The discovery of oestrogen receptor b (ERb/ESR2) was a landmark discovery. Its reported expressio... more The discovery of oestrogen receptor b (ERb/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ERa (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ERb antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ERb in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ERb protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.
Purpose: Increased expression of the adhesion molecule CD44 has been associated with an unfavoura... more Purpose: Increased expression of the adhesion molecule CD44 has been associated with an unfavourable clinical outcome in lymphomas. We evaluated the prognostic value of soluble CD44 in B-cell chronic lymphocytic leukaemia (B-CLL) and analysed the source and regulation of CD44 secretion in B-CLL clones in vitro. Patients and methods: Levels of soluble CD44 standard (sCD44s) and of the soluble variant isoform CD44v6 (sCD44v6) were analysed by enzyme linked immuno sorbent assay. Highly purified B-CLL cells (98% CD19 + CD3− cells) were stimulated in vitro by different combinations of thioredoxin (Trx), Staphylococcus aureus Cowan strain 1 (SAC), IL-2, IL-4, IL-10, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by anti-CD40 mAbs presented on irradiated CD32L cells. Results: Serum levels of sCD44s and of sCD44v6 are significantly elevated in B-CLL patients (n = 90) in comparison with normal persons (n = 44) (P < 0.001). Elevated levels of sCD44s and sCD44v6 are associated with an advanced disease as reflected by an extended lymph node involvement (P < 0.02), an advanced Binet (P < 0.03) and Rai stage (P < 0.04) and chemotherapy requirement (P < 0.02). High levels of sCD44s are associated with high leukocyte counts (P < 0.04) and increased sCD44v6 is significantly associated with splenomegaly (P < 0.002). In B-CLL sCD44s as well as sCD44v6 is shed from leukaemia cells as shown by in vitro cultures. Stimulation of B-CLL clones results in a proliferation-associated increased secretion of sCD44s (rho = 0.7; P = 0.0001) and of sCD44v6 (rho = 0.5; P = 0.005). B-CLL clones from advanced stage patients are characterised by an increased capacity for proliferation and CD44 production in comparison with early stage patients. Conclusions: Both sCD44s and sCD44v6 represent a reliable prognostic marker in B-CLL and may be involved in the pathogenesis of B-CLL.
Cellular processes can only be understood as the dynamic interplay of molecules. There is a need ... more Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at singlemolecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-c (IFN-c) signaling and low-molecular-weight inhibitors.
Improved methods are needed for in situ characterization of post-translational modifications in c... more 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  (PDGFR) 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 PDGFR in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFR in porcine aortic endothelial cells transfected with the -receptor, but not in cells transfected with the ␣-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFR. We furthermore visualized tyrosine phosphorylated PDGFR 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. Molecular & Cellular Proteomics 6:1500 -1509, 2007.
Gene expression -a key feature for modulating cell fate -is regulated in part by histone modifica... more Gene expression -a key feature for modulating cell fate -is regulated in part by histone modifications, which modulate accessibility of the chromatin to transcription factors. Until now, protein-DNA interactions (PDIs) have mostly been studied in bulk without retrieving spatial information from the sample or with poor sequence resolution. New tools are needed to reveal proteins interacting with specific DNA sequences in situ for further understanding of the orchestration of transcriptional control within the nucleus. We present herein an approach to visualise individual PDIs within cells, based on the in situ proximity ligation assay (PLA). This assay, previously used for the detection of protein-protein interactions in situ, was adapted for analysis of target PDIs, using padlock probes to identify unique DNA sequences in complex genomes. As a proof-of-principle we detected histone H3 interacting with a 26 bp consensus sequence of the Alu-repeat abundantly expressed in the human genome, but absent in mice. However, the mouse genome contains a highly similar sequence, providing a model system to analyse the selectivity of the developed methods. Although efficiency of detection currently is limiting, we conclude that in situ PLA can be used to achieve a highly selective analysis of PDIs in single cells.
The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Ac... more The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGFβ. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGFβ (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGFβ stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGFβ induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ-Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP-Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGFβ signaling pathways.
In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events ... more In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, β-catenin, PIPKIγ and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work, we demonstrated that the study of the interplay between E-cadherin and its binding partners, using PLA, is an easy, rapid, quantitative and highly reproducible technique that can be applied in routine labs to verify the pathogenicity of E-cadherin missense mutants for HDGC diagnosis, especially those located in the intracellular domain of the protein.
Cellular functions are regulated and executed by complex protein interaction networks. Accordingl... more Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.
Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifica... more Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le a (SLe a ) and Sialyla /SLe x -MUC6-were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or to the O-glycan haptens alone.
P-cadherin overexpression is associated with worse breast cancer survival, being a poor prognosti... more P-cadherin overexpression is associated with worse breast cancer survival, being a poor prognostic marker as well as a putative therapeutic target for the aggressive triple-negative and basal-like carcinomas (TNBCs). Previously, we have shown that P-cadherin promotes breast cancer invasion of cells where membrane E-cadherin was maintained; however, it suppresses invasion in models without endogenous cadherins, like melanomas. Here, we investigated if P-cadherin expression would interfere with the normal adhesion complex and which were the cellular/molecular consequences, constituting, in this way, a new mechanism by which E-cadherin invasive-suppressor function was disrupted. Using breast TNBC models, we demonstrated, for the first time, that P-cadherin co-localizes with E-cadherin, promoting cell invasion due to the disruption caused in the interaction between E-cadherin and cytoplasmic catenins. P-cadherin also induces cell migration and survival, modifying the expression profile of cells expressing wild-type E-cadherin and contributing to alter their cellular behaviour. Additionally, E- and P-cadherin co-expressing cells significantly enhanced in vivo tumour growth, compared with cells expressing only E- or only P-cadherin. Finally, we still found that co-expression of both molecules was significantly correlated with high-grade breast carcinomas, biologically aggressive, and with poor patient survival, being a strong prognostic factor in this disease. Our results show a role for E- and P-cadherin co-expression in breast cancer progression and highlight the potential benefit of targeting P-cadherin in the aggressive tumours expressing high levels of this protein.
The immunoglobulin VH gene mutation status is a strong prognostic indicator in B-cell chronic lym... more The immunoglobulin VH gene mutation status is a strong prognostic indicator in B-cell chronic lymphocytic leukemia (CLL), since unmutated VH genes are correlated with short survival. However, the traditional cut-off level dividing mutated and unmutated cases, i.e. more or less than 2% mutations, has been questioned and other cut-offs have been suggested. We investigated whether an alternative cut-off should be applied and the relation of mutational status to another prognostic marker, Binet staging. VH gene mutation status was assessed in 332 CLL cases by polymerase chain reaction amplification and nucleotide sequencing and was further correlated with overall survival using different VH mutation cut-offs (1-7%) and Binet stage. After testing different mutation borders, the 2% cut-off remained the best discriminative level for determining prognosis. Interestingly, prognostic stratification was improved by combining the information on VH gene mutation status with that of Binet stage: unmutated cases (all stages, n=151, mutated cases with stage A (n=77), and mutated cases with stage B or C (n=37) had a median survival of 82, 179 and 74 months, respectively. CLL cases displaying mutated VH genes with Binet stage B or C had a survival similar to that of unmutated cases and significantly shorter than that of mutated stage A CLL. Our result reveals clinical heterogeneity within the VH mutated CLL group by inclusion of Binet stage data, a finding which is of importance when considering surrogate marker(s) for VH mutation status.
Current Topics in Microbiology and Immunology, 2013
The fate of the cell is governed by interactions among proteins, nucleic acids, and other biomole... more The fate of the cell is governed by interactions among proteins, nucleic acids, and other biomolecules. It is vital to look at these interactions in a cellular environment if we want to increase our understanding of cellular processes. Herein we will describe how the in situ proximity ligation assay (in situ PLA) can be used to visualize protein interactions in fixed cells and tissues. In situ PLA is a novel technique that uses DNA, together with DNA modifying processes such as ligation, cleavage, and polymerization, as tools to create surrogate markers for protein interactions of interest. Different in situ PLA designs make it possible not only to detect protein-protein interactions but also post-translational modifications and interactions of proteins with nucleic acids. Flexibility in DNA probe design and the multitude of different DNA modifying enzymes provide the basis for modifications of the method to make it suitable to use in many applications. Furthermore, examples of how in situ PLA can be combined with other methods for a comprehensive view of the cellular activity status are discussed.
In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events ... more In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, β-catenin, PIPKIγ and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work, we demonstrated that the study of the interplay between E-cadherin and its binding partners, using PLA, is an easy, rapid, quantitative and highly reproducible technique that can be applied in routine labs to verify the pathogenicity of E-cadherin missense mutants for HDGC diagnosis, especially those located in the intracellular domain of the protein.
The discovery of oestrogen receptor b (ERb/ESR2) was a landmark discovery. Its reported expressio... more The discovery of oestrogen receptor b (ERb/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ERa (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ERb antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ERb in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ERb protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.
Purpose: Increased expression of the adhesion molecule CD44 has been associated with an unfavoura... more Purpose: Increased expression of the adhesion molecule CD44 has been associated with an unfavourable clinical outcome in lymphomas. We evaluated the prognostic value of soluble CD44 in B-cell chronic lymphocytic leukaemia (B-CLL) and analysed the source and regulation of CD44 secretion in B-CLL clones in vitro. Patients and methods: Levels of soluble CD44 standard (sCD44s) and of the soluble variant isoform CD44v6 (sCD44v6) were analysed by enzyme linked immuno sorbent assay. Highly purified B-CLL cells (98% CD19 + CD3− cells) were stimulated in vitro by different combinations of thioredoxin (Trx), Staphylococcus aureus Cowan strain 1 (SAC), IL-2, IL-4, IL-10, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by anti-CD40 mAbs presented on irradiated CD32L cells. Results: Serum levels of sCD44s and of sCD44v6 are significantly elevated in B-CLL patients (n = 90) in comparison with normal persons (n = 44) (P < 0.001). Elevated levels of sCD44s and sCD44v6 are associated with an advanced disease as reflected by an extended lymph node involvement (P < 0.02), an advanced Binet (P < 0.03) and Rai stage (P < 0.04) and chemotherapy requirement (P < 0.02). High levels of sCD44s are associated with high leukocyte counts (P < 0.04) and increased sCD44v6 is significantly associated with splenomegaly (P < 0.002). In B-CLL sCD44s as well as sCD44v6 is shed from leukaemia cells as shown by in vitro cultures. Stimulation of B-CLL clones results in a proliferation-associated increased secretion of sCD44s (rho = 0.7; P = 0.0001) and of sCD44v6 (rho = 0.5; P = 0.005). B-CLL clones from advanced stage patients are characterised by an increased capacity for proliferation and CD44 production in comparison with early stage patients. Conclusions: Both sCD44s and sCD44v6 represent a reliable prognostic marker in B-CLL and may be involved in the pathogenesis of B-CLL.
Cellular processes can only be understood as the dynamic interplay of molecules. There is a need ... more Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at singlemolecule resolution. Proximity probes-oligonucleotides attached to antibodies against the two target proteins-guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-c (IFN-c) signaling and low-molecular-weight inhibitors.
Improved methods are needed for in situ characterization of post-translational modifications in c... more 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  (PDGFR) 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 PDGFR in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFR in porcine aortic endothelial cells transfected with the -receptor, but not in cells transfected with the ␣-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFR. We furthermore visualized tyrosine phosphorylated PDGFR 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. Molecular & Cellular Proteomics 6:1500 -1509, 2007.
Gene expression -a key feature for modulating cell fate -is regulated in part by histone modifica... more Gene expression -a key feature for modulating cell fate -is regulated in part by histone modifications, which modulate accessibility of the chromatin to transcription factors. Until now, protein-DNA interactions (PDIs) have mostly been studied in bulk without retrieving spatial information from the sample or with poor sequence resolution. New tools are needed to reveal proteins interacting with specific DNA sequences in situ for further understanding of the orchestration of transcriptional control within the nucleus. We present herein an approach to visualise individual PDIs within cells, based on the in situ proximity ligation assay (PLA). This assay, previously used for the detection of protein-protein interactions in situ, was adapted for analysis of target PDIs, using padlock probes to identify unique DNA sequences in complex genomes. As a proof-of-principle we detected histone H3 interacting with a 26 bp consensus sequence of the Alu-repeat abundantly expressed in the human genome, but absent in mice. However, the mouse genome contains a highly similar sequence, providing a model system to analyse the selectivity of the developed methods. Although efficiency of detection currently is limiting, we conclude that in situ PLA can be used to achieve a highly selective analysis of PDIs in single cells.
The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Ac... more The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGFβ. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGFβ (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGFβ stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGFβ induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ-Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP-Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGFβ signaling pathways.
In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events ... more In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, β-catenin, PIPKIγ and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work, we demonstrated that the study of the interplay between E-cadherin and its binding partners, using PLA, is an easy, rapid, quantitative and highly reproducible technique that can be applied in routine labs to verify the pathogenicity of E-cadherin missense mutants for HDGC diagnosis, especially those located in the intracellular domain of the protein.
Cellular functions are regulated and executed by complex protein interaction networks. Accordingl... more Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.
Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifica... more Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le a (SLe a ) and Sialyla /SLe x -MUC6-were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or to the O-glycan haptens alone.
P-cadherin overexpression is associated with worse breast cancer survival, being a poor prognosti... more P-cadherin overexpression is associated with worse breast cancer survival, being a poor prognostic marker as well as a putative therapeutic target for the aggressive triple-negative and basal-like carcinomas (TNBCs). Previously, we have shown that P-cadherin promotes breast cancer invasion of cells where membrane E-cadherin was maintained; however, it suppresses invasion in models without endogenous cadherins, like melanomas. Here, we investigated if P-cadherin expression would interfere with the normal adhesion complex and which were the cellular/molecular consequences, constituting, in this way, a new mechanism by which E-cadherin invasive-suppressor function was disrupted. Using breast TNBC models, we demonstrated, for the first time, that P-cadherin co-localizes with E-cadherin, promoting cell invasion due to the disruption caused in the interaction between E-cadherin and cytoplasmic catenins. P-cadherin also induces cell migration and survival, modifying the expression profile of cells expressing wild-type E-cadherin and contributing to alter their cellular behaviour. Additionally, E- and P-cadherin co-expressing cells significantly enhanced in vivo tumour growth, compared with cells expressing only E- or only P-cadherin. Finally, we still found that co-expression of both molecules was significantly correlated with high-grade breast carcinomas, biologically aggressive, and with poor patient survival, being a strong prognostic factor in this disease. Our results show a role for E- and P-cadherin co-expression in breast cancer progression and highlight the potential benefit of targeting P-cadherin in the aggressive tumours expressing high levels of this protein.
The immunoglobulin VH gene mutation status is a strong prognostic indicator in B-cell chronic lym... more The immunoglobulin VH gene mutation status is a strong prognostic indicator in B-cell chronic lymphocytic leukemia (CLL), since unmutated VH genes are correlated with short survival. However, the traditional cut-off level dividing mutated and unmutated cases, i.e. more or less than 2% mutations, has been questioned and other cut-offs have been suggested. We investigated whether an alternative cut-off should be applied and the relation of mutational status to another prognostic marker, Binet staging. VH gene mutation status was assessed in 332 CLL cases by polymerase chain reaction amplification and nucleotide sequencing and was further correlated with overall survival using different VH mutation cut-offs (1-7%) and Binet stage. After testing different mutation borders, the 2% cut-off remained the best discriminative level for determining prognosis. Interestingly, prognostic stratification was improved by combining the information on VH gene mutation status with that of Binet stage: unmutated cases (all stages, n=151, mutated cases with stage A (n=77), and mutated cases with stage B or C (n=37) had a median survival of 82, 179 and 74 months, respectively. CLL cases displaying mutated VH genes with Binet stage B or C had a survival similar to that of unmutated cases and significantly shorter than that of mutated stage A CLL. Our result reveals clinical heterogeneity within the VH mutated CLL group by inclusion of Binet stage data, a finding which is of importance when considering surrogate marker(s) for VH mutation status.
Current Topics in Microbiology and Immunology, 2013
The fate of the cell is governed by interactions among proteins, nucleic acids, and other biomole... more The fate of the cell is governed by interactions among proteins, nucleic acids, and other biomolecules. It is vital to look at these interactions in a cellular environment if we want to increase our understanding of cellular processes. Herein we will describe how the in situ proximity ligation assay (in situ PLA) can be used to visualize protein interactions in fixed cells and tissues. In situ PLA is a novel technique that uses DNA, together with DNA modifying processes such as ligation, cleavage, and polymerization, as tools to create surrogate markers for protein interactions of interest. Different in situ PLA designs make it possible not only to detect protein-protein interactions but also post-translational modifications and interactions of proteins with nucleic acids. Flexibility in DNA probe design and the multitude of different DNA modifying enzymes provide the basis for modifications of the method to make it suitable to use in many applications. Furthermore, examples of how in situ PLA can be combined with other methods for a comprehensive view of the cellular activity status are discussed.
In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events ... more In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, β-catenin, PIPKIγ and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work, we demonstrated that the study of the interplay between E-cadherin and its binding partners, using PLA, is an easy, rapid, quantitative and highly reproducible technique that can be applied in routine labs to verify the pathogenicity of E-cadherin missense mutants for HDGC diagnosis, especially those located in the intracellular domain of the protein.
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Papers by Ola Söderberg