Multiple myeloma in humans is frequently associated with mast cell infiltration and neovascularization, which correlate directly with disease severity, but the mechanisms underlying this relationship remain unclear. Here, we report that primary murine mast cells express angiopoietin-1 (Ang-1) and low levels of VEGF-A but not Ang-2 and that 2 established murine plasmacytoma cell lines express high levels of VEGF-A but little or no Ang-1 or Ang-2. An in vivo angiogenesis assay using extracellular matrix components shows that mast cells and plasmacytoma cells, together, promote marked neovascularization composed of dilated vessels, which is prevented by neutralization of VEGF-A and Ang-1 but is only partially reduced by neutralization of either VEGF-A or Ang-1. Mast cells within extracellular matrix components express Ang-1, and recombinant Ang-1 together with plasmacytoma cells promotes extracellular matrix neovascularization similar to that induced by mast cells. A transplantation assay shows that primary mast cells accelerate tumor growth by established plasmacytoma cell lines and that neutralization of Ang-1 alone or with VEGF-A reduces significantly the growth of plasmacytomas containing mast cells. These results demonstrate that mast cell–derived Ang-1 promotes the growth of plasmacytomas by stimulating neovascularization and provide further evidence supporting a causal relationship between inflammation and tumor growth.
Takayuki Nakayama, Lei Yao, Giovanna Tosato
Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics.
Mona D. Lee, Yuhong She, Michael J. Soskis, Christopher P. Borella, Jeffrey R. Gardner, Paula A. Hayes, Benzon M. Dy, Mark L. Heaney, Mark R. Philips, William G. Bornmann, Francis M. Sirotnak, David A. Scheinberg
Evasion of apoptosis is a hallmark of cancer, but the molecular circuitries of this process are not understood. Here we show that survivin, a member of the inhibitor of apoptosis gene family that is overexpressed in cancer, exists in a novel mitochondrial pool in tumor cells. In response to cell death stimulation, mitochondrial survivin is rapidly discharged in the cytosol, where it prevents caspase activation and inhibits apoptosis. Selective targeting of survivin to mitochondria enhances colony formation in soft agar, accelerates tumor growth in immunocompromised animals, and abolishes tumor cell apoptosis in vivo. Therefore, mitochondrial survivin orchestrates a novel pathway of apoptosis inhibition, which contributes to tumor progression.
Takehiko Dohi, Elena Beltrami, Nathan R. Wall, Janet Plescia, Dario C. Altieri
Phosphorylation of the cell adhesion protein CEACAM1 increases insulin sensitivity and decreases insulin-dependent mitogenesis in vivo. Here we show that CEACAM1 is a substrate of the EGFR and that upon being phosphorylated, CEACAM1 reduces EGFR-mediated growth of transfected Cos-7 and MCF-7 cells in response to EGF. Using transgenic mice overexpressing a phosphorylation-defective CEACAM1 mutant in liver (L-SACC1), we show that the effect of CEACAM1 on EGF-dependent cell proliferation is mediated by its ability to bind to and sequester Shc, thus uncoupling EGFR signaling from the ras/MAPK pathway. In L-SACC1 mice, we also show that impaired CEACAM1 phosphorylation leads to ligand-independent increase of EGFR-mediated cell proliferation. This appears to be secondary to visceral obesity and the metabolic syndrome, with increased levels of output of free fatty acids and heparin-binding EGF-like growth factor from the adipose tissue of the mice. Thus, L-SACC1 mice provide a model for the mechanistic link between increased cell proliferation in states of impaired metabolism and visceral obesity.
George A. Abou-Rjaily, Sang Jun Lee, Denisa May, Qusai Y. Al-Share, Anthony M. DeAngelis, Randall J. Ruch, Michael Neumaier, Holger Kalthoff, Sue-Hwa Lin, Sonia M. Najjar
A mouse model involving the human papillomavirus type-16 oncogenes develops cervical cancers by lesional stages analogous to those in humans. In this study the angiogenic phenotype was characterized, revealing intense angiogenesis in high-grade cervical intraepithelial neoplasias (CIN-3) and carcinomas. MMP-9, a proangiogenic protease implicated in mobilization of VEGF, appeared in the stroma concomitant with the angiogenic switch, expressed by infiltrating macrophages, similar to what has been observed in humans. Preclinical trials sought to target MMP-9 and angiogenesis with a prototypical MMP inhibitor and with a bisphosphonate, zoledronic acid (ZA), revealing both to be antiangiogenic, producing effects comparable to a Mmp9 gene KO in impairing angiogenic switching, progression of premalignant lesions, and tumor growth. ZA therapy increased neoplastic epithelial and endothelial cell apoptosis without affecting hyperproliferation, indicating that ZA was not antimitotic. The analyses implicated cellular and molecular targets of ZA’s actions: ZA suppressed MMP-9 expression by infiltrating macrophages and inhibited metalloprotease activity, reducing association of VEGF with its receptor on angiogenic endothelial cells. Given its track record in clinical use with limited toxicity, ZA holds promise as an “unconventional” MMP-9 inhibitor for antiangiogenic therapy of cervical cancer and potentially for additional cancers and other diseases where MMP-9 expression by infiltrating macrophages is evident.
Enrico Giraudo, Masahiro Inoue, Douglas Hanahan
Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a wide spectrum of human malignancies. Here, we have assessed the effect of Stat3 deficiency on skin tumor development using the 2-stage chemical carcinogenesis model. The epidermis of Stat3-deficient mice showed a significantly reduced proliferative response following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) because of a defect in G1-to-S-phase cell cycle progression. Treatment with the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) resulted in a significant increase in the number of keratinocyte stem cells undergoing apoptosis in the bulge region of hair follicles of Stat3-deficient mice compared with nontransgenic littermates. Notably, Stat3-deficient mice were completely resistant to skin tumor development when DMBA was used as the initiator and TPA as the promoter. Abrogation of Stat3 function using a decoy oligonucleotide inhibited the growth of initiated keratinocytes possessing an activated Ha-ras gene, both in vitro and in vivo. In addition, injection of Stat3 decoy into skin tumors inhibited their growth. To our knowledge, these data provide the first evidence that Stat3 is required for de novo epithelial carcinogenesis, through maintaining the survival of DNA-damaged stem cells and through mediating and maintaining the proliferation necessary for clonal expansion of initiated cells during tumor promotion. Collectively, these data suggest that, in addition to its emerging role as a target for cancer therapy, Stat3 may also be a target for cancer prevention strategies.
Keith Syson Chan, Shigetoshi Sano, Kaoru Kiguchi, Joanne Anders, Nobuyasu Komazawa, Junji Takeda, John DiGiovanni
CD4+ T cells play important roles in orchestrating host immune responses against cancer and infectious diseases. Although EBV-encoded nuclear antigen 1–specific (EBNA1-specific) CD4+ T cells have been implicated in controlling the growth of EBV-associated tumors such as Burkitt lymphoma (BL) in vitro, direct evidence for their in vivo function remains elusive due to the lack of an appropriate experimental BL model. Here, we describe the development of a mouse EBNA1-expressing BL tumor model and the identification of 2 novel MHC H-2I-Ab–restricted T cell epitopes derived from EBNA1. Using our murine BL tumor model and the relevant peptides, we show that vaccination of mice with EBNA1 peptide–loaded DCs can elicit CD4+ T cell responses. These EBNA1-specific CD4+ T cells recognized peptide-pulsed targets as well as EBNA1-expressing tumor cells and were necessary and sufficient for suppressing tumor growth in vivo. By contrast, EBNA1 peptide–reactive CD8+ T cells failed to recognize tumor cells and did not contribute to protective immunity. These studies represent what we believe to be the first demonstration that EBNA1-specific CD4+ T cells can suppress tumor growth in vivo, which suggests that CD4+ T cells play an important role in generating protective immunity against EBV-associated cancer.
Tihui Fu, Kui Shin Voo, Rong-Fu Wang
The MHC class I chain–related molecules (MICs) have previously been shown to be induced on most epithelial tumor cells. Engagement of MIC by the activating immune receptor NKG2D triggers NK cells and augments antigen-specific CTL anti-tumor immunity. The MIC-NKG2D system was proposed to participate in epithelial tumor immune surveillance. Paradoxically, studies suggest that tumors may evade MIC-NKG2D–mediated immunity by MIC shedding–induced impairment of effector cell function. Here we demonstrate the first evidence to our knowledge of a significant correlation of MIC shedding and deficiency in NK cell function with the grade of disease in prostate cancer. MIC is widely expressed in prostate carcinoma. The presence of surface target MIC, however, is counteracted by shedding. A significant increase in serum levels of soluble MIC (sMIC) and deficiency in NK cell function was shown in patients with advanced cancer. Finally, the deficiency in NK cell function can be overcome by treatment with IL-2 or IL-15 in vitro. Our results suggest that (a) deficiency in MIC-NKG2D immune surveillance may contribute to prostate cancer progression, (b) sMIC may be a novel biomarker for prostate cancer, and (c) using cytokines to restore MIC-NKG2D–mediated immunity may have clinical significance for prostate cancer in cell-based adaptive immunotherapy.
Jennifer D. Wu, Lily M. Higgins, Alexander Steinle, David Cosman, Kathy Haugk, Stephen R. Plymate
The transcription factor NF-κB is activated in a range of human cancers and is thought to promote tumorigenesis, mainly due to its ability to protect transformed cells from apoptosis. To investigate the role of NF-κB in epithelial plasticity and metastasis, we utilized a well-characterized in vitro/in vivo model of mammary carcinogenesis that depends on the collaboration of the Ha-Ras oncoprotein and TGF-β. We show here that the IKK-2/IκBα/NF-κB pathway is required for the induction and maintenance of epithelial-mesenchymal transition (EMT). Inhibition of NF-κB signaling prevented EMT in Ras-transformed epithelial cells, while activation of this pathway promoted the transition to a mesenchymal phenotype even in the absence of TGF-β. Furthermore, inhibition of NF-κB activity in mesenchymal cells caused a reversal of EMT, suggesting that NF-κB is essential for both the induction and maintenance of EMT. In line with the importance of EMT for invasion, blocking of NF-κB activity abrogated the metastatic potential of mammary epithelial cells in a mouse model system. Collectively, these data provide evidence of an essential role for NF-κB during distinct steps of breast cancer progression and suggest that the cooperation of Ras- and TGF-β–dependent signaling pathways in late-stage tumorigenesis depends critically on NF-κB activity.
Margit A. Huber, Ninel Azoitei, Bernd Baumann, Stefan Grünert, Andreas Sommer, Hubert Pehamberger, Norbert Kraut, Hartmut Beug, Thomas Wirth
Cachexia is a syndrome characterized by wasting of skeletal muscle and contributes to nearly one-third of all cancer deaths. Cytokines and tumor factors mediate wasting by suppressing muscle gene products, but exactly which products are targeted by these cachectic factors is not well understood. Because of their functional relevance to muscle architecture, such targets are presumed to represent myofibrillar proteins, but whether these proteins are regulated in a general or a selective manner is also unclear. Here we demonstrate, using in vitro and in vivo models of muscle wasting, that cachectic factors are remarkably selective in targeting myosin heavy chain. In myotubes and mouse muscles, TNF-α plus IFN-γ strongly reduced myosin expression through an RNA-dependent mechanism. Likewise, colon-26 tumors in mice caused the selective reduction of this myofibrillar protein, and this reduction correlated with wasting. Under these conditions, however, loss of myosin was associated with the ubiquitin-dependent proteasome pathway, which suggests that mechanisms used to regulate the expression of muscle proteins may be cachectic factor specific. These results shed new light on cancer cachexia by revealing that wasting does not result from a general downregulation of muscle proteins but rather is highly selective as to which proteins are targeted during the wasting state.
Swarnali Acharyya, Katherine J. Ladner, Lori L. Nelsen, Jeffrey Damrauer, Peter J. Reiser, Steven Swoap, Denis C. Guttridge