Stewart Fleming

Summary: The aim if this study was to investigate the hypothesis that K‐RAS 4A is upregulated in a mineralocorticoid‐dependent manner in renal cell carcinoma and that this supports the proliferation and survival of some renal cancers. Expression of the K‐RAS in renal tumour tissues and cell lines was examined by real‐time PCR and Western blot and mineralocorticoid receptor, and its gatekeeper enzyme 11β‐hydroxysteroid dehydrogenase‐2 was examined by immunocytochemistry on a tissue microarray of 27 cases of renal cell carcinoma. Renal cancer cells lines 04A018 (RCC4 plus VHL) and 04A019 (RCC4 plus vector alone) were examined for the expression of K‐RAS4A and for the effect on K‐RAS expression of spironolactone blockade of the mineralocorticoid receptor. K‐RAS4A was suppressed by siRNA, and the effect on cell survival, proliferation and activation of the Akt and Raf signalling pathways was investigated in vitro. K‐RAS4A was expressed in RCC tissue and in the renal cancer cell lines but K‐RAS was downregulated by spironolactone and upregulated by aldosterone. Spironolactone treatment and K‐RAS suppression both led to a reduction in cell number in vitro. Both Akt and Raf pathways showed activation which was dependent on K‐RAS expression. K‐RAS expression in renal cell carcinoma is at least partially induced by aldosterone. Aldosterone supports the survival and proliferation of RCC cells by upregulation of K‐RAS acting through the Akt and Raf pathways.

There have been recent reports of a rare variant of renal cell carcinoma associated with upregulation of the anaplastic lymphoma kinase gene (ALK) arising as a consequence of chromosomal translocations. The tumours were described as having a characteristic morphology. Here, we describe a case with similar morphology characterised by eosinophilic cells, abundant intracytoplasmic lumina and scattered large ganglion-like tumour cells. There was focal staining for ALK demonstrated by immunohistochemistry. However, rather than exhibiting a chromosomal translocation involving ALK, the use of FISH and a break-apart probe demonstrated that there was increased copy number of intact 2p23, the chromosomal region containing the ALK gene. Furthermore, the use of comparative genomic hybridisation showed increase of the whole of chromosome 2 along with chromosomes 6 and 17. There was no evidence of loss of 3p nor of trisomy of 7 associated with clear cell and papillary carcinoma, respectively. We suggest that this demonstrates a novel mechanism of upregulation of ALK activity by increased copy number occurring during the development of a renal carcinoma with the characteristic ALK-associated morphology.

Renal tumours have recently been described in association with mutations in the gene encoding the B subunit of succinate dehydrogenase, a mitochondrial Krebs cycle and electron transport chain enzyme (SDHB‐associated renal cell carcinomas). The aim of this study was to investigate the roles of different signalling pathways in the pathogenesis of these tumours.

AMP-activated protein kinase (AMPK) acts as a cellular fuel gauge that responds to energy stress by suppressing cell growth and biosynthetic processes, thus ensuring that energy-consuming processes proceed only if there are sufficient metabolic resources. Malfunction of the AMPK pathway may allow cancer cells to undergo uncontrolled proliferation irrespective of their molecular energy levels. The aim of this study was to examine the state of AMPK phosphorylation histologically in primary breast cancer in relation to clinical and pathological parameters.

Background: Signals from the extracellular environment control many aspects of cell behaviour including proliferation, survival, differentiation, adhesion and migration. It is increasingly evident that these signals can be modulated by a group of matricellular proteins called the CCN family. CCN proteins have multiple domains through which they regulate the activities of a variety of signalling molecules including TGFβ, BMPs and integrins, thereby influencing a wide range of processes in development and disease. Whilst the developmental roles of CCN1 and CCN2 have been elucidated, very little is known about the function of CCN3 (NOV). To investigate this, we have generated mice carrying a targeted mutation in the Nov gene (Nov ^del3 ) which reveal for the first time its diverse functions in embryos and adults. Results: By replacing Nov exon 3 with a TKneomycin cassette, we have generated Nov ^del3 -/- mice which produce no full length NOV protein and express at a barely detectable level a mutant NOV protein that lacks the VWC domain. In Nov ^del3 -/- embryos, and to a lesser extent in Nov ^del3 +/- embryos, development of the appendicular and axial skeleton was affected with enlarged vertebrae, elongated long bones and digits, delayed ossification, increased bone mineralization and severe joint malformations. Primary embryo fibroblasts from Nov ^del3 -/- mutant embryos showed enhanced chondrogenesis and osteogenesis. Cardiac development was also influenced leading to enlargement and abnormal modelling of the endocardial cushions, associated with septal defects and delayed fusion. In adults, cardiomyopathy was apparent, with hypertrophy and calcification of the septum and left ventricle dilation. Muscle atrophy was seen by 5 months of age, associated with transdifferentiation to fat. Premature tissue degeneration was also seen in the lens, with cataracts present from 6 months. Conclusion: We have generated the first mice with a mutation in the Nov gene (Nov ^del3 ). Our data demonstrate that NOV is a regulator of skeletal and cardiac development, and implicates NOV in various disease processes including cardiomyopathy, muscle atrophy and cataract formation. Nov ^del3 mutants represent a valuable resource for studying NOV's role in the modulation and co-ordination of multiple signalling pathways that underpin organogenesis and tissue homeostasis.

Renal tumours constitute 2.5% of all malignancies and are among the 10 most common malignancies in the UK. Most of these are renal cell carcinomas (RCC) of various subtypes. Although historically RCC has been shown to be resistant to radiotherapy and chemotherapy, recent data suggest that the use of biological treatments, such as adjuvants, may be beneficial in patients with disease that has progressed at the time of presentation. The accurate diagnosis, staging, and grading of RCC is now a crucial element in optimal patient management. There are data to support the importance of histological type, tumour size, stage (especially patterns of extrarenal spread), and grade in determining outcome, and these data have been used to develop the published classification (Heidelberg/Rochester), staging (TNM), and grading (Fuhrman) systems. This article describes a dissection and histological sampling protocol that has been shown to increase the yield of staging information, a guide to histological classification and grading, and finally a minimum dataset for the completion of a satisfactory pathology report.