2004 Summer Meeting - Amsterdam - The Pathological Society of ...

41
Septin 9 interacts with cytoskeletal factors leading to altered
cell migration and resistance to microtubule-disrupting drugs
P A Hall , A D Chacko , S Chanduloy , S W Church , R D
Kennedy , S E H Russell
Centre for Cancer Research, QUB, Belfast, United Kingdom
Septins are an evolutionarily conserved gene family of GTP binding proteins
associated with cytokinesis and cell polarity. Several lines of evidence
implicate human septins with neoplasia including their involvement as fusion
partners with MLL, allelic imbalance and over–expression in tumour cells. We
have established cell lines expressing the Sept9-v4 isoform, and a constitutively
active GTPase mutant G144V. We observe profound morphological changes
with an increased number and length of actin-rich filopodia. In cell wounding
experiments of confluent monolayers we observed differences in the kinetics of
healing, data confirmed by time-lapse microscopy and Boyden chamber assay.
The morphology of cell movement also differed. Wild type and G144V mutants
bind to Cdc42 and N-WASP and the role of Cdc42 is confirmed by abrogation
of the response by dominant negative Cdc42. Sept9 has been previously shown
to interact with microtubules, and here we show that Sept9 over-expressing cell
lines display increased resistance to the microtubule interacting drugs paclitaxel
and vinorelbine without increased resistance to DNA damaging agents. Such
properties indicate that Sept9 has a role in both tumour cell migration as well as
drug resistance. We propose that the effects of Sept9 on the actin and tubulin
cytoskeletons reflect a novel signalling role for septins via Cdc42. Our data
makes a novel connection between the structural and biochemical properties of
Sept9 and the genetic studies that have linked Sept9 to human cancer.
42
Blottin: A Novel TFF2-Binding Protein of Foveolar Cells
MD Evans 1 , WR Otto 1 , K Patel 2 , I McKinnell 2 , C-Y Lee 1 , D
Frith 1 , S Hanrahan 1 , N Blin 3 , T Kayademir 3 , R Poulsom 1 , R
Jeffery 1 , T Hunt 1 , NA Wright 1 , F McGregor 1 , K Oien 1
1 Cancer Research UK, London & Glasgow, United Kingdom, 2 Royal
Veterinary College, London, United Kingdom, 3 University of Tubingen,
Tubingen, Germany
Purpose: Trefoil factor family (TFF) peptide receptors remain elusive. In
experiments to find such a molecule, we identified a novel murine TFF2-
binding protein which we have named Blottin. Methods: We made a fusion
protein of mouse TFF2 with secretory embryonic alkaline phosphatase (AP),
expressed it in A293 cells, and probed tissue sections for binding. Substantial
binding to gastric foveolum was seen. Results: A single protein was found by 2-
D Western blots using the same ligand, and identified by mass spectrometry.
This has only mammalian homologues, and is undescribed at the protein level.
By RACE, the full-length mRNA averages 800bp in human, mouse, rat and
cow, and these show much evolutionary conservation. The protein has
molecular weight of 21kDa, a hydrophobic signal peptide and a pI of 6.9. Using
rabbit antibodies, and mRNA in situ hybridisation, we found large signals in
gastric foveolar cells. It is mainly cytoplasmic, but with some membrane
associations, consistent with its N-, and particularly C-, termini being of
hydrophobic amino acids; the latter has 3 potential myristylation sites. SAGE
analyses report that the mRNA is reduced in human gastric cancers. Four
human GI cell lines, including gastric (HGT-101, Kato-3) and colorectal (HT-
29, Col-1), show positivity for Blottin by PCR and immunocytochemistry. Pilot
data using in vitro wounding assays suggest antibody to blottin may increase
wound recovery in HT-29 cells. Blottin ISH on human GI pathological sections
shows high expression in surface UACL epithelium, gastric metaplasia of
duodenum and pseudopyloric metaplasia of the stomach, mimicking some sites
for TFF1 expression. Conclusions: A new TFF2 binding protein is reported in
mammalian gastric foveolum, which may have expression patterns similar to
TFF1.
43
S100A11 Protein is Overexpressed in Common Human
Cancers With Spatial Translocation From the Nucleus to
Cytoplasm
SS Cross 1 , FC Hamdy 2 , JC Deloulme 3 , I Rehman 2
1 Academic Unit of Pathology, School of Medicine & Biomedical Sciences,
University of Sheffield, Sheffield, United Kingdom, 2 Academic Urology
Unit, School of medicine & Biomedical Sciences, University of Sheffield,
Sheffield, United Kingdom, 3 Departemente de Biologie Moleculaire et
Structurale du Commissariat a l'Energie Atomique, INSERM Unite 244,
Commissariat a l'Energie Atomique, Grenoble, France
S100A11 (S100C, calgizzarin) is a member of the EF-hand type calcium
binding protein family which includes some proteins that have abnormal
patterns of expression in human cancer. S100A11 has been shown to be
expressed in most human epithelia and there is a report of reduced expression in
carcinomas, but the data was only on 14 cancers. There is also an in vitro report
of S100A11 being a key mediator of calcium-induced growth inhibition of
human epidermal keratinocytes. We have performed an immunohistochemical
survey of S100A11 expression on a custom made tissue array containing 291
tissue cores representing 28 human tissue types and 21 different tumour types.
There was exclusively nuclear expression in virtually all epithelia except
proliferative phase endometrium. There was expression in the majority of
cancers including 40/45 breast cancers, 4/4 colorectal cancers, 8/8 cutaneous
basal cell cancers, and 5/5 gynaecological cancers. However the expression in
the tumours was cytoplasmic as well as nuclear. Other studies have shown that
in the nucleus S100A11 increases the transcription of p21 which inhibits cell
proliferation. Our results suggest that the cytoplasmic translocation of S100A11
in cancers could lead to increased proliferation in these cells. The mechanism
for the translocation requires further investigation.
44
Bone Marrow contributes to Tumour Stroma
NC Direkze 1 , K Hodivala-Dilke 3 , R Jeffery 1 , T Hunt 1 , D
Oukrif 4 , SL Preston 1 , MR Alison 2 , NA Wright 1
1 Cancer Research UK, London, United Kingdom, 2 Imperial College,
London, United Kingdom, 3 Barts and the London, Queen Mary School of
Medicine and Dentistry, London, United Kingdom, 4 University College,
London, United Kingdom
Background. We have previously shown that not only can bone marrow
contribute to myofibroblast populations in the mouse and human gut but that
this is an example of a more generalised phenomenon that is exacerbated by
injury. We now report that the bone marrow contributes to myofibroblast
populations in tumour stroma.
Method. RIPTag mice develop cell tumours of the pancreas. Female
RIPTag mice were transplanted with male bone marrow. The fate of the bone
marrow derived cells was followed by detection of the Y chromosome by in
situ hybridisation, combined with immunohistochemistry for myofibroblast
markers such as smooth muscle actin. We also examined samples from patients
who developed tumours after sex-mismatched bone marrow transplant in a
similar way.
Results. Approximately 25% of myofibroblasts were found to be bone
marrow-derived in pancreatic tumours post sex-mismatched bone marrow
transplant. These tended to be concentrated within 1 high power field of the
tumour edge (p