The retinoblastoma tumor suppressor (RB) protein is functionally inactivated in the majority of human cancers and is aberrant in one-third of all breast cancers. RB regulates G1/S-phase cell-cycle progression and is a critical mediator of antiproliferative signaling. Here the specific impact of RB deficiency on E2F-regulated gene expression, tumorigenic proliferation, and the response to 2 distinct lines of therapy was investigated in breast cancer cells. RB knockdown resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the RB deficiency compromised the short-term cell-cycle inhibition following cisplatin, ionizing radiation, and antiestrogen therapy. In the context of DNA-damaging agents, this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of antiestrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of aberrations in RB function were recapitulated by ectopic E2F expression, indicating that control of downstream target genes was an important determinant of the observed responses. Specific analyses of an RB gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy. Thus, because the RB pathway is a critical determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for directing therapy in the treatment of breast cancer.
Emily E. Bosco, Ying Wang, Huan Xu, Jack T. Zilfou, Karen E. Knudsen, Bruce J. Aronow, Scott W. Lowe, Erik S. Knudsen
The TGF-β signaling pathway has a complex role in regulating mammary carcinogenesis. Here we demonstrate that the type III TGF-β receptor (TβRIII, or betaglycan), a ubiquitously expressed TGF-β coreceptor, regulated breast cancer progression and metastasis. Most human breast cancers lost TβRIII expression, with loss of heterozygosity of the TGFBR3 gene locus correlating with decreased TβRIII expression. TβRIII expression decreased during breast cancer progression, and low TβRIII levels predicted decreased recurrence-free survival in breast cancer patients. Restoring TβRIII expression in breast cancer cells dramatically inhibited tumor invasiveness in vitro and tumor invasion, angiogenesis, and metastasis in vivo. TβRIII appeared to inhibit tumor invasion by undergoing ectodomain shedding and producing soluble TβRIII, which binds and sequesters TGF-β to decrease TGF-β signaling and reduce breast cancer cell invasion and tumor-induced angiogenesis. Our results indicate that loss of TβRIII through allelic imbalance is a frequent genetic event during human breast cancer development that increases metastatic potential.
Mei Dong, Tam How, Kellye C. Kirkbride, Kelly J. Gordon, Jason D. Lee, Nadine Hempel, Patrick Kelly, Benjamin J. Moeller, Jeffrey R. Marks, Gerard C. Blobe
Anaplastic large cell lymphomas (ALCLs) represent a subset of lymphomas in which the anaplastic lymphoma kinase (ALK) gene is frequently fused to the nucleophosmin (NPM) gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo and that ALK activity is strictly required for the survival of ALK-positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK-positive ALCL cell lines, abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell-permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPβ and the antiapoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK-positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions.
Roberto Piva, Elisa Pellegrino, Michela Mattioli, Luca Agnelli, Luigia Lombardi, Francesco Boccalatte, Giulia Costa, Bruce A. Ruggeri, Mangeng Cheng, Roberto Chiarle, Giorgio Palestro, Antonino Neri, Giorgio Inghirami
Piyali Dasgupta, Shipra Rastogi, Smitha Pillai, Dalia Ordonez-Ercan, Mark Morris, Eric Haura, Srikumar Chellappan
Overexpression of pituitary tumor–transforming 1 (PTTG1) is associated with thyroid cancer. We found elevated PTTG1 levels in the thyroid tumors of a mouse model of follicular thyroid carcinoma (TRβPV/PV mice). Here we examined the molecular mechanisms underlying elevated PTTG1 levels and the contribution of increased PTTG1 to thyroid carcinogenesis. We showed that PTTG1 was physically associated with thyroid hormone β receptor (TRβ) as well as its mutant, designated PV. Concomitant with thyroid hormone–induced (T3-induced) degradation of TRβ, PTTG1 proteins were degraded by the proteasomal machinery, but no such degradation occurred when PTTG1 was associated with PV. The degradation of PTTG1/TRβ was activated by the direct interaction of the liganded TRβ with steroid receptor coactivator 3 (SRC-3), which recruits proteasome activator PA28γ. PV, which does not bind T3, could not interact directly with SRC-3/PA28γ to activate proteasome degradation, resulting in elevated PTTG1 levels. The accumulated PTTG1 impeded mitotic progression in cells expressing PV. Our results unveil what we believe to be a novel mechanism by which PTTG1, an oncogene, is regulated by the liganded TRβ. The loss of this regulatory function in PV led to an aberrant accumulation of PTTG1 disrupting mitotic progression that could contribute to thyroid carcinogenesis.
Hao Ying, Fumihiko Furuya, Li Zhao, Osamu Araki, Brian L. West, John A. Hanover, Mark C. Willingham, Sheue-yann Cheng
Cylindromatosis (CYLD) is a deubiquitinating enzyme that is altered in patients with familial cylindromatosis, a condition characterized by numerous benign adnexal tumors. However, the regulatory function of CYLD remains unsettled. Here we show that the development of B cells, T cells, and myeloid cells was unaffected in CYLD-deficient mice, but that the activation of these cells with mediators of innate and adaptive immunity resulted in enhanced NF-κB and JNK activity associated with increased TNF receptor–associated factor 2 (TRAF2) and NF-κB essential modulator (NEMO) ubiquitination. CYLD-deficient mice were more susceptible to induced colonic inflammation and showed a dramatic increase in the incidence of tumors compared with controls in a colitis-associated cancer model. These results suggest that CYLD limits inflammation and tumorigenesis by regulating ubiquitination in vivo.
Jun Zhang, Brigid Stirling, Stephane T. Temmerman, Chi A. Ma, Ivan J. Fuss, Jonathan M.J. Derry, Ashish Jain
Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%–60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less α–SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.
Michael R. Mancuso, Rachel Davis, Scott M. Norberg, Shaun O’Brien, Barbara Sennino, Tsutomu Nakahara, Virginia J. Yao, Tetsuichiro Inai, Peter Brooks, Bruce Freimark, David R. Shalinsky, Dana D. Hu-Lowe, Donald M. McDonald
We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%–7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. MllPTD/WT mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. MllPTD/WT mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML.
Adrienne M. Dorrance, Shujun Liu, Weifeng Yuan, Brian Becknell, Kristy J. Arnoczky, Martin Guimond, Matthew P. Strout, Lan Feng, Tatsuya Nakamura, Li Yu, Laura J. Rush, Michael Weinstein, Gustavo Leone, Lizhao Wu, Amy Ferketich, Susan P. Whitman, Guido Marcucci, Michael A. Caligiuri
EGFR is frequently mutated and amplified in lung adenocarcinomas sensitive to EGFR inhibitors gefitinib and erlotinib. A secondary mutation, T790M, has been associated with acquired resistance but has not been shown to be sufficient to render EGFR mutant/amplified lung cancers resistant to EGFR inhibitors. We created a model for studying acquired resistance to gefitinib by prolonged exposure of a gefitinib-sensitive lung carcinoma cell line (H3255; EGFR mutated and amplified) to gefitinib in vitro. The resulting resistant cell line acquired a T790M mutation in a small fraction of the amplified alleles that was undetected by direct sequencing and identified only by a highly sensitive HPLC-based technique. In gefitinib-sensitive lung cancer cells with EGFR mutations and amplifications, exogenous introduction of EGFR T790M effectively conferred resistance to gefitinib and continued ErbB-3/PI3K/Akt signaling when in cis to an activating mutation. Moreover, continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110α E545K, was sufficient to abrogate gefitinib-induced apoptosis. These findings suggest that allelic dilution of biologically significant resistance mutations may go undetected by direct sequencing in cancers with amplified oncogenes and that restoration of PI3K activation via either a T790M mutation or other mechanisms can provide resistance to gefitinib.
Jeffrey A. Engelman, Toru Mukohara, Kreshnik Zejnullahu, Eugene Lifshits, Ana M. Borrás, Christopher-Michael Gale, George N. Naumov, Beow Y. Yeap, Emily Jarrell, Jason Sun, Sean Tracy, Xiaojun Zhao, John V. Heymach, Bruce E. Johnson, Lewis C. Cantley, Pasi A. Jänne
Active suppression of tumor-specific T lymphocytes can limit the efficacy of immune surveillance and immunotherapy. While tumor-recruited CD11b+ myeloid cells are known mediators of tumor-associated immune dysfunction, the true nature of these suppressive cells and the fine biochemical pathways governing their immunosuppressive activity remain elusive. Here we describe a population of circulating CD11b+IL-4 receptor α+ (CD11b+IL-4Rα+), inflammatory-type monocytes that is elicited by growing tumors and activated by IFN-γ released from T lymphocytes. CD11b+IL-4Rα+ cells produced IL-13 and IFN-γ and integrated the downstream signals of these cytokines to trigger the molecular pathways suppressing antigen-activated CD8+ T lymphocytes. Analogous immunosuppressive circuits were active in CD11b+ cells present within the tumor microenvironment. These suppressor cells challenge the current idea that tumor-conditioned immunosuppressive monocytes/macrophages are alternatively activated. Moreover, our data show how the inflammatory response elicited by tumors had detrimental effects on the adaptive immune system and suggest novel approaches for the treatment of tumor-induced immune dysfunctions.
Giovanna Gallina, Luigi Dolcetti, Paolo Serafini, Carmela De Santo, Ilaria Marigo, Mario P. Colombo, Giuseppe Basso, Frank Brombacher, Ivan Borrello, Paola Zanovello, Silvio Bicciato, Vincenzo Bronte