Tobias Sjöblom

The Consensus Coding Sequences of Human Breast and Colorectal Cancers

The elucidation of the human genome sequence has made it possible to identify genetic alterations in cancers in unprecedented detail. To begin a systematic analysis of such alterations, we determined the sequence of well-annotated human protein-coding genes in two common tumor types. Analysis of 13,023 genes in 11 breast and 11 colorectal cancers revealed that individual tumors accumulate an average of ∼90 mutant genes but that only a subset of these contribute to the neoplastic process. Using stringent criteria to delineate this subset, we identified 189 genes (average of 11 per tumor) that were mutated at significant frequency. The vast majority of these genes were not known to be genetically altered in tumors and are predicted to affect a wide range of cellular functions, including transcription, adhesion, and invasion. These data define the genetic landscape of two human cancer types, provide new targets for diagnostic and therapeutic intervention, and open fertile avenues for basic research in tumor biology.

The genomic landscapes of human breast and colorectal cancers.

Human cancer is caused by the accumulation of mutations in oncogenes and tumor suppressor genes. To catalog the genetic changes that occur during tumorigenesis, we isolated DNA from 11 breast and 11 colorectal tumors and determined the sequences of the genes in the Reference Sequence database in these samples. Based on analysis of exons representing 20,857 transcripts from 18,191 genes, we conclude that the genomic landscapes of breast and colorectal cancers are composed of a handful of commonly mutated gene “mountains” and a much larger number of gene “hills” that are mutated at low frequency. We describe statistical and bioinformatic tools that may help identify mutations with a role in tumorigenesis. These results have implications for understanding the nature and heterogeneity of human cancers and for using personal genomics for tumor diagnosis and therapy.

PDGF receptors as cancer drug targets.

The benefits of combining PDGF antagonists and VEGF antagonists in a mouse model of pancreatic islet cancer are shown in a report by Bergers et al.: Bergers, G., Song, S., Meyer-Morse, N., Bergsland, E., and Hanahan, D. (2003). Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J. Clin. Invest. 111, 1287–1295.

Integrated analysis of homozygous deletions, focal amplifications, and sequence alterations in breast and colorectal cancers

We have performed a genome-wide analysis of copy number changes in breast and colorectal tumors using approaches that can reliably detect homozygous deletions and amplifications. We found that the number of genes altered by major copy number changes, deletion of all copies or amplification to at least 12 copies per cell, averaged 17 per tumor. We have integrated these data with previous mutation analyses of the Reference Sequence genes in these same tumor types and have identified genes and cellular pathways affected by both copy number changes and point alterations. Pathways enriched for genetic alterations included those controlling cell adhesion, intracellular signaling, DNA topological change, and cell cycle control. These analyses provide an integrated view of copy number and sequencing alterations on a genome-wide scale and identify genes and pathways that could prove useful for cancer diagnosis and therapy.

In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method.

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor β (PDGFRβ) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRβ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRβ in porcine aortic endothelial cells transfected with the β-receptor, but not in cells transfected with the α-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRβ. We furthermore visualized tyrosine phosphorylated PDGFRβ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

Prognostic Significance of Stromal Platelet-Derived Growth Factor beta-Receptor Expression in Human Breast Cancer

This study systematically analyzes platelet-derived growth factor (PDGF) receptor expression in six types of common tumors as well as examines associations between PDGF beta-receptor status and clinicopathological characteristics in breast cancer. PDGF receptor expression was determined by immunohistochemistry on tumor tissue microarrays. Breast tumor data were combined with prognostic factors and related to outcome endpoints. PDGF alpha- and beta-receptors were independently expressed, at variable frequencies, in the tumor stroma of all tested tumor types. There was a significant association between PDGF beta-receptor expression on fibroblasts and perivascular cells in individual colon and prostate tumors. In breast cancer, high stromal PDGF beta-receptor expression was significantly associated with high histopathological grade, estrogen receptor negativity, and high HER2 expression. High stromal PDGF beta-receptor expression was correlated with significantly shorter recurrence-free and breast cancer-specific survival. The prognostic significance of stromal PDGF beta-receptor expression was particularly prominent in tumors from premenopausal women. Stromal PDGF alpha- and beta-receptor expression is a common, but variable and independent, property of solid tumors. In breast cancer, stromal PDGF beta-receptor expression significantly correlates with less favorable clinicopathological parameters and shorter survival. These findings highlight the prognostic significance of stromal markers and should be considered in ongoing clinical development of PDGF receptor inhibitors.

Sustained TGF beta exposure suppresses Smad and non-Smad signalling in mammary epithelial cells, leading to EMT and inhibition of growth arrest and apoptosis.

To better understand the dual, tumour-suppressive and tumour-promoting function of transforming growth factor-β (TGFβ), we analysed mammary epithelial NMuMG cells in response to short and long-term TGFβ exposure. NMuMG cells became proliferation-arrested and apoptotic after exposure to TGFβ for 2–5 days, whereas surviving cells underwent epithelial–mesenchymal transition (EMT). After chronic TGFβ exposure (2–3 weeks), however, NMuMG cells became resistant to proliferation arrest and apoptosis, showing sustained EMT instead (TD cells). EMT was fully reversed by a pharmacologic TGFβ-receptor-I kinase inhibitor or withdrawal of TGFβ for 6–12 days. Interestingly, both cell cycle arresting/proapoptotic (Smads, p38 kinase) and antiapoptotic, proliferation and EMT-promoting signalling pathways (PI3K–PKB/Akt, ERK) were co-suppressed to low, but significant levels. Except for PI3K-Akt, TGFβ-dependent downregulation of these signalling pathways in transdifferentiated (TD) cells was fully reversed upon TGFβ withdrawal, together with partial re-induction of proliferation arrest and apoptosis. Co-injection of non-tumorigenic NMuMG cells with tumour-forming CHO cells oversecreting exogenous TGFβ1 (CHO-TGFβ1) allowed outgrowth of epithelioid cells in CHO-TGFβ1 cell-induced tumours. These epithelial islands enhanced CHO-TGFβ1 tumour cell proliferation, possibly due to chemokines (for example, JE/MCP-1) secreted by NMuMG/TD cells. We conclude that suppression of antiproliferative, proapoptotic TGFβ signalling in TD cells may permit TGFβ-dependent proliferation, survival and EMT-enhancing signalling pathways to act at low levels. Thus, TGFβ may modulate its own signalling to facilitate switching from tumour suppression to tumour progression.

Platelet-Derived Growth Factor Production by B16 Melanoma Cells Leads to Increased Pericyte Abundance in Tumors and an Associated Increase in Tumor Growth Rate

Platelet-derived growth factor (PDGF) receptor signaling participates in different processes in solid tumors, including autocrine stimulation of tumor cell growth, recruitment of tumor stroma fibroblasts, and stimulation of tumor angiogenesis. In the present study, the B16 mouse melanoma tumor model was used to investigate the functional consequences of paracrine PDGF stimulation of host-derived cells. Production of PDGF-BB or PDGF-DD by tumor cells was associated with an increased tumor growth rate. Characterization of tumors revealed an increase in pericyte abundance in tumors derived from B16 cells producing PDGF-BB or PDGF-DD. The increased tumor growth rate associated with PDGF-DD production was not seen in mice expressing an attenuated PDGF beta-receptor and was thus dependent on host PDGF beta-receptor signaling. The increased pericyte abundance was not associated with an increased tumor vessel density. However, tumor cell apoptosis, but not proliferation, was reduced in tumors displaying PDGF-induced increased pericyte coverage. Our findings thus demonstrate that paracrine PDGF production stimulates pericyte recruitment to tumor vessels and suggest that pericyte abundance influences tumor cell apoptosis and tumor growth.

Preferential oxidation of the second phosphatase domain of receptor-like PTP-α revealed by an antibody against oxidized protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) constitute a large enzyme family with important biological functions. Inhibition of PTP activity through reversible oxidation of the active-site cysteine residue is emerging as a general, yet poorly characterized, regulatory mechanism. In this study, we describe a generic antibody-based method for detection of oxidation-inactivated PTPs. Previous observations of oxidation of receptor-like PTP (RPTP) α after treatment of cells with H2O2 were confirmed. Platelet-derived growth factor (PDGF)-induced oxidation of endogenous SHP-2, sensitive to treatment with the phosphatidylinositol 3-kinase inhibitor LY294002, was demonstrated. Furthermore, oxidation of RPTPα was shown after UV-irradiation. Interestingly, the catalytically inactive second PTP domain of RPTPα demonstrated higher susceptibility to oxidation. The experiments thus demonstrate previously unrecognized intrinsic differences between PTP domains to susceptibility to oxidation and suggest mechanisms for regulation of RPTPs with tandem PTP domains. The antibody strategy for detection of reversible oxidation is likely to facilitate further studies on regulation of PTPs and might be applicable to analysis of redox regulation of other enzyme families with active-site cysteine residues.

Transgenic overexpression of platelet-derived growth factor-C in the mouse heart induces cardiac fibrosis, hypertrophy, and dilated cardiomyopathy.

The platelet-derived growth factors are implicated in development of fibrotic reactions and disease in several organs. We have overexpressed platelet-derived growth factor-C in the heart using the alpha-myosin heavy chain promoter and created a transgenic mouse that exhibits cardiac fibrosis followed by hypertrophy with sex-dependent phenotypes. The transgenic mice developed several pathological changes including cardiac fibroblast proliferation and deposition of collagen, hypertrophy, vascular defects, and the presence of Anitschkow cells in the adult myocardium. Male mice developed a hypertrophic phenotype, whereas female mice were more severely affected and developed dilated cardiomyopathy, leading to heart failure and sudden death. The vascular defects initially included dilation of microvessels and vascular leakage. Subsequently, a marked loss of microvessels, formation of large vascular sac-like structures, and an increased density of smooth muscle-coated vessels were observed in the myocardium. In part, the observed vascular changes may be because of an up-regulation of vascular endothelial growth factor in cardiac fibroblasts of the transgenic hearts. This unique animal model reveals that a potent mitogen for cardiac fibroblasts result in an expansion of the interstitium that induce a secondary sex-dependent hypertrophic response in the cardiomyocytes.