CTL-associated antigen 4 (CTLA4) blockade releases inhibitory controls on T cell activation and proliferation, inducing antitumor immunity in both preclinical and early clinical trials. We examined the mechanisms of action of anti-CTLA4 and a GM-CSF–transduced tumor cell vaccine (Gvax) and their impact on the balance of effector T cells (Teffs) and Tregs in an in vivo model of B16/BL6 melanoma. Tumor challenge increased the frequency of Tregs in lymph nodes, and untreated tumors became infiltrated by CD4+Foxp3– and CD4+Foxp3+ T cells but few CD8+ T cells. Anti-CTLA4 did not deplete Tregs or permanently impair their function but acted in a cell-intrinsic manner on both Tregs and Teffs, allowing them to expand, most likely in response to self antigen. While Gvax primed the tumor-reactive Teff compartment, inducing activation, tumor infiltration, and a delay in tumor growth, the combination with CTLA4 blockade induced greater infiltration and a striking change in the intratumor balance of Tregs and Teffs that directly correlated with tumor rejection. The data suggest that Tregs control both CD4+ and CD8+ T cell activity within the tumor, highlight the importance of the intratumor ratio of effectors to regulators, and demonstrate inversion of the ratio and correlation with tumor rejection during Gvax/anti-CTLA4 immunotherapy.
Sergio A. Quezada, Karl S. Peggs, Michael A. Curran, James P. Allison
Metastasis is the major cause of cancer morbidity, but strategies for direct interference with invasion processes are lacking. Dedifferentiated, late-stage tumor cells secrete multiple factors that represent attractive targets for therapeutic intervention. Here we show that metastatic potential of oncogenic mammary epithelial cells requires an autocrine PDGF/PDGFR loop, which is established as a consequence of TGF-β–induced epithelial-mesenchymal transition (EMT), a faithful in vitro correlate of metastasis. The cooperation of autocrine PDGFR signaling with oncogenic Ras hyperactivates PI3K and is required for survival during EMT. Autocrine PDGFR signaling also contributes to maintenance of EMT, possibly through activation of STAT1 and other distinct pathways. Inhibition of PDGFR signaling interfered with EMT and caused apoptosis in murine and human mammary carcinoma cell lines. Consequently, overexpression of a dominant-negative PDGFR or application of the established cancer drug STI571 interfered with experimental metastasis in mice. Similarly, in mouse mammary tumor virus–Neu (MMTV-Neu) transgenic mice, TGF-β enhanced metastasis of mammary tumors, induced EMT, and elevated PDGFR signaling. Finally, expression of PDGFRα and -β correlated with invasive behavior in human mammary carcinomas. Thus, autocrine PDGFR signaling plays an essential role during cancer progression, suggesting a novel application of STI571 to therapeutically interfere with metastasis.
Martin Jechlinger, Andreas Sommer, Richard Moriggl, Peter Seither, Norbert Kraut, Paola Capodiecci, Michael Donovan, Carlos Cordon-Cardo, Hartmut Beug, Stefan Grünert
Identification of specific gene expression signatures characteristic of oncogenic pathways is an important step toward molecular classification of human malignancies. Aberrant activation of the Met signaling pathway is frequently associated with tumor progression and metastasis. In this study, we defined the Met-dependent gene expression signature using global gene expression profiling of WT and Met-deficient primary mouse hepatocytes. Newly identified transcriptional targets of the Met pathway included genes involved in the regulation of oxidative stress responses as well as cell motility, cytoskeletal organization, and angiogenesis. To assess the importance of a Met-regulated gene expression signature, a comparative functional genomic approach was applied to 242 human hepatocellular carcinomas (HCCs) and 7 metastatic liver lesions. Cluster analysis revealed that a subset of human HCCs and all liver metastases shared the Met-induced expression signature. Furthermore, the presence of the Met signature showed significant correlation with increased vascular invasion rate and microvessel density as well as with decreased mean survival time of HCC patients. We conclude that the genetically defined gene expression signatures in combination with comparative functional genomics constitute an attractive paradigm for defining both the function of oncogenic pathways and the clinically relevant subgroups of human cancers.
Pal Kaposi-Novak, Ju-Seog Lee, Luis Gòmez-Quiroz, Cédric Coulouarn, Valentina M. Factor, Snorri S. Thorgeirsson
T cells recognizing self antigens expressed by cancer cells are prevalent in the immune repertoire. However, activation of these autoreactive T cells is limited by weak signals that are incapable of fully priming naive T cells, creating a state of tolerance or ignorance. Even if T cell activation occurs, immunity can be further restricted by a dominant response directed at only a single epitope. Enhanced antigen presentation of multiple epitopes was investigated as a strategy to overcome these barriers. Specific point mutations that create altered peptide ligands were introduced into the gene encoding a nonimmunogenic tissue self antigen expressed by melanoma, tyrosinase-related protein-1 (Tyrp1). Deficient asparagine-linked glycosylation, which was caused by additional mutations, produced altered protein trafficking and fate that increased antigen processing. Immunization of mice with mutated Tyrp1 DNA elicited cross-reactive CD8+ T cell responses against multiple nonmutated epitopes of syngeneic Tyrp1 and against melanoma cells. These multispecific anti-Tyrp1 CD8+ T cell responses led to rejection of poorly immunogenic melanoma and prolonged survival when immunization was started after tumor challenge. These studies demonstrate how rationally designed DNA vaccines directed against self antigens for enhanced antigen processing and presentation reveal novel self epitopes and elicit multispecific T cell responses to nonimmunogenic, nonmutated self antigens, enhancing immunity against cancer self antigens.
José A. Guevara-Patiño, Manuel E. Engelhorn, Mary Jo Turk, Cailian Liu, Fei Duan, Gabrielle Rizzuto, Adam D. Cohen, Taha Merghoub, Jedd D. Wolchok, Alan N. Houghton
We have used a novel conditional transgenic system to study the mechanisms of angioproliferation induced by viral G protein–coupled receptor (vGPCR), the constitutively active chemokine receptor encoded by human herpesvirus 8 (HHV8, also known as Kaposi sarcoma herpesvirus). Using this system, we were able to control temporal expression of vGPCR and to monitor its expression in situ via the use of the surrogate marker LacZ. Upon treatment with doxycycline (DOX), cells expressing vGPCR and LacZ (vGPCR/LacZ+ cells) progressively accumulated in areas where angioproliferation was observed. Sorted vGPCR/LacZ+ cells from angiogenic lesions expressed markers characteristic of endothelial progenitor cells, produced angiogenic factors, and proliferated in vitro. Prolonged treatment of transgenic mice with DOX led to development of tumors in the skin of ears, tail, nose, and paws. vGPCR/LacZ+ cells were frequent in early lesions but scarce within these tumors. Finally, transfer of vGPCR/LacZ+ cells into Rag1–/– mice treated with DOX led to angioproliferation and, with time, to development of tumors containing both vGPCR/LacZ+ and vGPCR/LacZ– cells. Taken together, these results indicate that vGPCR triggers angioproliferation directly and suggest a novel role for this molecule in the pathogenesis of Kaposi sarcoma.
Marcos G. Grisotto, Alexandre Garin, Andrea P. Martin, Kristian K. Jensen, PokMan Chan, Stuart C. Sealfon, Sergio A. Lira
Previously we observed that neural cell adhesion molecule (NCAM) deficiency in β tumor cells facilitates metastasis into distant organs and local lymph nodes. Here, we show that NCAM-deficient β cell tumors grew leaky blood vessels with perturbed pericyte-endothelial cell-cell interactions and deficient perivascular deposition of ECM components. Conversely, tumor cell expression of NCAM in a fibrosarcoma model (T241) improved pericyte recruitment and increased perivascular deposition of ECM molecules. Together, these findings suggest that NCAM may limit tumor cell metastasis by stabilizing the microvessel wall. To directly address whether pericyte dysfunction increases the metastatic potential of solid tumors, we studied β cell tumorigenesis in primary pericyte-deficient Pdgfbret/ret mice. This resulted in β tumor cell metastases in distant organs and local lymph nodes, demonstrating a role for pericytes in limiting tumor cell metastasis. These data support a new model for how tumor cells trigger metastasis by perturbing pericyte-endothelial cell-cell interactions.
Xiaojie Xian, Joakim Håkansson, Anders Ståhlberg, Per Lindblom, Christer Betsholtz, Holger Gerhardt, Henrik Semb
The pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphomas is associated with independent chromosomal translocations that lead to the upregulation of either BCL10 or MALT1 or the generation of a fusion protein, cIAP2-MALT1. While both BCL10 and MALT1 are critically involved in antigen receptor–mediated NF-κB activation, the role of cIAP2 is not clear. Here we show that cIAP2 is a ubiquitin ligase (E3) of BCL10 and targets it for degradation, inhibiting antigen receptor–mediated cytokine production. cIAP2-MALT1 lacks E3 activity, and concomitantly, the BCL10 protein is stabilized in MALT lymphomas harboring this fusion. Furthermore, BCL10 and cIAP2-MALT1 synergistically activate NF-κB. These results reveal cIAP2 as an inhibitor of antigenic signaling and implicate its dysfunction in MALT lymphomas.
Shimin Hu, Ming-Qing Du, Sun-Mi Park, Allison Alcivar, Like Qu, Sanjeev Gupta, Jun Tang, Mathijs Baens, Hongtao Ye, Tae H. Lee, Peter Marynen, James L. Riley, Xiaolu Yang
Topoisomerase II (Topo II) inhibitors are cell cycle–specific DNA-damaging agents and often correlate with secondary leukemia with chromosomal translocations involving the mixed-lineage leukemia/myeloid lymphoid leukemia (MLL) gene on chromosome 11 band q23 (11q23). In spite of the clinical importance, the molecular mechanism for this chromosomal translocation has yet to be elucidated. In this study, we employed 2-color FISH and detected intracellular chromosomal translocations induced by etoposide treatment. Cells such as ataxia-telangiectasia mutated–deficient fibroblasts and U2OS cells, in which the early G2/M checkpoint after treatment with low concentrations of etoposide has been lost, executed mitosis with etoposide-induced DNA double-strand breaks, and 2-color FISH signals located on either side of the MLL gene were segregated in the postmitotic G1 phase. Long-term culture of cells that had executed mitosis under etoposide treatment showed frequent structural abnormalities of chromosome 11. These findings provide convincing evidence for Topo II inhibitor–induced 11q23 translocation. Our study also suggests an important role of the early G2/M checkpoint in preventing fixation of chromosomal abnormalities and reveals environmental and genetic risk factors for the development of chromosome 11 translocations, namely, low concentrations of Topo II inhibitors and dysfunctional early G2/M checkpoint control.
Shinichiro Nakada, Yoko Katsuki, Issei Imoto, Tetsuji Yokoyama, Masayuki Nagasawa, Johji Inazawa, Shuki Mizutani
Recent gene profiling studies have identified a new breast cancer subtype, the basal-like group, which expresses genes characteristic of basal epithelial cells and is associated with poor clinical outcomes. However, the genes responsible for the aggressive behavior observed in this group are largely unknown. Here we report that the small heat shock protein α-basic–crystallin (αB-crystallin) was commonly expressed in basal-like tumors and predicted poor survival in breast cancer patients independently of other prognostic markers. We also demonstrate that overexpression of αB-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, αB-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing αB-crystallin by RNA interference inhibited these abnormalities. αB-Crystallin overexpression also induced EGF- and anchorage-independent growth, increased cell migration and invasion, and constitutively activated the MAPK kinase/ERK (MEK/ERK) pathway. Moreover, the transformed phenotype conferred by αB-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing αB-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that αB-crystallin is a novel oncoprotein expressed in basal-like breast carcinomas that independently predicts shorter survival. Our data also implicate the MEK/ERK pathway as a potential therapeutic target for these tumors.
Jose V. Moyano, Joseph R. Evans, Feng Chen, Meiling Lu, Michael E. Werner, Fruma Yehiely, Leslie K. Diaz, Dmitry Turbin, Gamze Karaca, Elizabeth Wiley, Torsten O. Nielsen, Charles M. Perou, Vincent L. Cryns
Recent studies have established distinctive serum polypeptide patterns through mass spectrometry (MS) that reportedly correlate with clinically relevant outcomes. Wider acceptance of these signatures as valid biomarkers for disease may follow sequence characterization of the components and elucidation of the mechanisms by which they are generated. Using a highly optimized peptide extraction and matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) MS–based approach, we now show that a limited subset of serum peptides (a signature) provides accurate class discrimination between patients with 3 types of solid tumors and controls without cancer. Targeted sequence identification of 61 signature peptides revealed that they fall into several tight clusters and that most are generated by exopeptidase activities that confer cancer type–specific differences superimposed on the proteolytic events of the ex vivo coagulation and complement degradation pathways. This small but robust set of marker peptides then enabled highly accurate class prediction for an external validation set of prostate cancer samples. In sum, this study provides a direct link between peptide marker profiles of disease and differential protease activity, and the patterns we describe may have clinical utility as surrogate markers for detection and classification of cancer. Our findings also have important implications for future peptide biomarker discovery efforts.
Josep Villanueva, David R. Shaffer, John Philip, Carlos A. Chaparro, Hediye Erdjument-Bromage, Adam B. Olshen, Martin Fleisher, Hans Lilja, Edi Brogi, Jeff Boyd, Marta Sanchez-Carbayo, Eric C. Holland, Carlos Cordon-Cardo, Howard I. Scher, Paul Tempst