Knockout and transgenic studies in mice demonstrate that normal somatic tissues redundantly express 3 cyclin D proteins, whereas tumor cells seem dependent on a single overexpressed cyclin D. Thus, selective suppression of the individual cyclin D deregulated in a tumor represents a biologically valid approach to targeted cancer therapy. In multiple myeloma, overexpression of 1 of the cyclin D proteins is a ubiquitous feature, unifying at least 7 different initiating genetic events. We demonstrate here that RNAi of genes encoding cyclin D1 and cyclin D2 (CCND1 and CCND2, respectively) inhibits proliferation and is progressively cytotoxic in human myeloma cells. By screening a chemical library using a cell-based assay for inhibition of CCND2 trans-activation, we identified the plant cytokinin kinetin riboside as an inhibitor of CCND2 trans-activation. Kinetin riboside induced marked suppression of CCND2 transcription and rapidly suppressed cyclin D1 and D2 protein expression in primary myeloma cells and tumor lines, causing cell-cycle arrest, tumor cell–selective apoptosis, and inhibition of myeloma growth in xenografted mice. Mechanistically, kinetin riboside upregulated expression of transcription repressor isoforms of cAMP-response element modulator (CREM) and blocked both trans-activation of CCND2 by various myeloma oncogenes and cis-activation of translocated CCND1, suggesting induction of an overriding repressor activity that blocks multiple oncogenic pathways targeting cyclin D genes. These data support targeted repression of cyclin D genes as a therapeutic strategy for human malignancies.
Rodger E. Tiedemann, Xinliang Mao, Chang-Xin Shi, Yuan Xiao Zhu, Stephen E. Palmer, Michael Sebag, Ron Marler, Marta Chesi, Rafael Fonseca, P. Leif Bergsagel, Aaron D. Schimmer, A. Keith Stewart
The molecular pathogenesis of pediatric astrocytomas is still poorly understood. To further understand the genetic abnormalities associated with these tumors, we performed a genome-wide analysis of DNA copy number aberrations in pediatric low-grade astrocytomas by using array-based comparative genomic hybridization. Duplication of the BRAF protooncogene was the most frequent genomic aberration, and tumors with BRAF duplication showed significantly increased mRNA levels of BRAF and a downstream target, CCND1, as compared with tumors without duplication. Furthermore, denaturing HPLC showed that activating BRAF mutations were detected in some of the tumors without BRAF duplication. Similarly, a marked proportion of low-grade astrocytomas from adult patients also had BRAF duplication. Both the stable silencing of BRAF through shRNA lentiviral transduction and pharmacological inhibition of MEK1/2, the immediate downstream phosphorylation target of BRAF, blocked the proliferation and arrested the growth of cultured tumor cells derived from low-grade gliomas. Our findings implicate aberrant activation of the MAPK pathway due to gene duplication or mutation of BRAF as a molecular mechanism of pathogenesis in low-grade astrocytomas and suggest inhibition of the MAPK pathway as a potential treatment.
Stefan Pfister, Wibke G. Janzarik, Marc Remke, Aurélie Ernst, Wiebke Werft, Natalia Becker, Grischa Toedt, Andrea Wittmann, Christian Kratz, Heike Olbrich, Rezvan Ahmadi, Barbara Thieme, Stefan Joos, Bernhard Radlwimmer, Andreas Kulozik, Torsten Pietsch, Christel Herold-Mende, Astrid Gnekow, Guido Reifenberger, Andrey Korshunov, Wolfram Scheurlen, Heymut Omran, Peter Lichter
Current anticancer therapy is a delicate balance between elimination of malignant cells and harmful side effects for the host. In this study, we used a tumor-homing peptide to engineer anti-CD40 agonist antibodies and recombinant IL-2 such that they were selectively delivered into spontaneously arising tumors in a transgenic mouse model of islet cell carcinogenesis. Intravenous injection of these agents, either separately or together, led to accumulation in the vicinity of tumor neovessels without toxic side effects. Although both molecules are critical for adaptive immunity, the most profound effects were seen in endothelial cells. Combined, local anti-CD40 and IL-2 therapy reduced tumor vascularity and significantly delayed tumor growth in mice. Remarkably, tumor-bearing mice remained disease-free long-term when targeted anti-CD40 and IL-2 were combined with transfers of preactivated antitumor immune cells. In this therapeutic setting, triggering of CD40 on endothelial cells induced an inflammatory response of the vessel wall and facilitated effector cell accumulation in the tumor parenchyma while IL-2 promoted antigen-specific immune cell persistence. We believe this is a novel and highly effective anticancer approach, whereby tumor stroma is “conditioned” for enhanced immune cell entry and survival, facilitating immune-mediated tumor destruction and leading to a sustained antitumor response.
Juliana Hamzah, Delia Nelson, Gerd Moldenhauer, Bernd Arnold, Günter J. Hämmerling, Ruth Ganss
The pathogenic nature of cancer is attributed, at least in part, to the ability of tumors cells to induce systemic and local mechanisms of immune tolerance. However, we previously reported that tumor-free survival in up to 100% of tolerized HER-2/neu transgenic mice can be achieved by administration of neu-specific mAb concurrently with a HER-2/neu–expressing, GM-CSF–secreting whole cell vaccine. In this report, we show that one mechanism of improved antitumor activity induced by the combination of these 2 neu-targeted interventions was enhanced Fc-mediated activation of APCs. Specifically, in vivo studies demonstrated localization of radiolabeled neu-specific mAb at the vaccine site. Subsequently, increased accumulation of neu-specific mAb at the vaccine-draining lymph node correlated with increased vaccine cell uptake by DCs in vivo. This led to enhancement of CD8+ neu-specific T cell function in terms of proliferation, cytokine production, and central memory development. Thus, the administration of a neu-specific mAb with a neu-targeted GM-CSF–secreting tumor vaccine enhanced induction of neu-specific CD8+ T cells through Fc-mediated activation of DCs. This multimodality attack on the same tumor antigen may have the potential to overcome tolerance to self antigens and weaken the immunosuppressive networks within the tumor microenvironment.
Peter S. Kim, Todd D. Armstrong, Hong Song, Matthew E. Wolpoe, Vivian Weiss, Elizabeth A. Manning, Lan Qing Huang, Satoshi Murata, George Sgouros, Leisha A. Emens, R. Todd Reilly, Elizabeth M. Jaffee
Kirsteen H. Maclean, Frank C. Dorsey, John L. Cleveland, Michael B. Kastan
Yoshiyuki Takahashi, Nanae Harashima, Sachiko Kajigaya, Hisayuki Yokoyama, Elena Cherkasova, J. Philip McCoy, Ken-ichi Hanada, Othon Mena, Roger Kurlander, Abdul Tawab, Ramaprasad Srinivasan, Andreas Lundqvist, Elizabeth Malinzak, Nancy Geller, Michael I. Lerman, Richard W. Childs
CTLs have the potential to attack tumors, and adoptive transfer of CTLs can lead to tumor regression in mouse models and human clinical settings. However, the dynamics of tumor cell elimination during efficient T cell therapy is unknown, and it is unclear whether CTLs act directly by destroying tumor cells or indirectly by initiating the recruitment of innate immune cells that mediate tumor damage. To address these questions, we report real-time imaging of tumor cell apoptosis in vivo using intravital 2-photon microscopy and a Förster resonance energy transfer–based (FRET-based) reporter of caspase 3 activity. In a mouse model of solid tumor, we found that tumor regression after transfer of in vitro–activated CTLs occurred primarily through the direct action of CTLs on each individual tumor cell, with a minimal bystander effect. Surprisingly, the killing of 1 target cell by an individual CTL took an extended period of time, 6 hours on average, which suggested that the slow rate of killing intrinsically limits the efficiency of antitumor T cell responses. The ability to visualize when, where, and how tumor cells are killed in vivo offers new perspectives for understanding how immune effectors survey cancer cells and how local tumor microenvironments may subvert immune responses.
Béatrice Breart, Fabrice Lemaître, Susanna Celli, Philippe Bousso
Most patients (80%) with ovarian cancer (OvCa) present with metastatic disease. Attachment of OvCa cells to peritoneum and omentum represents the first rate-limiting step for metastatic spread. Therefore, identifying factors regulating cell attachment in the abdominal cavity is critical to the development of therapeutic agents. We show here that MMP-2 expression was upregulated in OvCa cells upon attachment to their microenvironment. Downregulation of MMP-2 mRNA or pharmacological inhibition of MMP-2 proteolytic function, in both human OvCa primary cells and cell lines, reduced attachment of OvCa cells to a 3D organotypic model of metastatic OvCa, full human omentum or peritoneum, and in vivo to mouse peritoneum and omentum. Absence of MMP-2 in the host did not alter OvCa adhesion, as determined utilizing mice harboring homozygous null mutations in either the Mmp2 or Mmp9 genes. Conversely, adhesion induced upregulation of MMP-2 mRNA in OvCa cells. MMP-2 inhibition in OvCa cells through pharmacological or antibody treatment prior to i.p. dissemination in nude mice significantly decreased tumor growth and metastasis and extended survival. MMP-2 enhanced peritoneal adhesion of OvCa cells through cleavage of ECM proteins fibronectin (FN) and vitronectin (Vn) into small fragments and increased binding of OvCa cells to these FN and Vn fragments and their receptors, α5β1 and αVβ3 integrin. These findings indicate that MMP-2 expressed by metastatic OvCa cells functionally regulates their attachment to peritoneal surfaces.
Hilary A. Kenny, Swayamjot Kaur, Lisa M. Coussens, Ernst Lengyel
Mobilization of endothelial progenitor cells (EPCs) from the bone marrow and their subsequent participation in neovessel formation are implicated in tumor growth and neovascularization. As the neurotransmitter dopamine (DA) modulates adult endothelial cell function, we hypothesized that DA might have a regulatory role in mobilization of EPCs from the bone marrow niche. We show that there was a significant decrease in bone marrow DA content and an increase in EPC mobilization in tumor-bearing mice associated with tumor neovascularization. DA treatment of tumor-bearing mice inhibited EPC mobilization and tumor growth through its D2 receptors, as DA treatment failed to inhibit EPC mobilization in tumor-bearing mice treated with a specific DA D2 receptor antagonist and in tumor-bearing mice lacking the D2 receptor. In addition, we found that DA, through D2 receptors, exerted its inhibitory effect on EPC mobilization through suppression of VEGFA-induced ERK1/ERK2 phosphorylation and MMP-9 synthesis. These findings reveal a new link between DA and EPC mobilization and suggest a novel use for DA and D2 agents in the treatment of cancer and other diseases involving neovessel formation.
Debanjan Chakroborty, Uttio Roy Chowdhury, Chandrani Sarkar, Rathindranath Baral, Partha Sarathi Dasgupta, Sujit Basu
Tumors elicit antitumor immune responses, but over time they evolve and can escape immune control through various mechanisms, including the loss of the antigen to which the response is directed. The escape of antigen-loss variants (ALVs) is a major obstacle to T cell–based immunotherapy for cancer. However, cancers can be cured if both the number of CTLs and the expression of antigen are high enough to allow targeting of not only tumor cells, but also the tumor stroma. Here, we showed that IFN-γ and TNF produced by CTLs were crucial for the elimination of established mouse tumors, including ALVs. In addition, both BM- and non-BM–derived stromal cells were required to express TNF receptors and IFN-γ receptors for the elimination of ALVs. Although IFN-γ and TNF were not required by CTLs for perforin-mediated killing of antigen-expressing tumor cells, the strong inference is that tumor antigen–specific CTLs must secrete IFN-γ and TNF for destruction of tumor stroma. Therefore, bystander killing of ALVs may result from IFN-γ and TNF acting on tumor stroma.
Bin Zhang, Theodore Karrison, Donald A. Rowley, Hans Schreiber