in an inducible transgenic rat model of hypertension (Cyp1a1-Ren2) as compared with normotensive litter mate controls. After 6 months of hypertension as compared with controls, a significant reduction in vascular width was paralleled by an increase in the protein levels of claudin-5, an endothelial tight junction protein. Notably, vascular alterations were associated with increased microglia, and these changes were preceded by increased eNOS expression. Investigation of global gene expression by microarray analysis indicated alterations in predominantly growth factor related genes. Herein, we show that modest, sustained levels of hypertension are sufficient to cause cerebrovascular alterations accompanied by endothelial and inflammatory changes. These changes are paralleled by alterations in growth factor expression suggestive of a mechanistic role.

Monitoring systemic amyloidosis using MRI measurements of the extracellular volume fraction

We report the in vivo evaluation, in a murine model, of MRI measurements of the extracellular volume fraction (ECV) for the detection and monitoring of systemic amyloidosis. A new inducible transgenic model was used, with increased production of mouse serum amyloid A protein controlled by oral administration of doxycycline, that causes both the usual hepatic and splenic amyloidosis and also cardiac deposits. ECV was measured in vivo by equilibrium contrast MRI in the heart and liver of 11 amyloidotic and 10 control mice. There was no difference in the cardiac function between groups, but ECV was significantly increased in the heart, mean (standard deviation) 0.20 (0.05) versus 0.14 (0.04), p < 0.005, and liver, 0.27 (0.04) versus 0.15 (0.04), p < 0.0005, of amyloidotic animals and was strongly correlated with the histological amyloid score, myocardium, ρ = 0.67, p < 0.01; liver, ρ = 0.87, p < 0.01. In a further four mice that received human serum amyloid P component (SAP) followed by anti-human SAP antibody, a treatment to eliminate visceral amyloid deposits, ECV in the liver and spleen returned to baseline after therapy (p < 0.01). MRI measurement of ECV is a sensitive marker of amyloid deposits with potential application for early detection and monitoring therapies promoting their clearance.

Abstract 1573: An inducible transgenic porcine model for human cancer.

Abstract Common rodent-based models have limitations in terms of modeling human cancers. Given that pigs share many genetic and physiological similarities with humans, we investigated the potential of developing genetic porcine models of cancer. In this regard, we previously reported that activation of oncogenes like Ras in conjunction with inhibiting tumor suppressor pathways like p53 were required, in part, to convert normal porcine cells to a tumorigenic state. Based on this, we chose to generate transgenic pigs that can be induced to express oncogenic Kras and dominant-negative p53. First, porcine Kras and p53 wild-type genes were cloned, sequenced and aligned with porcine, human and murine homologues to identify porcine-specific mutation sites corresponding to those commonly found in human cancers. Porcine Kras mutation occurs at the 12th glycine (G) to aspartic acid (D), whereas p53 arginine (R) at 167th position was mutated to histidine (H). KrasG12D and p53R167H mutants were linked by internal ribosome entry sites (IRES) for their simultaneous expression and then inserted into a vector following the LoxP-polyA(STOP)-LoxP sequence (LSL). Porcine fetal fibroblasts were transfected in vitro with this vector construct and infected by adenovirus (Ad) vectors encoding Cre recombinase (Ad-Cre-GFP), which deletes the LSL sequence and permits transgene expression, or control Ad vectors without the inserted Cre transgene (Ad-GFP). Cre recombinase-mediated KrasG12D and p53R167H expression was significantly induced in porcine fibroblasts transfected with Ad-Cre-GFP virus compared with Ad-GFP control, which provides an in vitro proof of functional test of the “oncopig” construct. We then transfected porcine fibroblasts with the aforementioned “oncopig” construct to produce donor cell lines for nuclear transfer cloning. Transgenic fibroblast cell lines generated from the cloned pig were subjected to a wound assay through which we observed a statistically significant difference of in vitro migration capability between Ad-Cre-GFP cells and Ad-GFP control cells. In a migration time of 24h, the number of cells in the wound area for the Ad-Cre-GFP cells was 184 as for the Ad-GFP cells was only 67 (p-value ≤ 0.01). A statistically significant difference was also observed between the cell cycle length for these cell lines by flow cytometry. Ad-Cre-GFP cells went through a greater number of cell divisions compared with Ad-GFP cells. Within a 73h time period, Ad-Cre-GFP cells divided twice as many times than Ad-GFP cells (p-value ≤ 0.01). Present results demonstrate that induction of the transgenes in these porcine cells triggered a transformed phenotype. In the future, these cells will be tested for growth in soft agar, tumor growth in mice, and then the pigs will be monitored for tumor incidence following site-specific transgene induction. Such an approach could provide a porcine model to cancer etiology and the development of anti-cancer therapy. Citation Format: Fernanda M. Rodrigues, Wenping Hu, Laurie Rund, Ying Liang, Christopher Counter, Lawrence Schook. An inducible transgenic porcine model for human cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1573. doi:10.1158/1538-7445.AM2013-1573

Hedgehog Pathway Inhibition Radiosensitizes Non-Small Cell Lung Cancers

Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntag and radiation. In a transgenic mouse model of Kras(G12D)-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radiosensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer.

Generation and characterization of an inducible transgenic model for studying mouse esophageal biology

BackgroundTo facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein.ResultsFirst, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the promoter (ED-L2) of the Epstein-Barr virus (EBV) gene encoding the latent membrane protein-1 (LMP-1). The newly generated ED-L2-rtTA2-M2 (ED-L2-rtTA) mice were then bred with the previously developed tetO-HIST1H2BJ/GFP (tetO-H2B-GFP) model to assess inducibility and tissue-specificity. Expression of the H2B-GFP fusion protein was observed upon doxycycline induction but was restricted to the terminally differentiated cells above the basal cell layer. To achieve expression in the basal compartment of the esophagus, we subsequently employed a different transgenic model expressing the reverse transactivator rtTA2S-M2 under the control of the ubiquitous, methylation-free CpG island of the human hnRNPA2B1-CBX3 gene (hnRNP-rtTA). Upon doxycycline administration to the compound hnRNP-rtTA/tetO-H2B-GFP mice, near-complete labeling of all esophageal cells was achieved. Pulse-chase experiments confirmed that complete turnover of the esophageal epithelium in the adult mouse is achieved within 7–10 days.ConclusionsWe show that the esophagus-specific promoter ED-L2 is expressed only in the differentiated cells above the basal layer. Moreover, we confirmed that esophageal turn-over in the adult mouse does not exceed 7–10 days.

Abstract 3294: A novel, inducible, mammary gland-specific PELP1 murine breast cancer model

Abstract Despite treatment advances, breast cancer remains the second most lethal malignant disease for women worldwide. Although pharmacologic agents that modulate estrogen receptor-alpha (ER) functions or reduce circulating estrogens levels significantly reduced mortality in patients with estrogen sensitive (ER positive) tumors, both de novo and acquired resistance limits efficacy. A critical need for identifying more precise diagnostic/prognostic biomarkers and novel therapeutic targets prompted deeper investigation into ER-coregulatory protein function and regulation. ER-coactivator PELP1, mediates both nuclear and extra-nuclear estrogen signaling and crosstalk with growth factors. Deregulated PELP1 expression occurs in hormone-driven cancers, associates with undifferentiated invasive breast adenocarcinomas and an independent prognostic biomarker in assessing clinical outcome of luminal-like breast cancer patients. Collectively, several studies suggest PELP1 is an ER coregulator with tumorigenic potential. However, the in vivo significance of PELP1 deregulation during initiation and progression of breast cancer remains unknown. To determine the role of PELP1 over-expression in mammary tumorigenesis, we generated an inducible transgenic murine model. Transgene construct (pTetOPELP1) consists of a full-length human PELP1 cDNA linked to luciferase gene reporter through an internal ribosomal entry site (IRES). pTetOPELP1 mice crossed with mammary gland-specific rtTA mice (MMTVrtTA) to establish two independent MMTVrtTA-TetOPELP1 transgenic lines. Transgene induction in adult nulliparous bitransgenic females was achieved with doxycycline administered in drinking water. Concurrent expression and activity of the luciferase gene reporter was detected specifically in the mammary gland by in vivo bioluminescence imaging, luciferase assay and RT-PCR. Mammary epithelial-specific expression of PELP1 was validated by immunohistochemistry and Western blot analysis. PELP1-mediated morphological and histological changes were analyzed by examining carmine-stained whole mounts and H&amp;E-stained paraffin embedded mammary glands sections. We observed an increase in proliferation, extensive side branching and precocious differentiation in PELP1 expressing mammary gland compared to controls. Aged MMTVrtTA-TetOPELP1 bitransgenic mice (n=42) revealed hyperplasia and preneoplastic changes as early as 12 weeks with ER-positive mammary gland tumors developing by 8 months following PELP1 induction. Taken together, we established PELP1 as an oncogenic in vivo. Our inducible mammary-specific PELP1 transgenic model will serve as a valuable tool for future in vivo investigation into molecular mechanisms of PELP1-mediated tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3294. doi:1538-7445.AM2012-3294

P4-03-05: Development of an Inducible Estrogen Receptor Co-Activator PELP1 Mammary Tumor Model.

Abstract Despite treatment advances, breast cancer remains the second most lethal malignant disease for women worldwide. Given the importance of estrogen receptor (ER) and hormone-dependent nature of breast cancer, pharmacologic agents were developed to either modulate ER functions or reduce circulating estrogens levels. Although targeted endocrine therapies significantly reduce mortality in patients with estrogen sensitive (ER+) tumors, both de novo and acquired resistance limits efficacy. A critical need for identifying more precise diagnostic/prognostic biomarkers and novel therapeutic targets prompted deeper investigation into ER-coregulatory protein function and regulation. ER-coactivator PELP1, mediates both nuclear and extra-nuclear estrogen signaling and crosstalk with growth factors. PELP1 is deregulated in hormone-driven cancers, associates with undifferentiated invasive breast adenocarcinomas and an independent prognostic biomarker in assessing clinical outcome of luminal-like breast cancer patients. Collectively, several studies suggest PELP1 is an ERα coregulator with tumorigenic potential. However, the in vivo significance of PELP1 deregulation during initiation and progression of breast cancer is unknown. To determine the role of PELP1 overexpression in mammary tumorigenesis, we generated an inducible transgenic murine model. Transgene construct (pTetOPELP1) consists of a full-length human PELP1 cDNA linked to luciferase gene reporter through an internal ribosomal entry site (IRES). PELP1 transgene was purified and microinjected into mouse zygotes to generate pTetOPELP1 mice. Founder mice were identified by Southern blot analysis, of genomic DNA extracted from tail biopsies, for transgene integration through germline transmission. pTetOPELP1 mice were breed with mammary gland-specific rtTA mice (MMTVrtTA) to produce MMTVrtTA-TetOPELP1 bitransgenic mice. Potential founder mice were identified by polymerase chain reaction and breed to establish two independent transgenic lines. Transgene expression was induced in adult female bitransgenic animals with 200mg/mL of doxycycline administered in drinking water. Concurrent expression and activity of the luciferase gene reporter was detected specifically in the mammary gland by in vivo bioluminescence imaging, luciferase assay and RT-PCR. Mammary epithelial-specific expression of PELP1 was validated by immunohistochemistry and Western blot analysis. PELP1-mediated morphological and histological changes were analyzed by examining carmine-stained whole mounts and H&amp;E-stained mammary glands sections. Our early findings with MMTVrtTA-TetOPELP1 bitransgenic mice (n=30) early preneoplastic changes and hyperplasia were evident as early as 12 weeks and the formation of mammary tumors by 8 months of age following PELP1 induction by doxycycline treatment. By utilizing the tetracycline-regulatory system, we created a novel, inducible and mammary gland-specific PELP1-expressing transgenic model for future in vivo studies into molecular mechanisms of PELP1-mediated mammary tumorigenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-03-05.

NF-kappaB activation within macrophages leads to an anti-tumor phenotype in a mammary tumor lung metastasis model

IntroductionMetastasis from primary tumor to the lungs is a major cause of the mortality associated with breast cancer. Both immune and inflammatory responses impact whether circulating mammary tumor cells successfully colonize the lungs leading to established metastases. Nuclear factor -kappaB (NF-κB) transcription factors regulate both immune and inflammatory responses mediated in part by the activities of macrophages. Therefore, NF-κB activity specifically within macrophages may be a critical determinant of whether circulating tumor cells successfully colonize the lungs.MethodsTo investigate NF-κB signaling within macrophages during metastasis, we developed novel inducible transgenic models which target expression of the reverse tetracycline transactivator (rtTA) to macrophages using the cfms promoter in combination with inducible transgenics that express either an activator (cIKK2) or an inhibitor (IκBα-DN). Doxycyline treatment led to activation or inhibition of NF-κB within macrophages. We used a tail vein metastasis model with mammary tumor cell lines established from MMTV-Polyoma Middle T-Antigen-derived tumors to investigate the effects of modulating NF-κB in macrophages during different temporal windows of the metastatic process.ResultsWe found that activation of NF-κB in macrophages during seeding leads to a reduction in lung metastases. The mechanism involved expression of inflammatory cytokines and reactive oxygen species, leading to apoptosis of tumor cells and preventing seeding in the lung. Activation of NF-κB within macrophages after the seeding phase has no significant impact on establishment of metastases.ConclusionsOur results have identified a brief, defined window in which activation of NF-κB has significant anti-metastatic effects and inhibition of NF-κB results in a worse outcome.