Chronic infection and associated inflammation are key contributors to human carcinogenesis. Ulcerative colitis (UC) is an oxyradical overload disease and is characterized by free radical stress and colon cancer proneness. Here we examined tissues from noncancerous colons of ulcerative colitis patients to determine (a) the activity of two base excision–repair enzymes , AAG, the major 3-methyladenine DNA glycosylase, and APE1, the major apurinic site endonuclease; and (b) the prevalence of microsatellite instability (MSI). AAG and APE1 were significantly increased in UC colon epithelium undergoing elevated inflammation and MSI was positively correlated with their imbalanced enzymatic activities. These latter results were supported by mechanistic studies using yeast and human cell models in which overexpression of AAG and/or APE1 was associated with frameshift mutations and MSI. Our results are consistent with the hypothesis that the adaptive and imbalanced increase in AAG and APE1 is a novel mechanism contributing to MSI in patients with UC and may extend to chronic inflammatory or other diseases with MSI of unknown etiology.
Lorne J. Hofseth, Mohammed A. Khan, Mark Ambrose, Olga Nikolayeva, Meng Xu-Welliver, Maria Kartalou, S. Perwez Hussain, Richard B. Roth, Xiaoling Zhou, Leah E. Mechanic, Irit Zurer, Varda Rotter, Leona D. Samson, Curtis C. Harris
Malignant cells often display defects in autophagy, an evolutionarily conserved pathway for degrading long-lived proteins and cytoplasmic organelles. However, as yet, there is no genetic evidence for a role of autophagy genes in tumor suppression. The beclin 1 autophagy gene is monoallelically deleted in 40–75% of cases of human sporadic breast, ovarian, and prostate cancer. Therefore, we used a targeted mutant mouse model to test the hypothesis that monoallelic deletion of beclin 1 promotes tumorigenesis. Here we show that heterozygous disruption of beclin 1 increases the frequency of spontaneous malignancies and accelerates the development of hepatitis B virus–induced premalignant lesions. Molecular analyses of tumors in beclin 1 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Furthermore, beclin 1 heterozygous disruption results in increased cellular proliferation and reduced autophagy in vivo. These findings demonstrate that beclin 1 is a haplo-insufficient tumor-suppressor gene and provide genetic evidence that autophagy is a novel mechanism of cell-growth control and tumor suppression. Thus, mutation of beclin 1 or other autophagy genes may contribute to the pathogenesis of human cancers.
Xueping Qu, Jie Yu, Govind Bhagat, Norihiko Furuya, Hanina Hibshoosh, Andrea Troxel, Jeffrey Rosen, Eeva-Liisa Eskelinen, Noboru Mizushima, Yoshinori Ohsumi, Giorgio Cattoretti, Beth Levine
Prostate cancer is one of the most diagnosed and mortal cancers in western countries. A major clinical problem is the development of androgen-independent prostate cancer (AIPC) during antihormonal treatment. The molecular mechanisms underlying the change from androgen dependence to independence of these tumors are poorly understood and represent a challenge to develop new therapies. Based on genetic data showing amplification of the c-myc gene in AIPC, we studied the ability of c-myc to confer AIPC cell growth. Human androgen-dependent prostate cancer cells overexpressing c-myc grew independently of androgens and presented tumorigenic properties in androgen-depleted conditions. Analysis of signalling pathways by pharmacological inhibitors of the androgen receptor (AR) or by RNA interference directed against AR or c-myc showed that c-myc acted downstream of AR through multiple growth effectors. Thus c-myc is required for androgen-dependent growth and following ectopic expression can induce androgen-independent growth. Moreover, RNA interference directed against c-myc showed that growth of human AIPC cells, AR-positive or -negative, required c-myc expression. Furthermore, we showed that c-myc–overexpressing cells retain a functional p53 pathway and thus respond to etoposide.
David Bernard, Albin Pourtier-Manzanedo, Jesús Gil, David H. Beach
Tuberous sclerosis (TSC) is a familial tumor syndrome due to mutations in TSC1 or TSC2, in which progression to malignancy is rare. Primary Tsc2–/– murine embryo fibroblast cultures display early senescence with overexpression of p21CIP1/WAF1 that is rescued by loss of TP53. Tsc2–/–TP53–/– cells, as well as tumors from Tsc2+/– mice, display an mTOR-activation signature with constitutive activation of S6K, which is reverted by treatment with rapamycin. Rapamycin also reverts a growth advantage of Tsc2–/–TP53–/– cells. Tsc1/Tsc2 does not bind directly to mTOR, however, nor does it directly influence mTOR kinase activity or cellular phosphatase activity. There is a marked reduction in Akt activation in Tsc2–/–TP53–/– and Tsc1–/– cells in response to serum and PDGF, along with a reduction in cell ruffling. PDGFRα and PDGFRβ expression is markedly reduced in both the cell lines and Tsc mouse renal cystadenomas, and ectopic expression of PDGFRβ in Tsc2-null cells restores Akt phosphorylation in response to serum, PDGF, EGF, and insulin. This activation of mTOR along with downregulation of PDGFR PI3K-Akt signaling in cells lacking Tsc1 or Tsc2 may explain why these genes are rarely involved in human cancer. This is in contrast to PTEN, which is a negative upstream regulator of this pathway.
Hongbing Zhang, Gregor Cicchetti, Hiroaki Onda, Henry B. Koon, Kirsten Asrican, Natalia Bajraszewski, Francisca Vazquez, Christopher L. Carpenter, David J. Kwiatkowski
Tumor-infiltrating blood vessels deviate morphologically and biochemically from normal vessels, raising the prospect of selective pharmacological targeting. Current antiangiogenic approaches focus mainly on endothelial cells, but recent data imply that targeting pericytes may provide additional benefits. Further development of these concepts will require deeper insight into mechanisms of pericyte recruitment and function in tumors. Here, we applied genetic tools to decipher the function of PDGF-B and PDGF-Rβ in pericyte recruitment in a mouse fibrosarcoma model. In tumors transplanted into PDGF-B retention motif–deficient (pdgf-bret/ret) mice, pericytes were fewer and were partially detached from the vessel wall, coinciding with increased tumor vessel diameter and hemorrhaging. Transgenic PDGF-B expression in tumor cells was able to increase the pericyte density in both WT and pdgf-bret/ret mice but failed to correct the pericyte detachment in pdgf-bret/ret mice. Coinjection of exogenous pericytes and tumor cells showed that pericytes require PDGF-Rβ for recruitment to tumor vessels, whereas endothelial PDGF-B retention is indispensable for proper integration of pericytes in the vessel wall. Our data support the notion that pericytes serve an important function in tumor vessels and highlight PDGF-B and PDGF-Rβ as promising molecular targets for therapeutic intervention.
Alexandra Abramsson, Per Lindblom, Christer Betsholtz
The TGF-β signaling network plays a complex role in carcinogenesis because it has the potential to act as either a tumor suppressor or a pro-oncogenic pathway. Currently, it is not known whether TGF-β can switch from tumor suppressor to pro-oncogenic factor during the course of carcinogenic progression in a single cell lineage with a defined initiating oncogenic event or whether the specific nature of the response is determined by cell type and molecular etiology. To address this question, we have introduced a dominant negative type II TGF-β receptor into a series of genetically related human breast–derived cell lines representing different stages in the progression process. We show that decreased TGF-β responsiveness alone cannot initiate tumorigenesis but that it can cooperate with an initiating oncogenic lesion to make a premalignant breast cell tumorigenic and a low-grade tumorigenic cell line histologically and proliferatively more aggressive. In a high-grade tumorigenic cell line, however, reduced TGF-β responsiveness has no effect on primary tumorigenesis but significantly decreases metastasis. Our results demonstrate a causal role for loss of TGF-β responsiveness in promoting breast cancer progression up to the stage of advanced, histologically aggressive, but nonmetastatic disease and suggest that at that point TGF-β switches from tumor suppressor to prometastatic factor.
Binwu Tang, Mary Vu, Timberly Booker, Steven J. Santner, Fred R. Miller, Miriam R. Anver, Lalage M. Wakefield
Cripto, a cell surface–associated protein belonging to the EGF-CFC family of growth factor–like molecules, is overexpressed in many human solid tumors, including 70–80% of breast and colon tumors, yet how it promotes cell transformation is unclear. During embryogenesis, Cripto complexes with Alk4 via its unique cysteine-rich CFC domain to facilitate signaling by the TGF-β ligand Nodal. We report, for the first time to our knowledge, that Cripto can directly bind to another TGF-β ligand, Activin B, and that Cripto overexpression blocks Activin B growth inhibition of breast cancer cells. This result suggests a novel mechanism for antagonizing Activin signaling that could promote tumorigenesis by deregulating growth homeostasis. We show that an anti–CFC domain antibody, A8.G3.5, both disrupts Cripto-Nodal signaling and reverses Cripto blockade of Activin B–induced growth suppression by blocking Cripto’s association with either Alk4 or Activin B. In two xenograft models, testicular and colon cancer, A8.G3.5 inhibited tumor cell growth by up to 70%. Both Nodal and Activin B expression was found in the xenograft tumor, suggesting that either ligand could be promoting tumorigenesis. These data validate that functional blockade of Cripto inhibits tumor growth and highlight antibodies that block Cripto signaling mediated through its CFC domain as an important class of antibodies for further therapeutic development.
Heather B. Adkins, Caterina Bianco, Susan G. Schiffer, Paul Rayhorn, Mohammad Zafari, Anne E. Cheung, Olivia Orozco, Dian Olson, Antonella De Luca, Ling Ling Chen, Konrad Miatkowski, Chris Benjamin, Nicola Normanno, Kevin P. Williams, Matthew Jarpe, Doreen LePage, David Salomon, Michele Sanicola
IFN-α activates the signal transducer and activator of transcription (STAT) family of proteins; however, it is unknown whether IFN-α exerts its antitumor actions primarily through a direct effect on malignant cells or by stimulating the immune system. To investigate the contribution of STAT1 signaling within the tumor, we generated a STAT1-deficient melanoma cell line, AGS-1. We reconstituted STAT1 into AGS-1 cells by retroviral gene transfer. The resulting cell line (AGS-1STAT1) showed normal regulation of IFN-α–stimulated genes (e.g., H2k, ISG-54) as compared with AGS-1 cells infected with the empty vector (AGS-1MSCV). However, mice challenged with the AGS-1, AGS-1STAT1, and AGS-1MSCV cell lines exhibited nearly identical survival in response to IFN-α treatment, indicating that restored STAT1 signaling within the tumor did not augment the antitumor activity of IFN-α. In contrast, STAT1–/– mice could not utilize exogenous IFN-α to inhibit the growth of STAT1+/+ melanoma cells in either an intraperitoneal tumor model or in the adjuvant setting. The survival of tumor-bearing STAT1–/– mice was identical regardless of treatment (IFN-α or PBS). Additional cell depletion studies demonstrated that NK cells mediated the antitumor effects of IFN-α. Thus, STAT1-mediated gene regulation within immune effectors was necessary for mediating the antitumor effects of IFN-α in this experimental system.
Gregory B. Lesinski, Mirela Anghelina, Jason Zimmerer, Timothy Bakalakos, Brian Badgwell, Robin Parihar, Yan Hu, Brian Becknell, Gerard Abood, Abhik Ray Chaudhury, Cynthia Magro, Joan Durbin, William E. Carson III
The present study evaluates the potential of third-generation lentivirus vectors with respect to their use as in vivo–administered T cell vaccines. We demonstrate that lentivector injection into the footpad of mice transduces DCs that appear in the draining lymph node and in the spleen. In addition, a lentivector vaccine bearing a T cell antigen induced very strong systemic antigen-specific cytotoxic T lymphocyte (CTL) responses in mice. Comparative vaccination performed in two different antigen models demonstrated that in vivo administration of lentivector was superior to transfer of transduced DCs or peptide/adjuvant vaccination in terms of both amplitude and longevity of the CTL response. Our data suggest that a decisive factor for efficient T cell priming by lentivector might be the targeting of DCs in situ and their subsequent migration to secondary lymphoid organs. The combination of performance, ease of application, and absence of pre-existing immunity in humans make lentivector-based vaccines an attractive candidate for cancer immunotherapy.
Christoph Esslinger, Laurence Chapatte, Daniela Finke, Isabelle Miconnet, Philippe Guillaume, Frédéric Lévy, H. Robson MacDonald
The secretory factor VEGF-C has been directly implicated in various physiological processes during embryogenesis and human cancers. However, the importance of the conversion of its precursor proVEGF-C to mature VEGF-C in tumorigenesis, and vessel formation and the identity of the protease(s) that regulate these processes is/are not known. The intracellular processing of proVEGF-C that occurs within the dibasic motif HSIIRR227SL suggests the involvement of the proprotein convertases (PCs) in this process. In addition, furin and VEGF-C were found to be coordinately expressed in adult mouse tissues. Cotransfection of the furin-deficient colon carcinoma cell line LoVo with proVEGF-C and different PC members revealed that furin, PC5, and PC7 are candidate VEGF-C convertases. This finding is consistent with the in vitro digestions of an internally quenched synthetic fluorogenic peptide mimicking the cleavage site of proVEGF-C (220Q-VHSIIRR↓SLP230). The processing of proVEGF-C is blocked by the inhibitory prosegments of furin, PC5, and PACE4, as well as by furin-motif variants of α2-macroglobulin and α1-antitrypsin. Subcutaneous injection of CHO cells stably expressing VEGF-C into nude mice enhanced angiogenesis and lymphangiogenesis, but not tumor growth. In contrast, expression of proVEGF-C obtained following mutation of the cleavage site (HSIIRR227SL to HSIISS227SL) inhibits angiogenesis and lymphangiogenesis as well as tumor growth. Our findings demonstrate the processing of proVEGF-C by PCs and highlight the potential use of PC inhibitors as agents for inhibiting malignancies induced by VEGF-C.
Geraldine Siegfried, Ajoy Basak, James A. Cromlish, Suzanne Benjannet, Jadwiga Marcinkiewicz, Michel Chrétien, Nabil G. Seidah, Abdel-Majid Khatib