Alexandra K. Bell

p53 polymorphism influences response in cancer chemotherapy via modulation of p73-dependent apoptosis

Intact p73 function is shown to be an important determinant of cellular sensitivity to anticancer agents. Inhibition of p73 function by dominant-negative proteins or by mutant p53 abrogates apoptosis and cytotoxicity induced by these agents. A polymorphism encoding either arginine (72R) or proline (72P) at codon 72 of p53 influences inhibition of p73 by a range of p53 mutants identified in squamous cancers. Clinical response following cisplatin-based chemo-radiotherapy for advanced head and neck cancer is influenced by this polymorphism, cancers expressing 72R mutants having lower response rates than those expressing 72P mutants. Polymorphism in p53 may influence individual responsiveness to cancer therapy.

Involution of the mouse mammary gland is associated with an immune cascade and an acute-phase response, involving LBP, CD14 and STAT3.

IntroductionInvolution of the mammary gland is a complex process of controlled apoptosis and tissue remodelling. The aim of the project was to identify genes that are specifically involved in this process.MethodsWe used Affymetrix oligonucleotide microarrays to perform a detailed transcript analysis on the mechanism of controlled involution after withdrawal of the pups at day seven of lactation. Some of the results were confirmed by semi-quantitative reverse transcriptase polymerase chain reaction, Western blotting or immunohistochemistry.ResultsWe identified 145 genes that were specifically upregulated during the first 4 days of involution; of these, 49 encoded immunoglobulin genes. A further 12 genes, including those encoding the signal transducer and activator of transcription 3 (STAT3), the lipopolysaccharide receptor (CD14) and lipopolysaccharide-binding protein (LBP), were involved in the acute-phase response, demonstrating that the expression of acute-phase response genes can occur in the mammary gland itself and not only in the liver. Expression of LBP and CD14 was upregulated, at both the RNA and protein level, immediately after pup withdrawal; CD14 was strongly expressed in the luminal epithelial cells. Other genes identified suggested neutrophil activation early in involution, followed by macrophage activation late in the process. Immunohistochemistry and histological staining confirmed the infiltration of the involuting mammary tissue with neutrophils, plasma cells, macrophages and eosinophils.ConclusionOligonucleotide microarrays are a useful tool for identifying genes that are involved in the complex developmental process of mammary gland involution. The genes identified are consistent with an immune cascade, with an early acute-phase response that occurs in the mammary gland itself and resembles a wound healing process.

Concomitant inactivation of p53 and Chk2 in breast cancer

The structure and expression of the human Rad53 homologue Chk2 was analysed in breast cancer. The previously described silent polymorphism at nucleotide 252 in codon 84 (GAA>GAG) was observed in 5/141 cases. Somatic Chk2 coding mutations were detected in 7/141 cases, these occurring in 4/18 BRCA1-associated breast cancers, 1/78 sporadic breast cancers and 2/25 typical medullary carcinomas. Each of the BRCA1-associated cancers with Chk2 mutations also contained mutations in p53, whereas the single sporadic cancer with Chk2 mutation was wild-type for p53. Expression of Chk2 was ubiquitously detected in normal ductal epithelium of the breast, but there was loss of expression in a significant proportion of breast carcinomas, and this occurred in cancers both with and without p53 mutation. A CpG island was identified 5′ of the Chk2 transcriptional start site, but there was no evidence of cytosine methylation in any of the cancers with down-regulated Chk2 expression. Analysis of the germ-line of 45 individuals with hereditary or early onset breast cancer revealed wild-type Chk2 sequence in all cases. Thus, despite the rarity of somatic mutations in Chk2 in sporadic breast carcinomas, our results nevertheless reveal that concomitant loss of function in Chk2 (via down-regulation of expression) and p53 (via mutation) occurs in a proportion of sporadic cases. However, consistent with other studies, we show that germ-line mutations in Chk2 are unlikely to account for a significant proportion of non BRCA1-, non BRCA2-associated hereditary breast cancers.

p73 is over-expressed in vulval cancer principally as the Delta 2 isoform.

p73 was studied in squamous cancers and precursor lesions of the vulva. Over-expression of p73 occurred commonly in both human papillomavirus (HPV)-positive and -negative squamous cell cancers (SCC) and high-grade premalignant lesions. Whereas expression in normal vulval epithelium was detected only in the basal and supra-basal layers, expression in neoplastic epithelium increased with grade of neoplasia, being maximal at both protein and RNA levels in SCC. p73 Δ2 was the principal over-expressed isoform in the majority of cases of vulval SCC and often the sole form expressed in SCC. Over-expression of p73 was associated with expression of HPV-encoded E7 or with hypermethylation or mutation of p16INK4a in HPV-negative cases. There was a close correlation between expression of p73 and p14ARF in cancers with loss of p53 function. The frequent over-expression of p73 Δ2 in neoplastic but not normal vulval epithelium, and its co-ordinate deregulation with other E2F-1 responsive genes suggests a role in the oncogenic process.

Annexin A8 Is Up-Regulated During Mouse Mammary Gland Involution and Predicts Poor Survival in Breast Cancer

Purpose: Microarray studies have linked Annexin A8 RNA expression to a “basal cell–like” subset of breast cancers, including BRCA1-related cancers, that are characterized by cytokeratin 5 (CK5) and CK17 expression and show poor prognosis. We assessed Annexin A8s contribution to the overall prognosis and its expression in normal, benign, and cancerous tissue and addressed Annexin A8s physiologic role in the mammary gland. Experimental Design: Using microarrays and reverse transcription-PCR, the Annexin A8 expression was studied during mouse mammary gland development and in isolated mammary structures. Reverse transcription-PCR on cultured human luminal and basal cells, along with immunocytochemistry on normal and benign breast tissues, was used for cellular localization. Annexin A8s prognostic relevance and its coexpression with CK5 were assessed on tissue arrays of 1,631 cases of invasive breast cancer. Coexpression was further evaluated on a small cohort of 14 BRCA1-related breast cancers. Results:Annexin A8 was up-regulated during mouse mammary gland involution and in pubertal ductal epithelium. Annexin A8 showed preferred expression in cultured basal cells but predominant luminal expression in normal human breast tissue in vivo. Hyperplasias and in situ carcinomas showed a strong staining of basal cells. Annexin A8 expression was significantly associated with grade (P < 0.0001), CK5 (P < 0.0001), and estrogen receptor status (P < 0.0001); 85.7% BRCA1-related breast tumors coexpressed Annexin A8 and CK5. Conclusion: Annexin A8 is involved in mouse mammary gland involution. In humans, it is a luminally expressed protein with basal expression in cell culture and in hyperplasia/ductal carcinoma in situ. Expression in invasive breast carcinomas has a significant effect on survival (P = 0.03) but is not independent of grade or CK5.

Involvement of axonal guidance proteins and their signaling partners in the developing mouse mammary gland

Mammary morphogenesis in the mouse is driven by specialized structures at the ends of the developing ducts, the terminal end buds (TEB). The mechanisms controlling the precise branching and spacing of the ducts are, as yet, unknown. To identify genes that are associated with migration of TEB and differentiation of the subtending ducts, we developed a novel method of isolating TEB and ducts free of stroma, and compared the gene expression profiles of these two isolates using oligonucleotide microarrays. Ninety one genes were upregulated in TEB compared to ducts. Three of these genes, Sprr1A, Sema3B, and BASP1, are associated with axonal growth and guidance. Two additional members of the Sprr family, Sprr2A and 2B, not previously associated with axonal growth, were also highly expressed in TEB. Expression of these genes was confirmed by RT‐PCR and Western blotting, and the cellular distribution of Sprr1A and BASP1 was demonstrated by immunohistochemistry. Other semaphorins, including Sema3C, 4A, 4F and the cancer invasion associated Sema 4D were also expressed in the mouse mammary gland along with the semaphorin receptors, Plexins A2, A3, B2, and D1, and Neuropilins 1 and 2. These results are discussed in the context of other proteins expressed in the developing gland that are known to be downstream effectors of these signaling molecules. We suggest that these genes may influence ductal growth and morphogenesis in the developing mammary gland.

Loss of Reelin Expression in Breast Cancer Is Epigenetically Controlled and Associated with Poor Prognosis

Reelin is a secreted, signaling protein associated with neuronal cell positioning and migration. Recently, reelin was found to be epigenetically silenced in gastric and pancreatic cancers in which down-regulation was associated with increased migratory ability and reduced survival. Here we analyzed reelin expression by immunohistochemistry in 17 normal breast tissue samples from reduction mammoplasties and in two independent tissue microarrays of 136 and more than 2000 breast cancer biopsy samples, respectively. Results were analyzed with regard to clinical parameters, including BRE (Bloom, Richardson, Elston) grade, nodal status, estrogen receptor and HER2 status, and overall survival. Reelin was expressed in the luminal epithelium and myoepithelium of the normal human breast but not in cancerous breasts. Loss of reelin protein expression correlated significantly with decreased survival (P=0.01) and positive lymph node status (P<0.001). By measuring reelin expression and promoter methylation status in 39 primary breast tumors, as well as in breast cancer-derived cell lines before and after decitabine treatment, we established that reelin expression levels correlated inversely with promoter methylation status, whereas demethylation increased reelin mRNA expression in vitro. Reelin overexpression in MDA-MB231 cells, as well as incubation with recombinant reelin, suppressed cell migration, invadopodia formation, and invasiveness in vitro. We conclude that reelin may play an important role in controlling invasiveness and metastatic potential of breast cancer cells and that its expression is controlled by promoter methylation.

Analysis of CHK2 in vulval neoplasia.

Structure and expression of the Rad53 homologue CHK2 were studied in vulval neoplasia. We identified the previously described silent polymorphism at codon 84 (A>G at nucleotide 252) in the germ-line of six out of 72, and somatic mutations in two out of 40 cases of vulval squamous cell carcinomas and none of 32 cases of vulval intraepithelial neoplasia. One mutation introduced a premature stop codon in the kinase domain of CHK2, whereas the second resulted in an amino acid substitution in the kinase domain. The two squamous cell carcinomas with mutations in CHK2 also expressed mutant p53. A CpG island was identified close to the putative CHK2 transcriptional start site, but methylation-specific PCR did not detect methylation in any of 40 vulval squamous cell carcinomas, irrespective of human papillomavirus or p53 status. Consistent with this observation, no cancer exhibited loss of CHK2 expression at mRNA or protein level. Taken together, these observations reveal that genetic but not epigenetic changes in CHK2 occur in a small proportion of vulval squamous cell carcinomas.

E7 proteins from oncogenic human papillomavirus types transactivate p73: role in cervical intraepithelial neoplasia

In common with other E2F1 responsive genes such as p14ARF and B-myb, the promoter of p73 is shown to be positively regulated in cell lines and primary human keratinocytes by E7 proteins from oncogenic human papillomavirus (HPV) types 16, 18, 31 and 33, but not HPV 6. Mutational analysis revealed that transactivation of the p73 promoter by HPV 16E7 requires association with pRb. Expression of p73 in normal cervical epithelium is confined to the basal and supra-basal layers. In contrast, expression in neoplastic lesions is detected throughout the epithelium and increases with grade of neoplasia, being maximal in squamous cell cancers (SCC). Deregulation of expression of the N-terminal splice variant p73Δ2 was observed in a significant proportion of cancers, but not in normal epithelium. The frequent over-expression of p73Δ2, which has recognized transdominant properties, in malignant and pre-malignant lesions suggests a role in the oncogenic process in cervical epithelium.

TSC-22D1 isoforms have opposing roles in mammary epithelial cell survival

Transforming growth factor β (TGFβ)-stimulated clone-22 domain family member 1 (TSC-22D1) has previously been associated with enhanced apoptosis in several cell systems. In an attempt to identify novel factors that are involved in the control of cell death during mammary gland involution, we found that the mRNA for isoform 2 of TSC-22D1 was highly upregulated 24 h after forced weaning, when a dramatic increase in cell death occurred, closely following the expression of the known inducer of cell death during involution, TGFβ3. This was paralleled by strongly increased TSC-22D1 isoform 2 protein levels in the luminal epithelium. In contrast, RNA and protein expression levels of the isoform 1 of TSC-22D1 did not change during development. Whereas isoform 2 induced cell death, isoform 1 suppressed TGFβ-induced cell death and enhanced proliferation in mammary epithelial cell lines. Furthermore, four distinct forms of isoform 2 protein were detected in the mammary gland, of which only a 15-kDa form was associated with early involution. Our data describe novel opposing functions of the two mammalian TSC-22D1 isoforms in cell survival and proliferation, and establish the TSC-22D1 isoform 2 as a potential regulator of cell death during mammary gland involution.