Regulatory T Cells in Tumor-Associated Tertiary Lymphoid Structures Suppress Anti-tumor T Cell Responses.

Infiltration of regulatory T (Treg) cells into many tumor types correlates with poor patient prognoses. However, mechanisms of intratumoral Treg cell function remain to be elucidated. We investigated Treg cell function in a genetically engineered mouse model of lung adenocarcinoma and found that Treg cells suppressed anti-tumor responses in tumor-associated tertiary lymphoid structures (TA-TLSs). TA-TLSs have been described in human lung cancers, but their function remains to be determined. TLSs in this model were spatially associated with >90% of tumors and facilitated interactions between T cells and tumor-antigen-presenting dendritic cells (DCs). Costimulatory ligand expression by DCs and T cell proliferation rates increased in TA-TLSs upon Treg cell depletion, leading to tumor destruction. Thus, we propose that Treg cells in TA-TLSs can inhibit endogenous immune responses against tumors, and targeting these cells might provide therapeutic benefit for cancer patients.

α5 and αv integrins cooperate to regulate vascular smooth muscle and neural crest functions in vivo.

The RGD-binding α5 and αv integrins have been shown to be key regulators of vascular smooth muscle cell (vSMC) function in vitro. However, their role on vSMCs during vascular development in vivo remains unclear. To address this issue, we have generated mice that lack α5, αv or both α5 and αv integrins on their vSMCs, using the SM22α-Cre transgenic mouse line. To our surprise, neither α5 nor αv mutants displayed any obvious vascular defects during embryonic development. By contrast, mice lacking both α5 and αv integrins developed interrupted aortic arches, large brachiocephalic/carotid artery aneurysms and cardiac septation defects, but developed extensive and apparently normal vasculature in the skin. Cardiovascular defects were also found, along with cleft palates and ectopically located thymi, in Wnt1-Cre α5/αv mutants, suggesting that α5 and αv cooperate on neural crest-derived cells to control the remodelling of the pharyngeal arches and the septation of the heart and outflow tract. Analysis of cultured α5/αv-deficient vSMCs suggests that this is achieved, at least in part, through proper assembly of RGD-containing extracellular matrix proteins and the correct incorporation and activation of latent TGF-ß.

Small RNA combination therapy for lung cancer.

MicroRNAs (miRNAs) and siRNAs have enormous potential as cancer therapeutics, but their effective delivery to most solid tumors has been difficult. Here, we show that a new lung-targeting nanoparticle is capable of delivering miRNA mimics and siRNAs to lung adenocarcinoma cells in vitro and to tumors in a genetically engineered mouse model of lung cancer based on activation of oncogenic Kirsten rat sarcoma viral oncogene homolog (Kras) and loss of p53 function. Therapeutic delivery of miR-34a, a p53-regulated tumor suppressor miRNA, restored miR-34a levels in lung tumors, specifically down-regulated miR-34a target genes, and slowed tumor growth. The delivery of siRNAs targeting Kras reduced Kras gene expression and MAPK signaling, increased apoptosis, and inhibited tumor growth. The combination of miR-34a and siRNA targeting Kras improved therapeutic responses over those observed with either small RNA alone, leading to tumor regression. Furthermore, nanoparticle-mediated small RNA delivery plus conventional, cisplatin-based chemotherapy prolonged survival in this model compared with chemotherapy alone. These findings demonstrate that RNA combination therapy is possible in an autochthonous model of lung cancer and provide preclinical support for the use of small RNA therapies in patients who have cancer.

CRISPR-mediated direct mutation of cancer genes in the mouse liver.

The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the ß-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of ß-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

Integrin-α5ß1 is not required for mural cell functions during development of blood vessels but is required for lymphatic-blood vessel separation and lymphovenous valve formation.

Integrin α5ß1 is essential for vascular development but it remains unclear precisely where and how it functions. Here, we report that deletion of the gene encoding the integrin-α5 subunit (Itga5) using the Pdgfrb-Cre transgenic mouse line, leads to oedema, haemorrhage and increased levels of embryonic lethality. Unexpectedly, these defects were not caused by loss of α5 from Pdgfrb-Cre expressing mural cells (pericytes and vascular smooth muscle cells), which wrap around the endothelium and stabilise blood vessels, nor by defects in the heart or great vessels, but were due to abnormal development of the lymphatic vasculature. Reminiscent of the pathologies seen in the human lymphatic malformation, fetal cystic hygroma, α5 mutants display defects both in the separation of their blood and lymphatic vasculature and in the formation of the lymphovenous valves. As a consequence, α5-deficient mice develop dilated, blood-filled lymphatic vessels and lymphatic capillaries that are ectopically covered with smooth muscle cells. Analysis of the expression of Pdgfrb during lymphatic development suggests that these defects probably arise from loss of α5ß1 integrin in subsets of specialised Prox1(+)Pdgfrb(+) venous endothelial cells that are essential for the separation of the jugular lymph sac from the cardinal vein and formation of the lymphovenous valve leaflets.

p53 constrains progression to anaplastic thyroid carcinoma in a Braf-mutant mouse model of papillary thyroid cancer.

Anaplastic thyroid carcinoma (ATC) has among the worst prognoses of any solid malignancy. The low incidence of the disease has in part precluded systematic clinical trials and tissue collection, and there has been little progress in developing effective therapies. v-raf murine sarcoma viral oncogene homolog B (BRAF) and tumor protein p53 (TP53) mutations cooccur in a high proportion of ATCs, particularly those associated with a precursor papillary thyroid carcinoma (PTC). To develop an adult-onset model of BRAF-mutant ATC, we generated a thyroid-specific CreER transgenic mouse. We used a Cre-regulated Braf(V600E) mouse and a conditional Trp53 allelic series to demonstrate that p53 constrains progression from PTC to ATC. Gene expression and immunohistochemical analyses of murine tumors identified the cardinal features of human ATC including loss of differentiation, local invasion, distant metastasis, and rapid lethality. We used small-animal ultrasound imaging to monitor autochthonous tumors and showed that treatment with the selective BRAF inhibitor PLX4720 improved survival but did not lead to tumor regression or suppress signaling through the MAPK pathway. The combination of PLX4720 and the mapk/Erk kinase (MEK) inhibitor PD0325901 more completely suppressed MAPK pathway activation in mouse and human ATC cell lines and improved the structural response and survival of ATC-bearing animals. This model expands the limited repertoire of autochthonous models of clinically aggressive thyroid cancer, and these data suggest that small-molecule MAPK pathway inhibitors hold clinical promise in the treatment of advanced thyroid carcinoma.

Nkx2-1 represses a latent gastric differentiation program in lung adenocarcinoma.

Tissue-specific differentiation programs become dysregulated during cancer evolution. The transcription factor Nkx2-1 is a master regulator of pulmonary differentiation that is downregulated in poorly differentiated lung adenocarcinoma. Here we use conditional murine genetics to determine how the identity of lung epithelial cells changes upon loss of their master cell-fate regulator. Nkx2-1 deletion in normal and neoplastic lungs causes not only loss of pulmonary identity but also conversion to a gastric lineage. Nkx2-1 is likely to maintain pulmonary identity by recruiting transcription factors Foxa1 and Foxa2 to lung-specific loci, thus preventing them from binding gastrointestinal targets. Nkx2-1-negative murine lung tumors mimic mucinous human lung adenocarcinomas, which express gastric markers. Loss of the gastrointestinal transcription factor Hnf4α leads to derepression of the embryonal proto-oncogene Hmga2 in Nkx2-1-negative tumors. These observations suggest that loss of both active and latent differentiation programs is required for tumors to reach a primitive, poorly differentiated state.

Nuclear factor I/B is an oncogene in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive cancer often diagnosed after it has metastasized. Despite the need to better understand this disease, SCLC remains poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation.

Uncoupling cancer mutations reveals critical timing of p53 loss in sarcomagenesis.

It is well accepted that cancer develops following the sequential accumulation of multiple alterations, but how the temporal order of events affects tumor initiation and/or progression remains largely unknown. Here, we describe a mouse model that allows for temporally distinct cancer mutations. By integrating a Flp-inducible allele of K-ras(G12D) with established methods for Cre-mediated p53 deletion, we were able to separately control the mutation of these commonly associated cancer genes in vitro and in vivo. We show that delaying p53 deletion relative to K-ras(G12D) activation reduced tumor burden in a mouse model of soft-tissue sarcoma, suggesting that p53 strongly inhibits very early steps of transformation in the muscle. Furthermore, using in vivo RNA interference, we implicate the p53 target gene p21 as a critical mediator in this process, highlighting cell-cycle arrest as an extremely potent tumor suppressor mechanism.

Suppression of lung adenocarcinoma progression by Nkx2-1.

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras(LSL-G12D/+);p53(flox/flox) mice initiates lung adenocarcinoma development. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor.

Endogenous T cell responses to antigens expressed in lung adenocarcinomas delay malignant tumor progression.

Neoantigens derived from somatic mutations in tumors may provide a critical link between the adaptive immune system and cancer. Here, we describe a system to introduce exogenous antigens into genetically engineered mouse lung cancers to mimic tumor neoantigens. We show that endogenous T cells respond to and infiltrate tumors, significantly delaying malignant progression. Despite continued antigen expression, T cell infiltration does not persist and tumors ultimately escape immune attack. Transplantation of cell lines derived from these lung tumors or prophylactic vaccination against the autochthonous tumors, however, results in rapid tumor eradication or selection of tumors that lose antigen expression. These results provide insight into the dynamic nature of the immune response to naturally arising tumors.

Endothelial alpha5 and alphav integrins cooperate in remodeling of the vasculature during development.

Integrin cell adhesion receptors and fibronectin, one of their extracellular matrix ligands, have been demonstrated to be important for angiogenesis using functional perturbation studies and complete knockout mouse models. Here, we report on the roles of the alpha5 and alphav integrins, which are the major endothelial fibronectin receptors, in developmental angiogenesis. We generated an integrin alpha5-floxed mouse line and ablated alpha5 integrin in endothelial cells. Unexpectedly, endothelial-specific knockout of integrin alpha5 has no obvious effect on developmental angiogenesis. We provide evidence for genetic interaction between mutations in integrin alpha5 and alphav and for overlapping functions and compensation between these integrins and perhaps others. Nonetheless, in embryos lacking both alpha5 and alphav integrins in their endothelial cells, initial vasculogenesis and angiogenesis proceed normally, at least up to E11.5, including the formation of apparently normal embryonic vasculature and development of the branchial arches. However, in the absence of endothelial alpha5 and alphav integrins, but not of either alone, there are extensive defects in remodeling of the great vessels and heart resulting in death at ~E14.5. We also found that fibronectin assembly is somewhat affected in integrin alpha5 knockout endothelial cells and markedly reduced in integrin alpha5/alphav double-knockout endothelial cell lines. Therefore, neither alpha5 nor alphav integrins are required in endothelial cells for initial vasculogenesis and angiogenesis, although they are required for remodeling of the heart and great vessels. These integrins on other cells, and/or other integrins on endothelial cells, might contribute to fibronectin assembly and vascular development.

Chronic cisplatin treatment promotes enhanced damage repair and tumor progression in a mouse model of lung cancer.

Chemotherapy resistance is a major obstacle in cancer treatment, yet the mechanisms of response to specific therapies have been largely unexplored in vivo. Employing genetic, genomic, and imaging approaches, we examined the dynamics of response to a mainstay chemotherapeutic, cisplatin, in multiple mouse models of human non-small-cell lung cancer (NSCLC). We show that lung tumors initially respond to cisplatin by sensing DNA damage, undergoing cell cycle arrest, and inducing apoptosis-leading to a significant reduction in tumor burden. Importantly, we demonstrate that this response does not depend on the tumor suppressor p53 or its transcriptional target, p21. Prolonged cisplatin treatment promotes the emergence of resistant tumors with enhanced repair capacity that are cross-resistant to platinum analogs, exhibit advanced histopathology, and possess an increased frequency of genomic alterations. Cisplatin-resistant tumors express elevated levels of multiple DNA damage repair and cell cycle arrest-related genes, including p53-inducible protein with a death domain (Pidd). We demonstrate a novel role for PIDD as a regulator of chemotherapy response in human lung tumor cells.

GPR56 plays varying roles in endogenous cancer progression.

GPR56, a non-classical adhesion receptor, was previously reported to suppress tumor growth and metastasis in xenograft models using human melanoma cell lines. To understand whether GPR56 plays similar roles in the development of endogenous tumors, we analyzed cancer progression in Gpr56 (-/-) mice using a variety of transgenic cancer models. Our results showed that GPR56 suppressed prostate cancer progression in the TRAMP model on a mixed genetic background, similar to its roles in progression of melanoma xenografts. However, its roles in other cancer types appeared to be complex. It had marginal effects on tumor onset of mammary tumors in the MMTV-PyMT model, but had no effects on subsequent tumor progression in either the MMTV-PyMT mice or the melanoma model, Ink4a/Arf (-/-) tyr-Hras. These results indicate diverse roles of GPR56 in cancer progression and provide the first genetic evidence for the involvement of an adhesion GPCR in endogenous cancer development.

Context-dependent transformation of adult pancreatic cells by oncogenic K-Ras.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies. To investigate the cellular origin(s) of this cancer, we determined the effect of PDAC-relevant gene mutations in distinct cell types of the adult pancreas. We show that a subpopulation of Pdx1-expressing cells is susceptible to oncogenic K-Ras-induced transformation without tissue injury, whereas insulin-expressing endocrine cells are completely refractory to transformation under these conditions. However, chronic pancreatic injury can alter their endocrine fate and allow them to serve as the cell of origin for exocrine neoplasia. These results suggest that one mechanism by which inflammation and/or tissue damage can promote neoplasia is by altering the fate of differentiated cells that are normally refractory to oncogenic stimulation.

Genetic ablation of alphav integrins in epithelial cells of the eyelid skin and conjunctiva leads to squamous cell carcinoma.

Integrin-mediated cell adhesion and signaling events are essential for the proper development and homeostasis of most epithelial tissues. Dysregulation of integrin expression and function can cause abnormal epithelial cell proliferation and/or differentiation, contributing to the pathogenesis of malignant epithelial cancers. Here we report on the use of a conditional knockout strategy exploiting the Cre/Lox technology to study the in vivo functions of alphav integrins during epithelial cell proliferation and differentiation. We show that genetic ablation of alphav integrin expression in basal epithelial cells of the eyelid skin and conjunctiva causes the formation of tumors that are strikingly similar to the malignant epithelial cancer, squamous cell carcinoma. These data suggest a mechanism whereby alphav integrins normally suppress epithelial cell proliferation, likely via adhesion to ECM ligands, as well as by the modulation of intracellular signaling cascades. We propose that alphav gene deletion eliminates normal integrin-mediated growth suppression, ultimately leading to cellular transformation and tumorigenesis. Hence, these studies reveal a novel tumor suppressor-like function of alphav integrins and provide a genetically tractable mouse model for studying the pathogenesis of squamous cell carcinoma and related cancers of epithelial origin, as well as to test and develop novel therapeutic compounds to treat or prevent squamous cell carcinoma of the skin.

Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters.

miR-17 approximately 92, miR-106b approximately 25, and miR-106a approximately 363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-1792 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17 approximately 92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17 approximately 92 cluster is also essential for B cell development. Absence of miR-17 approximately 92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b approximately 25 or miR-106a approximately 363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b approximately 25 and miR-17 approximately 92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17 approximately 92 and its functions during B lymphopoiesis and lung development.

Analyses of the role of endogenous SPARC in mouse models of prostate and breast cancer.

Secreted protein, acidic and rich in cysteine (SPARC, also known as osteonectin or BM-40) is a glycoprotein component of the extracellular matrix that has been reported to be involved with a variety of cellular processes. Although SPARC expression levels are frequently altered in a variety of tumor types, the exact implications of deregulated SPARC expression--whether it promotes, inhibits or has no effect on tumor progression--have remained unclear. Our recent gene expression analyses have shown that SPARC is significantly downregulated in highly metastatic human prostate cancer cells. To test the role of endogenous SPARC in tumorigenesis directly, we examined cancer progression and metastasis in SPARC(+/-) and SPARC(-/-) mice using two separate transgenic mouse tumor models: transgenic adenocarcinoma of the mouse prostate (TRAMP) and murine mammary tumor virus-polyoma middle T (MMTV-PyMT). Surprisingly, in both instances, we found that loss of SPARC had no significant effects on tumor initiation, progression or metastasis. Tumor angiogenesis and collagen deposition were also largely unaffected. Our results indicate that, although differential SPARC expression may be a useful marker of aggressive, metastasis-prone tumors, loss of SPARC is not sufficient either to promote or to inhibit cancer progression in two spontaneous mouse tumor models.

Ulcerative colitis and autoimmunity induced by loss of myeloid alphav integrins.

The gastrointestinal tract is constantly challenged by foreign antigens and commensal bacteria but nonetheless is able to maintain a state of immunological quiescence. Recent advances have highlighted the importance of active suppression by regulatory lymphocytes and immunosuppressive cytokines in controlling mucosal immunity. Failures of these mechanisms contribute to the development of inflammatory bowel disease, but how these regulatory networks are established remains unclear. Here, we demonstrate key roles for alphav integrins in regulation of mucosal immunity. We report that deletion of alphav in the immune system causes severe colitis, autoimmunity, and cancer. Mice lacking immune cell alphav have fewer regulatory T (Treg) cells in the colon and corresponding increases in activated T cells and T cell cytokine production, leading to colitis. Using conditional gene targeting, we demonstrate that this is specifically attributable to loss of alphav from myeloid cells. Furthermore, we show that gut-associated macrophages and dendritic cells fail both to remove apoptotic cells efficiently and to induce Treg cells. Our results identify a vital role for myeloid alphav integrins in generating mucosal Treg cells and emphasize the importance of antigen-presenting cells in establishing immune tolerance.