Severine Roselli

Deletion of Cd151 Results in a Strain-Dependent Glomerular Disease Due to Severe Alterations of the Glomerular Basement Membrane

Alterations in CD151 have been associated with primary glomerular disease in both humans and mice, implicating CD151 as a key component of the glomerular filtration barrier. CD151 belongs to the tetraspanin family and associates with cell-matrix adhesion complexes such as alpha3beta1-integrin. Here we show that Cd151-deficient mice develop severe kidney disease on an FVB background but are healthy on a B6 background, providing a new and unique tool for the identification of genes that modulate the onset of proteinuria. To better understand the function of CD151 in the kidney, we studied its expression pattern and characterized early ultrastructural defects in Cd151-null kidneys. CD151 is expressed in podocytes of the mouse kidney and co-localizes with alpha3-integrin at the base of podocyte foot processes, at the site of anchorage to the glomerular basement membrane (GBM). Interestingly, the first ultrastructural lesions seen at the onset of proteinuria in Cd151-null kidneys were severe alterations of the GBM, reminiscent of Alport syndrome and consisting of massive thickening and splitting of the GBM. These lesions are associated with increased expression of GBM components. Podocyte abnormalities, effacement of foot processes, and podocyte loss appear to occur consequently to the GBM damage. In conclusion, CD151 appears to be involved in the establishment, maturation, and/or maintenance of the GBM structure in addition to its role in integrin-mediated adhesion strengthening.

Nerve–Cancer Cell Cross-talk: A Novel Promoter of Tumor Progression

Recent studies have revealed the essential role played by nerves in tumor progression. Nerves have been shown to infiltrate the tumor microenvironment and actively stimulate cancer cell growth and dissemination. This mechanism involves the release of neurotransmitters, such as catecholamines and acetylcholine, directly into the vicinity of cancer and stromal cells to activate corresponding membrane receptors. Conversely, the secretion of neurotrophic growth factors by cancer cells drives the outgrowth of nerves in solid tumors. This reciprocal interaction between nerves and cancer cells provides new insights into the cellular and molecular bases of tumorigenesis and points to the potential utility of antineurogenic therapies. This review will discuss our evolving understanding of the cross-talk between nerves and cancer cells.

Nerve fibers infiltrate the tumor microenvironment and are associated with nerve growth factor production and lymph node invasion in breast cancer

Infiltration of the tumor microenvironment by nerve fibers is an understudied aspect of breast carcinogenesis. In this study, the presence of nerve fibers was investigated in a cohort of 369 primary breast cancers (ductal carcinomas in situ, invasive ductal and lobular carcinomas) by immunohistochemistry for the neuronal marker PGP9.5. Isolated nerve fibers (axons) were detected in 28% of invasive ductal carcinomas as compared to only 12% of invasive lobular carcinomas and 8% of ductal carcinomas in situ (p = 0.0003). In invasive breast cancers, the presence of nerve fibers was observed in 15% of lymph node negative tumors and 28% of lymph node positive tumors (p = 0.0031), indicating a relationship with the metastatic potential. In addition, there was an association between the presence of nerve fibers and the expression of nerve growth factor (NGF) in cancer cells (p = 0.0001). In vitro, breast cancer cells were able to induce neurite outgrowth in PC12 cells, and this neurotrophic activity was partially inhibited by anti‐NGF blocking antibodies. In conclusion, infiltration by nerve fibers is a feature of the tumor microenvironment that is associated with aggressiveness and involves NGF production by cancer cells. The potential participation of nerve fibers in breast cancer progression needs to be further considered.

Deletion of Cd151 reduces mammary tumorigenesis in the MMTV/PyMT mouse model

BackgroundTetraspanins are transmembrane proteins that serve as scaffolds for multiprotein complexes containing, for example, integrins, growth factor receptors and matrix metalloproteases, and modify their functions in cell adhesion, migration and transmembrane signaling. CD151 is part of the tetraspanin family and it forms tight complexes with β1 and β4 integrins, both of which have been shown to be required for tumorigenesis and/or metastasis in transgenic mouse models of breast cancer. High levels of the tetraspanin CD151 have been linked to poor patient outcome in several human cancers including breast cancer. In addition, CD151 has been implicated as a promoter of tumor angiogenesis and metastasis in various model systems.MethodsHere we investigated the effect of Cd151 deletion on mammary tumorigenesis by crossing Cd151-deficient mice with a spontaneously metastasising transgenic model of breast cancer induced by the polyoma middle T antigen (PyMT) driven by the murine mammary tumor virus promoter (MMTV).ResultsCd151 deletion did not affect the normal development and differentiation of the mammary gland. While there was a trend towards delayed tumor onset in Cd151−/− PyMT mice compared to Cd151+/+ PyMT littermate controls, this result was only approaching significance (Log-rank test P-value =0.0536). Interestingly, Cd151 deletion resulted in significantly reduced numbers and size of primary tumors but did not appear to affect the number or size of metastases in the MMTV/PyMT mice. Intriguingly, no differences in the expression of markers of cell proliferation, apoptosis and blood vessel density was observed in the primary tumors.ConclusionThe findings from this study provide additional evidence that CD151 acts to enhance tumor formation initiated by a range of oncogenes and strongly support its relevance as a potential therapeutic target to delay breast cancer progression.

ProNGF is a potential diagnostic biomarker for thyroid cancer

The precursor for nerve growth factor (proNGF) is expressed in some cancers but its clinicopathological significance is unclear. The present study aimed to define the clinicopathological significance of proNGF in thyroid cancer. ProNGF expression was analysed by immunohistochemistry in two cohorts of cancer versus benign tumors (adenoma) and normal thyroid tissues. In the first cohort (40 thyroid cancers, 40 thyroid adenomas and 80 normal thyroid tissues), proNGF was found overexpressed in cancers compared to adenomas and normal samples (p<0.0001). The area under the receiver-operating characteristic (ROC) curve was 0.84 (95% CI 0.75-0.93, p<0.0001) for cancers versus adenomas, and 0.99 (95% CI 0.98-1.00, p<0.0001) for cancers versus normal tissues. ProNGF overexpression was confirmed in a second cohort (127 cancers of various histological types and 55 normal thyroid tissues) and using a different antibody (p<0.0001). ProNGF staining intensity was highest in papillary carcinomas compared to other histological types (p<0.0001) and there was no significant association with age, gender, tumor size, stage and lymph node status. In conclusion, proNGF is increased in thyroid cancer and should be considered as a new potential diagnostic biomarker.

The neurotrophic tyrosine kinase receptor TrkA and its ligand NGF are increased in squamous cell carcinomas of the lung

The neurotrophic tyrosine kinase receptor TrkA (NTRK1) and its ligand nerve growth factor (NGF) are emerging promoters of tumor progression. In lung cancer, drugs targeting TrkA are in clinical trials, but the clinicopathological significance of TrkA and NGF, as well as that of the precursor proNGF, the neurotrophin co-receptor p75NTR and the proneurotrophin co-receptor sortilin, remains unclear. In the present study, analysis of these proteins was conducted by immunohistochemistry and digital quantification in a series of 204 lung cancers of different histological subtypes versus 121 normal lung tissues. TrkA immunoreactivity was increased in squamous cell carcinoma compared with benign and other malignant lung cancer histological subtypes (p < 0.0001). NGF and proNGF were also increased in squamous cell carcinoma, as well as in adenocarcinoma (p < 0.0001). In contrast, p75NTR was increased across all lung cancer histological subtypes compared to normal lung (p < 0.0001). Sortilin was higher in adenocarcinoma and small cell carcinoma (p < 0.0001). Nerves in the tumor microenvironment were negative for TrkA, NGF, proNGF, p75NTR and sortilin. In conclusion, these data suggest a preferential therapeutic value of targeting the NGF-TrkA axis in squamous cell carcinomas of the lung.

Functional importance of PP2A regulatory subunit loss in breast cancer

PurposeProtein phosphatase 2A (PP2A) is a family of serine/threonine phosphatases that regulate multiple cellular signalling pathways involved in proliferation, survival and apoptosis. PP2A inhibition occurs in many cancers and is considered a tumour suppressor. Deletion/downregulation of PP2A genes has been observed in breast tumours, but the functional role of PP2A subunit loss in breast cancer has not been investigated.MethodsPP2A subunit expression was examined by immunohistochemistry in human breast tumours, and by qPCR and immunoblotting in breast cancer cell lines. PP2A subunits were inhibited by shRNA, and mutant PP2A genes overexpressed, in MCF10A and MCF7 cells, and growth and signalling in standard and three-dimensional cultures were assessed.ResultsExpression of PP2A-Aα, PP2A-Bα and PP2A-B′α subunits was significantly lower in primary human breast tumours and lymph node metastases, compared to normal mammary tissue. PP2A-Aα and the regulatory subunits PP2A-Bα, -Bδ and -B′γ were also reduced in breast cancer cell lines compared to normal mammary epithelial cells. Functionally, shRNA-mediated knockdown of PP2A-Bα, -B′α and -B′γ, but not PP2A-Aα, induced hyper-proliferation and large multilobular acini in MCF10A 3D cultures, characterised by activation of ERK. Expression of a breast cancer-associated PP2A-A mutant, PP2A-Aα-E64G, which inhibits binding of regulatory subunits to the PP2A core, induced a similar hyper-proliferative phenotype. Knockdown of PP2A-Bα also induced hyper-proliferation in MCF7 breast cancer cells.ConclusionThese results suggest that loss of specific PP2A regulatory subunits is functionally important in breast tumourigenesis, and support strategies to enhance PP2A activity as a therapeutic approach in breast cancer.

Characterization of the early molecular changes in the glomeruli of Cd151 −/− mice highlights induction of mindin and MMP-10

In humans and FVB/N mice, loss of functional tetraspanin CD151 is associated with glomerular disease characterised by early onset proteinuria and ultrastructural thickening and splitting of the glomerular basement membrane (GBM). To gain insight into the molecular mechanisms associated with disease development, we characterised the glomerular gene expression profile at an early stage of disease progression in FVB/N Cd151−/− mice compared to Cd151+/+ controls. This study identified 72 up-regulated and 183 down-regulated genes in FVB/N Cd151−/− compared to Cd151+/+ glomeruli (p < 0.05). Further analysis highlighted induction of the matrix metalloprotease MMP-10 and the extracellular matrix protein mindin (encoded by Spon2) in the diseased FVB/N Cd151−/− GBM that did not occur in the C57BL/6 diseased-resistant strain. Interestingly, mindin was also detected in urinary samples of FVB/N Cd151−/− mice, underlining its potential value as a biomarker for glomerular diseases associated with GBM alterations. Gene set enrichment and pathway analysis of the microarray dataset showed enrichment in axon guidance and actin cytoskeleton signalling pathways as well as activation of inflammatory pathways. Given the known function of mindin, its early expression in the diseased GBM could represent a trigger of both further podocyte cytoskeletal changes and inflammation, thereby playing a key role in the mechanisms of disease progression.

Abstract 2375: Functional role of the tumor suppressor protein phosphatase, PP2A-B55α, in breast cancer

Breast cancer is the most common cancer in women and a leading cause of death. Dysregulation of cellular signalling pathways controlling proliferation, survival and migration, such as the PI3K/Akt and Ras/MAPK pathways, are key features of breast cancer. Protein phosphatase 2A (PP2A) negatively regulates many components of these pathways. PP2A is a family of trimeric serine/threonine phosphatases, each consisting of a structural, a catalytic and a regulatory subunit of which there are multiple isoforms. The addition of specific regulatory subunits provides subcellular targeting and substrate specificity to the enzyme. While PP2A is generally considered a tumor suppressor, a specific role for individual PP2A subunits in breast cancer has not been described. To address this, we first examined PP2A subunit expression in human breast tumors. Immunohistochemical analysis revealed significantly lower expression of the structural subunit, PP2A-A, and regulatory subunits PP2A-B55α and PP2A-B56α, in primary tumors and metastases, compared to adjacent normal mammary tissue. We further found an association of low PP2A-B55α with aggressive breast cancer subtypes, and with worse disease-free and overall survival. Functionally, shRNA-mediated knockdown of PP2A-B55α in normal mammary epithelial 3D cultures induced a tumorigenic phenotype, characterised by increased proliferation and enlarged multi-lobular acini. In contrast, overexpression of PP2A-B55α in breast cancer cells inhibited proliferation. Thus PP2A inactivation, in particular loss of B55α, is functionally important in breast tumorigenesis. PP2A-B55α complexes play an important role in DNA damage repair, and we found B55α knockdown impaired DNA damage repair. Thus low PP2A-B55α may contribute to genomic instability. To examine the functional role of PP2A-B55α in vivo, we have generated the first PP2A-B55α (Ppp2r2a) knockout mouse. Constitutive knockout of Ppp2r2a is embryonic lethal, with embryos dying during late development, post 14.5 days p.c. Heterozygous Pppr2ra mice (Ppp2r2a+/-) are viable, despite expressing only ~10% of PP2A-B55α protein levels compared to WT mice. Interestingly, Ppp2r2a+/- mice have significantly reduced branching in the developing mammary gland, similar to that observed in mice with mammary-specific loss of the breast tumor susceptibility gene, Brca1. Analysis of breast tumor formation in these mice, either alone or when crossed with MMTV-Neu animals, is underway. Finally, we show that pharmacological activation of PP2A, using FTY720, or the non-phosphorylatable analogue, AAL(S), inhibits tumor growth and metastases in an orthotopic xenograft model of aggressive, triple negative breast cancer (MDA-MB-231). Together this work demonstrates the importance of PP2A as a tumor suppressor in breast cancer, and suggests that targeting PP2A is a potential therapeutic strategy for poor outcome patients. Citation Format: Nikita Panicker, Abdul Mannan, Lauren F. Watt, Ben Copeland, Matt D. Dun, Simon King, Megan Clarke, Kathryn Skelding, Severine Roselli, Nicole M. Verrills. Functional role of the tumor suppressor protein phosphatase, PP2A-B55α, in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2375. doi:10.1158/1538-7445.AM2017-2375

Abstract P6-01-11: The neuronal protein sortilin is expressed in aggressive breast cancers and participates in tumor cell growth and invasion

The membrane protein sortilin is involved in intracellular trafficking and has emerged as a key player in the regulation of neuronal viability and function. Few studies have suggested that sortilin may also be implicated in cancer, but its expression in human tumors and potential value as a therapeutic target is unknown. In this study, the level of sortilin was analyzed in a series of 318 clinically annotated breast cancers and 53 normal adjacent tissues by immunohistochemistry. Sortilin was specifically localized in epithelial cells and was detected in 65% of cancers compared to 46% of normal tissues (p=0.0088). Sortilin was detected in 79% of invasive ductal carcinomas compared to only 54% of invasive lobular carcinomas (p Citation Format: Jay Pundavela, Severine Roselli, Yohann Demont, Sam Faulkner, John Attia, Sheridan Keene, Marjorie M Walker, Hubert Hondermarck. The neuronal protein sortilin is expressed in aggressive breast cancers and participates in tumor cell growth and invasion [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-11.