Vimentin Is Required for Lung Adenocarcinoma Metastasis via Heterotypic Tumor Cell-Cancer-Associated Fibroblast Interactions during Collective Invasion.

Purpose: Vimentin is an epithelial-to-mesenchymal transition (EMT) biomarker and intermediate filament protein that functions during cell migration to maintain structure and motility. Despite the abundance of clinical data linking vimentin to poor patient outcome, it is unclear if vimentin is required for metastasis or is a correlative biomarker. We developed a novel genetically engineered mouse model (GEMM) to probe vimentin in lung adenocarcinoma metastasis.Experimental Design: We used the LSL-KrasG12D/Lkb1fl/fl/Vim-/- model (KLV-/-), which incorporates a whole-body knockout of vimentin and is derived from the Cre-dependent LSL-KrasG12D/Lkb1fl/fl model (KLV+/+). We compared the metastatic phenotypes of the GEMMs and analyzed primary tumors from the KLV models and lung adenocarcinoma patients to assess vimentin expression and function.Results: Characterization of KLV+/+ and KLV-/- mice shows that although vimentin is not required for primary lung tumor growth, vimentin is required for metastasis, and vimentin loss generates lower grade primary tumors. Interestingly, in the KLV+/+ mice, vimentin was not expressed in tumor cells but in cancer-associated fibroblasts (CAFs) surrounding collective invasion packs (CIPs) of epithelial tumor cells, with significantly less CIPs in KLV-/- mice. CIPs correlate with tumor grade and are vimentin-negative and E-cadherin-positive, indicating a lack of cancer cell EMT. A similar heterotypic staining pattern was observed in human lung adenocarcinoma samples. In vitro studies show that vimentin is required for CAF motility to lead tumor cell invasion, supporting a vimentin-dependent model of collective invasion.Conclusions: These data show that vimentin is required for lung adenocarcinoma metastasis by maintaining heterotypic tumor cell-CAF interactions during collective invasion. Clin Cancer Res; 24(2); 420-32. ©2017 AACR.

Targeting adhesion signaling in KRAS, LKB1 mutant lung adenocarcinoma.

Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre-induced Kras and Lkb1 mutant genetically engineered mouse model (KLLenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KLLenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.

SOX4 Is Essential for Prostate Tumorigenesis Initiated by PTEN Ablation.

Understanding remains incomplete of the mechanisms underlying initiation and progression of prostate cancer, the most commonly diagnosed cancer in American men. The transcription factor SOX4 is overexpressed in many human cancers, including prostate cancer, suggesting it may participate in prostate tumorigenesis. In this study, we investigated this possibility by genetically deleting Sox4 in a mouse model of prostate cancer initiated by loss of the tumor suppressor Pten. We found that specific homozygous deletion of Sox4 in the adult prostate epithelium strongly inhibited tumor progression initiated by homozygous loss of Pten. Mechanistically, Sox4 ablation reduced activation of AKT and ß-catenin, leading to an attenuated invasive phenotype. Furthermore, SOX4 expression was induced by Pten loss as a result of the activation of PI3K-AKT-mTOR signaling, suggesting a positive feedback loop between SOX4 and PI3K-AKT-mTOR activity. Collectively, our findings establish that SOX4 is a critical component of the PTEN/PI3K/AKT pathway in prostate cancer, with potential implications for combination-targeted therapies against both primary and advanced prostate cancers.

Mutation of murine Sox4 untranslated regions results in partially penetrant perinatal lethality.

BACKGROUND: Sox4 is an essential gene, and genetic deletion results in embryonic lethality. In an effort to develop mice with tissue-specific deletion, we bred conditional knockout mice bearing LoxP recombination sites flanking the Sox4 gene, with the LoxP sites located in the Sox4 5'UTR and 3'UTR. RESULTS: The number of mice homozygous for this LoxP-flanked conditional knockout allele was far below the expected number, suggesting embryonic lethality with reduced penetrance. From over 200 animals bred, only 11% were homozygous Sox4(flox/flox) mice, compared to the expected Mendelian ratio of 25% (p<0.001). Moreover, there was a significant reduction in the number of female Sox4(flox/flox) mice (26%) relative to male Sox4(flox/flox) mice (p=0.0371). Reduced Sox4 expression in homozygous embryos was confirmed by in-situ hybridization and Quantitative real-time polymerase chain reaction (QPCR). CONCLUSION: LoxP sites in the 5' and 3' UTR of both alleles of Sox4 resulted in reduced, but variable expression of Sox4 message.

Mitochondrial genome instability and ROS enhance intestinal tumorigenesis in APC(Min/+) mice.

Alterations in mitochondrial oxidative phosphorylation have long been documented in tumors. Other types of mitochondrial dysfunction, including altered reactive oxygen species (ROS) production and apoptosis, also can contribute to tumorigenesis and cancer phenotypes. Furthermore, mutation and altered amounts of mitochondrial DNA (mtDNA) have been observed in cancer cells. However, how mtDNA instability per se contributes to cancer remains largely undetermined. Mitochondrial transcription factor A (TFAM) is required for expression and maintenance of mtDNA. Tfam heterozygous knock-out (Tfam(+/-)) mice show mild mtDNA depletion, but have no overt phenotypes. We show that Tfam(+/-) mouse cells and tissues not only possess less mtDNA but also increased oxidative mtDNA damage. Crossing Tfam(+/-) mice to the adenomatous polyposis coli multiple intestinal neoplasia (APC(Min/+)) mouse cancer model revealed that mtDNA instability increases tumor number and growth in the small intestine. This was not a result of enhancement of Wnt/ß-catenin signaling, but rather appears to involve a propensity for increased mitochondrial ROS production. Direct involvement of mitochondrial ROS in intestinal tumorigenesis was shown by crossing APC(Min/+) mice to those that have catalase targeted to mitochondria, which resulted in a significant reduction in tumorigenesis in the colon. Thus, mitochondrial genome instability and ROS enhance intestinal tumorigenesis and Tfam(+/-) mice are a relevant model to address the role of mtDNA instability in disease states in which mitochondrial dysfunction is implicated, such as cancer, neurodegeneration, and aging.

p120-Catenin is required for mouse vascular development.

RATIONALE: p120-catenin (p120) is an armadillo family protein that binds to the cytoplasmic domain of classical cadherins and prevents cadherin endocytosis. The role of p120 in vascular development is unknown. OBJECTIVE: The purpose of this study is to examine the role of p120 in mammalian vascular development by generating a conditionally mutant mouse lacking endothelial p120 and determining the effects of the knockout on vasculogenesis, angiogenic remodeling, and the regulation of endothelial cadherin levels. METHODS AND RESULTS: A conditional Cre/loxP gene deletion strategy was used to ablate p120 expression, using the Tie2 promoter to drive endothelial Cre recombinase expression. Mice lacking endothelial p120 died embryonically beginning at embryonic day 11.5. Major blood vessels appeared normal at embryonic day 9.5. However, both embryonic and extraembryonic vasculature of mutant animals were disorganized and displayed decreased microvascular density by embryonic day 11.5. Importantly, both vascular endothelial cadherin and N-cadherin levels were significantly reduced in vessels lacking p120. This decrease in cadherin expression was accompanied by reduced pericyte recruitment and hemorrhaging. Furthermore, p120-null cultured endothelial cells exhibited proliferation defects that could be rescued by exogenous expression of vascular endothelial cadherin. CONCLUSIONS: These findings reveal a fundamental role for p120 in regulating endothelial cadherin levels during vascular development, as well as microvascular patterning, vessel integrity, and endothelial cell proliferation. Loss of endothelial p120 results in lethality attributable to decreased microvascular density and hemorrhages.

Loss of extracellular superoxide dismutase leads to acute lung damage in the presence of ambient air: a potential mechanism underlying adult respiratory distress syndrome.

The extracellular superoxide dismutase 3 (SOD3) is highly expressed in both blood vessels and lungs. In different models of pulmonary injury, SOD3 is reduced; however, it is unclear whether this contributes to lung injury. To study the role of acute SOD3 reduction in lung injury, the SOD3 gene was deleted in adult mice by using the Cre-Lox technology. Acute reduction of SOD3 led to a fivefold increase in lung superoxide, marked inflammatory cell infiltration, a threefold increase in the arterial-alveolar gradient, respiratory acidosis, histological changes similar to those observed in adult respiratory distress syndrome, and 85% mortality. Treatment with the SOD mimetic MnTBAP and intranasal administration of SOD-containing polyketal microparticles reduced mortality, prevented the histological alterations, and reduced lung superoxide levels. To understand how mice with the SOD3 embryonic deletion survived without lung injury, gene array analysis was performed. These data demonstrated the up-regulation of 37 genes and down-regulation of nine genes, including those involved in cell signaling, inflammation, and gene transcription in SOD3-/- mice compared with either mice with acute SOD3 reduction or wild-type controls. These studies show that SOD3 is essential for survival in the presence of ambient oxygen and that acute loss of this enzyme can lead to severe lung damage. Strategies either to prevent SOD3 inactivation or to augment its levels might prove useful in the treatment of acute lung injury.

Identification of thioredoxin-2 as a regulator of the mitochondrial permeability transition.

Thioredoxin-2 (Trx2) is a multifunctional, mitochondria-specific protein, which inhibits cell death. The mitochondrial permeability transition (MPT) is a distinct mechanism for cell death activated by oxidants and linked to both necrotic and apoptotic morphologies. We studied mitochondria from Trx2 transgenic mice to determine whether Trx2 protects against oxidant-induced MPT. All experiments were performed in isolated mitochondria. Results showed that Trx2 protected against MPT induced by exogenously added peroxide. Unexpectedly, Trx2 also protected against the MPT induced by Ca(2+) in the absence of added peroxide. The results indicate that in addition to protecting against oxidative stress, Trx2 is an endogenous regulator of the MPT.

NFAR-1 and -2 modulate translation and are required for efficient host defense.

We report here that the alternatively spliced nuclear factors associated with double-stranded RNA, NFAR-1 (90 kDa) and -2 (110 kDa), are involved in retaining cellular transcripts in intranuclear foci and can regulate the export of mRNA to the cytoplasm. Furthermore, the NFAR proteins were found to remain associated with exported ribonucleoprotein complexes. Loss of NFAR function, which was embryonic-lethal, caused an increase in protein synthesis rates, an effect augmented by the presence of the mRNA export factors TAP, p15, or Rae1. Significantly, NFAR depletion in normal murine fibroblasts rendered these cells dramatically susceptible to vesicular stomatitis virus replication. Collectively, our data demonstrate that the NFARs exert influence on mRNA trafficking and the modulation of translation rates and may constitute an innate immune translational surveillance mechanism important in host defense countermeasures against virus infection.

Non-invasive bioluminescent detection of prostate cancer growth and metastasis in a bigenic transgenic mouse model.

BACKGROUND: We previously established a bioluminescent transgenic mouse model, sPSA-Luc, with luciferase gene expression restricted to the prostate under the control of the supra prostate-specific antigen (sPSA) promoter. We now assess the feasibility of generating bigenic mice, TRAMP-Luc, with the sPSA-Luc as the founder strain crossbred with TRAMP (transgenic adenocarcinoma mouse prostate) mice, to evaluate non-invasively the metastatic potential of prostate tumors. METHODS: TRAMP-Luc mice were obtained as [C57BL/6 TRAMP x FVB sPSA-Luc] F1 offspring. Tumor development in 10 TRAMP-Luc males was followed by bioluminescence imaging from 8 to 24 weeks of age. Immunohistochemical (IHC) staining for T antigen (Tg), androgen receptor (AR), luciferase and/or pathological analysis verified the tumor distribution in the imaged tissues including prostate gland, lymph node and bone. RESULTS: Group I animals that presented with no grossly visible tumors showed prostate-confined bioluminescence with slightly increased signal intensity with age. Group II animals that developed large tumors displayed a widely distributed and biphasic bioluminescence pattern. The peak was reached between 10 and 14 weeks of age, then markedly decreased or even disappeared beyond week 16, except for one mouse that showed an increased bioluminescence signal at the jaw bone and hind limbs at week 22. These tumors were shown by IHC to contain Tg but lost AR and luciferase beyond week 16 in poorly differentiated prostate tumors. CONCLUSION: A direct correlation between bioluminescence emission and AR expression was found in TRAMP-Luc tumor progression model. This model allows non-invasive imaging of prostate cancer metastases to bone and soft tissues.

Conditional expression of human 15-lipoxygenase-1 in mouse prostate induces prostatic intraepithelial neoplasia: the FLiMP mouse model.

The incidence and mortality of prostate cancer (PCa) vary greatly in different geographic regions, for which lifestyle factors, such as dietary fat intake, have been implicated. Human 15-lipoxygenase-1 (h15-LO-1), which metabolizes polyunsaturated fatty acids, is a highly regulated, tissue-specific, lipid-peroxidating enzyme that functions in physiological membrane remodeling and in the pathogenesis of atherosclerosis, inflammation, and carcinogenesis. We have shown that aberrant overexpression of 15-LO-1 occurs in human PCa, particularly high-grade PCa, and in high-grade prostatic intraepithelial neoplasia (HGPIN), and that the murine orthologue is increased in SV40-based genetically engineered mouse (GEM) models of PCa, such as LADY and TRansgenic Adenocarcinoma of Mouse Prostate. To further define the role of 15-LO-1 in prostate carcinogenesis, we established a novel GEM model with targeted overexpression of h15-LO-1 in the prostate [human fifteen lipoxygenase-1 in mouse prostate (FLiMP)]. We used a Cre- mediated and a loxP-mediated recombination strategy to target h15-LO-1 specifically to the prostate of C57BL/6 mice. Wild-type (wt), FLiMP+/-, and FLiMP+/+ mice aged 7 to 21, 24 to 28, and 35 weeks were characterized by histopathology, immunohistochemistry (IHC), and DNA/RNA and enzyme analyses. Compared to wt mice, h15-LO-1 enzyme activity was increased similarly in both homozygous FLiMP+/+ and hemizygous FLiMP+/- prostates. Dorsolateral and ventral prostates of FLiMP mice showed focal and progressive epithelial hyperplasia with nuclear atypia, indicative of the definition of mouse prostatic intraepithelial neoplasia (mPIN) according to the National Cancer Institute. These foci showed increased proliferation by Ki-67 IHC. No progression to invasive PCa was noted up to 35 weeks. By IHC, h15-LO-1 expression was limited to luminal epithelial cells, with increased expression in mPIN foci (similar to human HGPIN). In summary, targeted overexpression of h15-LO-1 (a gene overexpressed in human PCa and HGPIN) to mouse prostate is sufficient to promote epithelial proliferation and mPIN development. These results support 15-LO-1 as having a role in prostate tumor initiation and as an early target for dietary or other prevention strategies. The FLiMP mouse model should also be useful in crosses with other GEM models to further define the combinations of molecular alterations necessary for PCa progression.

Disabled-2 is essential for endodermal cell positioning and structure formation during mouse embryogenesis.

The signal transduction adapter protein Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The brain-specific Disabled-1 (Dab1) functions in positional organization of brain cells during development. Dab2 is widely distributed and is highly expressed in many epithelial cell types. The dab2 gene was interrupted by in-frame insertion of beta-galactosidase (LacZ) in embryonic stem cells and transgenic mice were produced. Dab2 expression was first observed in the primitive endoderm at E4.5, immediately following implantation. The homozygous Dab2-deficient mutant is embryonic lethal (earlier than E6.5) due to defective cell positioning and structure formation of the visceral endoderm. In E5.5 dab2 (-/-) conceptus, visceral endoderm-like cells are present in the deformed primitive egg cylinder; however, the visceral endoderm cells are not organized, the cells of the epiblast have not expanded, and the proamniotic cavity fails to form. Disorganization of the visceral endodermal layer is evident, as cells with positive visceral endoderm markers are scattered throughout the dab2 (-/-) conceptus. Only degenerated remains were observed at E6.5 for dab2 (-/-) embryos, and by E7.5, the defective embryos were completely reabsorbed. In blastocyst in vitro culture, initially cells with characteristics of endoderm, trophectoderm, and inner cell mass were observed in the outgrowth of the hatched dab2 (-/-) blastocysts. However, the dab2 (-/-) endodermal cells are much more dispersed and disorganized than those from wild-type blastocysts, the inner cell mass fails to expand, and the outgrowth degenerates by day 7. Thus, Dab2 is required for visceral endodermal cell organization during early mouse development. The absence of an organized visceral endoderm in Dab2-deficient conceptus leads to the growth failure of the inner cell mass. We suggest that Dab2 functions in a signal pathway to regulate endodermal cell organization using endocytosis of ligands from the blastocoel cavity as a positioning cue.

CCR6 expression distinguishes mouse myeloid and lymphoid dendritic cell subsets: demonstration using a CCR6 EGFP knock-in mouse.

The homing properties of subsets of lymphocytes and dendritic cells (DC) are regulated in part by the profile of chemokine receptors expressed. To determine how CCR6 influences cell trafficking, a mutant allele of the mouse CCR6 gene was produced that includes an enhanced green fluorescent protein (EGFP) reporter under the control of the CCR6 promoter. In mice heterozygous for the EGFP/CCR6 knock-in, CCR6 expression was detected on all mature B cells, subpopulations of splenic CD4(+) and CD8(+) T cells, and on some CD11c(+) DC. Most CD11b(+) myeloid DC expressed CCR6, but CD8alpha(+) lymphoid DC were negative for CCR6. Among myeloid DC, the CD4(+) subset was uniformly positive for CCR6 expression and the CD4(-) subset was mostly CCR6 positive. Epidermal Langerhans cells (LC) also expressed CCR6, but at lower levels than splenic myeloid DC. Culture of bone marrow precursors from the knock-in mice with GM-CSF for 4 to 6 days led to the appearance of a subset of CD11c(+) DC expressing CCR6. The differences in CCR6 expression among the major DC subsets indicate that CCR6 and its chemokine ligand MIP-3alpha participate in determining the positioning of DC subsets in epithelial and lymphoid tissues.