B cell chronic lymphocytic leukemia (CLL) is a disease of expanding monoclonal B cells whose B cell receptor (BCR) mutational status defines 2 subgroups; patients with mutated BCRs have a more favorable prognosis than those with unmutated BCRs. CLL B cells express a restricted BCR repertoire including antibodies with quasi-identical complementarity-determining region 3 (CDR3), which suggests specific antigen recognition. The antigens recognized by CLL antibodies may include autoantigens since about half of CLL B cells produce autoreactive antibodies. However, the distribution of autoreactive antibodies between Ig heavy-chain variable–unmutated (IgV-unmutated) CLL (UM-CLL) and IgV-mutated CLL (M-CLL) is unknown. To determine the role of antibody reactivity and the impact of somatic hypermutation (SHM) on CLL antibody specificity, we cloned and expressed in vitro recombinant antibodies from M- and UM-CLL B cells and tested their reactivity by ELISA. We found that UM-CLL B cells expressed highly polyreactive antibodies whereas most M-CLL B cells did not. When mutated nonautoreactive CLL antibody sequences were reverted in vitro to their germline counterparts, they encoded polyreactive and autoreactive antibodies. We concluded that both UM-CLLs and M-CLLs originate from self-reactive B cell precursors and that SHM plays an important role in the development of the disease by altering original BCR autoreactivity.
Maxime Hervé, Kai Xu, Yen-Shing Ng, Hedda Wardemann, Emilia Albesiano, Bradley T. Messmer, Nicholas Chiorazzi, Eric Meffre
We used a proteomic approach for identifying molecules involved in the pathogenesis of chronic lymphocytic leukemia (CLL). We investigated 14 patients who were completely concordant for IgVH mutational status (unmutated vs. mutated), CD38 expression (positive vs. negative), and clinical behavior (progressive vs. stable); these patients were characterized as having either poor or good prognoses. The 2 patient subsets differed in the expression of hematopoietic lineage cell-specific protein 1 (HS1). In patients with poor prognoses, most HS1 protein was constitutively phosphorylated, whereas only a fraction was phosphorylated in patients with good prognoses. This difference was investigated in a larger cohort of 26 unselected patients. The survival curve of all 40 patients analyzed revealed that patients with predominately phosphorylated HS1 experience a significantly shorter median survival time. As HS1 is a protein pivotal in the signal cascade triggered by B cell receptor (BCR) stimulation, we studied its pattern of expression following BCR engagement. Normal mature B cells stimulated by anti-IgM shifted the non- or less-phosphorylated form of HS1 toward the more phosphorylated form. Naive B cells showed both HS1 forms while memory B cells expressed mainly the phosphorylated fraction. These data indicate a central role for antigen stimulation in CLL and suggest a new therapeutic target for patients with aggressive disease.
Cristina Scielzo, Paolo Ghia, Antonio Conti, Angela Bachi, Giuseppe Guida, Massimo Geuna, Massimo Alessio, Federico Caligaris-Cappio
The interactions of transformed cells with the surrounding stromal cells are of importance for tumor progression and metastasis. The relevance of adipocyte-derived factors to breast cancer cell survival and growth is well established. However, it remains unknown which specific adipocyte-derived factors are most critical in this process. Collagen VI is abundantly expressed in adipocytes. Collagen–/– mice in the background of the mouse mammary tumor virus/polyoma virus middle T oncogene (MMTV-PyMT) mammary cancer model demonstrate dramatically reduced rates of early hyperplasia and primary tumor growth. Collagen VI promotes its growth-stimulatory and pro-survival effects in part by signaling through the NG2/chondroitin sulfate proteoglycan receptor expressed on the surface of malignant ductal epithelial cells to sequentially activate Akt and β-catenin and stabilize cyclin D1. Levels of the carboxyterminal domain of collagen VIα3, a proteolytic product of the full-length molecule, are dramatically upregulated in murine and human breast cancer lesions. The same fragment exerts potent growth-stimulatory effects on MCF-7 cells in vitro. Therefore, adipocytes play a vital role in defining the ECM environment for normal and tumor-derived ductal epithelial cells and contribute significantly to tumor growth at early stages through secretion and processing of collagen VI.
Puneeth Iyengar, Virginia Espina, Terence W. Williams, Ying Lin, David Berry, Linda A. Jelicks, Hyangkyu Lee, Karla Temple, Reed Graves, Jeffrey Pollard, Neeru Chopra, Robert G. Russell, Ram Sasisekharan, Bruce J. Trock, Marc Lippman, Valerie S. Calvert, Emanuel F. Petricoin III, Lance Liotta, Ekaterina Dadachova, Richard G. Pestell, Michael P. Lisanti, Paolo Bonaldo, Philipp E. Scherer
In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade.
Rosa Marina Melillo, Maria Domenica Castellone, Valentina Guarino, Valentina De Falco, Anna Maria Cirafici, Giuliana Salvatore, Fiorina Caiazzo, Fulvio Basolo, Riccardo Giannini, Mogens Kruhoffer, Torben Orntoft, Alfredo Fusco, Massimo Santoro
Lipid rafts are cholesterol- and sphingolipid-enriched microdomains in cell membranes that regulate phosphorylation cascades originating from membrane-bound proteins. In this study, we tested whether alteration of the cholesterol content of lipid rafts in prostate cancer (PCa) cell membranes affects cell survival mechanisms in vitro and in vivo. Simvastatin, a cholesterol synthesis inhibitor, lowered raft cholesterol content, inhibited Akt1 serine-threonine kinase (protein kinase Bα)/protein kinase B (Akt/PKB) pathway signaling, and induced apoptosis in caveolin- and PTEN-negative LNCaP PCa cells. Replenishing cell membranes with cholesterol reversed these inhibitory and apoptotic effects. Cholesterol also potentiated Akt activation in normal prostate epithelial cells, which were resistant to the apoptotic effects of simvastatin. Elevation of circulating cholesterol in SCID mice increased the cholesterol content and the extent of protein tyrosine phosphorylation in lipid rafts isolated from LNCaP/sHB xenograft tumors. Cholesterol elevation also promoted tumor growth, increased phosphorylation of Akt, and reduced apoptosis in the xenografts. Our results implicate membrane cholesterol in Akt signaling in both normal and malignant cells and provide evidence that PCa cells can become dependent on a cholesterol-regulated Akt pathway for cell survival.
Liyan Zhuang, Jayoung Kim, Rosalyn M. Adam, Keith R. Solomon, Michael R. Freeman
The mainstay in the management of invasive bladder cancer continues to be radical cystectomy. With regard to improvement of quality of life, however, therapies that preserve the bladder are desirable. We investigated the use of intravesical PLK-1 small interfering RNA (siRNA) against bladder cancer. Patients with bladder cancers expressing high levels of PLK-1 have a poor prognosis compared with patients with low expression. Using siRNA/cationic liposomes, the expression of endogenous PLK-1 could be suppressed in bladder cancer cells in a time- and dose-dependent manner. As a consequence, PLK-1 functions were disrupted. Inhibition of bipolar spindle formation, accumulation of cyclin B1, reduced cell proliferation, and induction of apoptosis were observed. In order to determine the efficacy of the siRNA/liposomes in vivo, we established an orthotopic mouse model using a LUC-labeled bladder cancer cell line, UM-UC-3LUC. PLK-1 siRNA was successfully transfected into the cells, reduced PLK-1 expression, and prevented the growth of bladder cancer in this mouse model. This is the first demonstration, to our knowledge, of inhibition of cancer growth in the murine bladder by intravesical siRNA/cationic liposomes. We believe intravesical siRNA instillation against bladder cancer will be useful as a therapeutic tool.
Masaki Nogawa, Takeshi Yuasa, Shinya Kimura, Motoyoshi Tanaka, Junya Kuroda, Kiyoshi Sato, Asumi Yokota, Hidekazu Segawa, Yoshinobu Toda, Susumu Kageyama, Tatsuhiro Yoshiki, Yusaku Okada, Taira Maekawa
We used a spheroid model of colon carcinoma to analyze integrin dynamics as a function of the epithelial-mesenchymal transition (EMT), a process that provides a paradigm for understanding how carcinoma cells acquire a more aggressive phenotype. This EMT involves transcriptional activation of the β6 integrin subunit and a consequent induction of αvβ6 expression. This integrin enhances the tumorigenic properties of colon carcinoma, including activation of autocrine TGF-β and migration on interstitial fibronectin. Importantly, this study validates the clinical relevance of the EMT. Kaplan-Meier analysis of β6 expression in 488 colorectal carcinomas revealed a striking reduction in median survival time of patients with high β6 expression. Elevated receptor expression did not simply reflect increasing tumor stage, since log-rank analysis showed a more significant impact on the survival of patients with early-stage, as opposed to late-stage, disease. Cox regression analysis confirmed that this integrin is an independent variable for these tumors. These findings define the αvβ6 integrin as an important risk factor for early-stage disease and a novel therapeutic candidate for colorectal cancer.
Richard C. Bates, David I. Bellovin, Courtney Brown, Elizabeth Maynard, Bingyan Wu, Hisaaki Kawakatsu, Dean Sheppard, Peter Oettgen, Arthur M. Mercurio
The ability of leukemia cells to accumulate methotrexate polyglutamate (MTXPG) is an important determinant of the antileukemic effects of methotrexate (MTX). We measured in vivo MTXPG accumulation in leukemia cells from 101 children with acute lymphoblastic leukemia (ALL) and established that B-lineage ALL with either TEL-AML1 or E2A-PBX1 gene fusion, or T-lineage ALL, accumulates significantly lower MTXPG compared with B-lineage ALL without these genetic abnormalities or compared with hyperdiploid (fewer than 50 chromosomes) ALL. To elucidate mechanisms underlying these differences in MTXPG accumulation, we used oligonucleotide microarrays to analyze expression of 32 folate pathway genes in diagnostic leukemia cells from 197 children. This revealed ALL subtype–specific patterns of folate pathway gene expression that were significantly related to MTXPG accumulation. We found significantly lower expression of the reduced folate carrier (SLC19A1, an MTX uptake transporter) in E2A-PBX1 ALL, significantly higher expression of breast cancer resistance protein (ABCG2, an MTX efflux transporter) in TEL-AML1 ALL, and lower expression of FPGS (which catalyzes formation of MTXPG) in T-lineage ALL, consistent with lower MTXPG accumulation in these ALL subtypes. These findings reveal distinct mechanisms of subtype-specific differences in MTXPG accumulation and point to new strategies to overcome these potential causes of treatment failure in childhood ALL.
Leo Kager, Meyling Cheok, Wenjian Yang, Gianluigi Zaza, Qing Cheng, John C. Panetta, Ching-Hon Pui, James R. Downing, Mary V. Relling, William E. Evans
We used bioluminescence imaging to reveal patterns of metastasis formation by human breast cancer cells in immunodeficient mice. Individual cells from a population established in culture from the pleural effusion of a breast cancer patient showed distinct patterns of organ-specific metastasis. Single-cell progenies derived from this population exhibited markedly different abilities to metastasize to the bone, lung, or adrenal medulla, which suggests that metastases to different organs have different requirements. Transcriptomic profiling revealed that these different single-cell progenies similarly express a previously described “poor-prognosis” gene expression signature. Unsupervised classification using the transcriptomic data set supported the hypothesis that organ-specific metastasis by breast cancer cells is controlled by metastasis-specific genes that are separate from a general poor-prognosis gene expression signature. Furthermore, by using a gene expression signature associated with the ability of these cells to metastasize to bone, we were able to distinguish primary breast carcinomas that preferentially metastasized to bone from those that preferentially metastasized elsewhere. These results suggest that the bone-specific metastatic phenotypes and gene expression signature identified in a mouse model may be clinically relevant.
Andy J. Minn, Yibin Kang, Inna Serganova, Gaorav P. Gupta, Dilip D. Giri, Mikhail Doubrovin, Vladimir Ponomarev, William L. Gerald, Ronald Blasberg, Joan Massagué
Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1–8 of the AKAP9 gene and exons 9–18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.
Raffaele Ciampi, Jeffrey A. Knauf, Roswitha Kerler, Manoj Gandhi, Zhaowen Zhu, Marina N. Nikiforova, Hartmut M. Rabes, James A. Fagin, Yuri E. Nikiforov