Kristina Magnusson

A Human Protein Atlas for Normal and Cancer Tissues Based on Antibody Proteomics

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, ∼400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Human tumors instigate granulin-expressing hematopoietic cells that promote malignancy by activating stromal fibroblasts in mice

Systemic instigation is a process by which endocrine signals sent from certain tumors (instigators) stimulate BM cells (BMCs), which are mobilized into the circulation and subsequently foster the growth of otherwise indolent carcinoma cells (responders) residing at distant anatomical sites. The identity of the BMCs and their specific contribution or contributions to responder tumor growth have been elusive. Here, we have demonstrated that Sca1+ cKit- hematopoietic BMCs of mouse hosts bearing instigating tumors promote the growth of responding tumors that form with a myofibroblast-rich, desmoplastic stroma. Such stroma is almost always observed in malignant human adenocarcinomas and is an indicator of poor prognosis. We then identified granulin (GRN) as the most upregulated gene in instigating Sca1+ cKit- BMCs relative to counterpart control cells. The GRN+ BMCs that were recruited to the responding tumors induced resident tissue fibroblasts to express genes that promoted malignant tumor progression; indeed, treatment with recombinant GRN alone was sufficient to promote desmoplastic responding tumor growth. Further, analysis of tumor tissues from a cohort of breast cancer patients revealed that high GRN expression correlated with the most aggressive triple-negative, basal-like tumor subtype and reduced patient survival. Our data suggest that GRN and the unique hematopoietic BMCs that produce it might serve as novel therapeutic targets.

SATB2 in Combination With Cytokeratin 20 Identifies Over 95% of all Colorectal Carcinomas

The special AT-rich sequence-binding protein 2 (SATB2), a nuclear matrix-associated transcription factor and epigenetic regulator, was identified as a tissue type-specific protein when screening protein expression patterns in human normal and cancer tissues using an antibody-based proteomics approach. In this respect, the SATB2 protein shows a selective pattern of expression and, within cells of epithelial lineages, SATB2 expression is restricted to glandular cells lining the lower gastrointestinal tract. The expression of SATB2 protein is primarily preserved in cancer cells of colorectal origin, indicating that SATB2 could function as a clinically useful diagnostic marker to distinguish colorectal cancer (CRC) from other types of cancer. The aim of this study was to further explore and validate the specific expression pattern of SATB2 as a clinical biomarker and to compare SATB2 with the well-known cytokeratin 20 (CK20). Immunohistochemistry was used to analyze the extent of SATB2 expression in tissue microarrays with tumors from 9 independent cohorts of patients with primary and metastatic CRCs (n=1882). Our results show that SATB2 is a sensitive and highly specific marker for CRC with distinct positivity in 85% of all CRCs, and that SATB2 and/or CK20 was positive in 97% of CRCs. In conclusion, the specific expression of SATB2 in a large majority of CRCs suggests that SATB2 can be used as an important complementary tool for the differential diagnosis of carcinoma of unknown primary origin.

The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase.

Mutations within the amyloid‐β (Aβ) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer’s disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Aβ protofibrils in vitro and intraneuronal Aβ aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased β‐secretase cleavage, as demonstrated by altered levels of N‐ and C‐terminal APP fragments. Although the Arctic mutation is located close to the α‐secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease‐10, a major α‐secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for α‐secretase cleavage. As a result, the extent and subcellular location of Aβ formation is changed, as revealed by increased Aβ levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra‐Aβ mutations, could relate to altered APP processing with increased steady‐state levels of Arctic Aβ, particularly at intracellular locations.

Nuclear expression of the RNA-binding protein RBM3 is associated with an improved clinical outcome in breast cancer.

Single-strand RNA-binding proteins (RBPs) are involved in many aspects of RNA metabolism and in the regulation of gene transcription. The RBP RBM3 was recently suggested to be a proto-oncogene in colorectal cancer; however, such a role has not been corroborated by previous studies in the colon or other tumor types, and the prognostic implications of tumor-specific RBM3 expression remain unclear. Mono-specific antibodies against RBM3 were generated. Antibody specificity was confirmed using siRNA gene silencing, western blotting and immunohistochemistry on a panel of breast cancer cell lines. Using tissue microarrays and IHC, RBM3 protein expression was examined in 48 normal tissues and in 20 common cancers. Additional analysis in two independent breast cancer cohorts (n=1016) with long-term follow-up was also carried out. RBM3 was upregulated in cancer compared to normal tissues. The nuclear expression of RBM3 in breast cancer was associated with low grade (P<0.001), small tumors (P<0.001), estrogen receptor (ER) positivity (P<0.001) and Ki-67 negativity (P<0.001) in both the breast cancer cohorts. An increased nuclear expression of RBM3 was associated with a prolonged overall and recurrence-free survival. The prognostic value was particularly pronounced in hormone receptor-positive tumors and remained significant in multivariate interaction analysis after controlling for tamoxifen treatment (HR: 0.49, 95% CI: 0.30–0.79, P=0.004). These data strongly indicate that nuclear RBM3 is an independent favorable prognostic factor in breast cancer, and seems to have a specific role in ER-positive tumors.

CDK-mediated activation of the SCFFBXO28 ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer

SCF (Skp1/Cul1/F‐box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F‐box protein, FBXO28 that controls MYC‐dependent transcription by non‐proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2‐mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F‐box mutant unable to support MYC ubiquitylation results in an impairment of MYC‐driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK‐FBXO28‐MYC axis as a potential molecular drug target in MYC‐driven cancers, including breast cancer.

Nandrolone decanoate administration elevates hippocampal prodynorphin mRNA expression and impairs Morris water maze performance in male rats.

The misuse of anabolic androgenic steroids has in several reports been associated with effects resulting in altered behavior. This study used the Morris water maze task to investigate the effect of high doses of the anabolic androgenic steroid nandrolone on spatial learning and memory in male rats. During the experiment, we observed a significantly impaired Morris water maze performance in the nandrolone-treated rats compared with controls. The hippocampus, a brain region associated with cognitive function, was analyzed for mRNA expression of prodynorphin, the precursor of dynorphinergic peptides. The results indicated that the transcription levels of prodynorphin were significantly elevated in the animals treated with nandrolone compared with controls. Thus, the findings suggest that administration of nandrolone to male rats impairs memory function, possibly via dynorphinergic actions.

Selective Expression of Syntaxin-7 Protein in Benign Melanocytes and Malignant Melanoma

To search for proteins expressed in human melanocytes and melanoma, we employed an antibody-based proteomics strategy to screen for protein expression in tissue microarrays containing normal tissues, cancer tissues and cell lines. Syntaxin-7 (STX7) was identified as a novel protein, not previously characterized in cells of melanocytic lineage, displaying a cell type-specific protein expression pattern. In tumor tissues, STX7 was expressed in malignant melanoma and lymphoma. The protein was further characterized regarding subcellular localization, specificity, tissue distribution pattern and potential as a diagnostic and prognostic marker using cell lines and tissue microarrays containing normal skin, melanocytic nevi and primary and metastatic melanoma. STX7 was expressed in normal melanocytes, various benign melanocytic nevi, atypical nevi and malignant melanoma. Analysis in two independent melanoma cohorts demonstrated STX7 expression in nearly all investigated tumors, although at varying levels (> 90% positive tumors). The expression level of STX7 protein was inversely correlated to tumor stage, suggesting that decreased expression of STX7 is associated with more aggressive tumors. In conclusion, we present protein profiling data for a novel protein showing high sensitivity and specificity for cells of the melanocytic lineage. The presented antibody-based proteomics approach can be used as an effective strategy to identify novel tumor markers and evaluate their potential clinical relevance.

Enzymatic conversion of dynorphin A in the rat brain is affected by administration of nandrolone decanoate

The misuse of anabolic androgenic steroids (AAS) seems to produce profound effects on the central nervous system, leading to aggressive behavior and increased sensitivity to other drugs of abuse. The present study addresses the effect on the enzymatic transformation, here called dynorphin converting enzyme-like activity. The formation of the mu/delta opioid peptide receptor-preferring Leu-enkephalin-Arg(6) from the kappa opioid peptide receptor-preferring dynorphin A was measured in rats treated with nandrolone decanoate. Significant variations in enzymatic transformation were observed in several brain regions. An altered receptor activation profile in these regions may be one contributory factor behind AAS-induced personality changes.

Nandrolone decanoate administration dose-dependently affects the density of kappa opioid peptide receptors in the rat brain determined by autoradiography

The kappa opioid receptor ligand [(3)H]CI-977 was used to autoradiographically determine the density of kappa opioid receptors in the male rat brain following chronic treatment with the anabolic androgenic steroid nandrolone decanoate at two different doses. As compared to controls, significantly lower densities of the kappa opioid receptor were encountered after two weeks of high dose nandrolone decanoate (15 mg/kg) in the nucleus accumbens shell (16%), lateral hypothalamic area (36%), ventromedial hypothalamic nucleus (37%), dorsomedial hypothalamic nucleus (49%), central amygdaloid nucleus, capsular part (28%), lateral globus pallidus (35%) and in the stria terminalis (24%). Furthermore, an up-regulation of the receptor level was observed in the caudate putamen (18%) and in the dorsal endopiriform nucleus (23%). These alterations in the kappa opioid receptor expression are possibly attributed to a previously observed pronounced impact of nandrolone decanoate on the dynorphinergic system and could also include involvement of the dopaminergic reward system.