Jim Heighway

Aurora Kinases as Anticancer Drug Targets

The human aurora family of serine-threonine kinases comprises three members, which act in concert with many other proteins to control chromosome assembly and segregation during mitosis. Aurora dysfunction can cause aneuploidy, mitotic arrest, and cell death. Aurora kinases are strongly expressed in a broad range of cancer types. Aurora A expression in tumors is often associated with gene amplification, genetic instability, poor histologic differentiation, and poor prognosis. Aurora B is frequently expressed at high levels in a variety of tumors, often coincidently with aurora A, and expression level has also been associated with increased genetic instability and clinical outcome. Further, aurora kinase gene polymorphisms are associated with increased risk or early onset of cancer. The expression of aurora C in cancer is less well studied. In recent years, several small-molecule aurora kinase inhibitors have been developed that exhibit preclinical activity against a wide range of solid tumors. Preliminary clinical data from phase I trials have largely been consistent with cytostatic effects, with disease stabilization as the best response achieved in solid tumors. Objective responses have been noted in leukemia patients, although this might conceivably be due to inhibition of the Abl kinase. Current challenges include the optimization of drug administration, the identification of potential biomarkers of tumor sensitivity, and combination studies with cytotoxic drugs. Here, we summarize the most recent preclinical and clinical data and discuss new directions in the development of aurora kinase inhibitors as antineoplastic agents.

Expression profiling of primary non-small cell lung cancer for target identification.

Using a panel of cDNA microarrays comprising 47 650 transcript elements, we have carried out a dual-channel analysis of gene expression in 39 resected primary human non-small cell lung tumours versus normal lung tissue. Whilst ∼11 000 elements were scored as differentially expressed at least twofold in at least one sample, 96 transcripts were scored as over-represented fourfold or more in at least seven out of 39 tumours and 30 sequences 16-fold in at least two out of 39 tumours, including 24 transcripts in common. Transcripts (178) were found under-represented fourfold in at least seven out of 39 tumours, 31 of which are under-represented 16-fold in at least two out of 39 lesions. The relative expression levels of representative genes from these lists were analysed by comparative multiplex RT–PCR and found to be broadly consistent with the microarray data. Two dramatically over-represented genes, previously designated as potential tumour suppressors in breast (maspin) and lung and breast (S100A2) cancers, were analysed more extensively and demonstrate the effectiveness of this approach in identifying potential lung cancer diagnostic or therapeutic targets. Whilst it has been reported that S100A2 is downregulated in NSCLC at an early stage, our microarray, cmRT–PCR, Western and immunohistochemistry data indicate that it is strongly expressed in the majority of tumours.

Expression of p16INK4a/p16alpha and p19ARF/p16beta is frequently altered in non-small cell lung cancer and correlates with p53 overexpression.

The CDKN2 locus expresses two different mRNA transcripts, designated α and β. The protein product of the α transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The β transcript is translated in an alternate reading frame (ARF) and in humans encodes a 15 kDa protein (p19ARF). Immunohistochemical and Western analysis of p16INK4a has shown that the protein is downregulated in a significant number of tumours, but less is known on the expression of the p19ARF. We have examined the expression of p16INK4a and p19ARF in resectable non-small cell lung cancer (NSCLC) by immunostaining (n=49) and multiplex RT–PCR (n=28). In order to investigate the mechanism responsible for p16INK4a downregulation, exon 1α methylation was analysed in a PCR-based assay. Of 49 tumours examined by immunostaining, 24 and 20 tumours expressed p16INK4a and p19ARF at nil to low levels, respectively. p19ARF was localized primarily to the nuclei of tumour cells, but was also seen to varying degrees in nuclei of lymphocytes, chondrocytes, fibroblasts, and epithelial cells. No tumour with normal p16INK4a had decreased p19ARF expression. Among 16 tumours with nil to low p16INK4a expression, 11 tumours exhibited full methylation of at least one site within exon 1α and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1α was observed in five tumours which also lacked p19ARF. In normal lung, p16INK4a and p19ARF were not expressed at detectable levels, the multiplex RT–PCR results were balanced, and sites within exon 1α were strongly methylated. In tumours, imbalanced multiplex RT–PCR data (p16INK4a<p19ARF) predicted methylation of exon 1α (P=0.0006) as well as downregulation of p16INK4a. p19ARF downregulation was inversely correlated with p53 overexpression (P=0.025), whilst negative immunostaining for p16INK4a was inversely correlated with pRb downregulation (P=0.003) and directly correlated with p53 overexpression as assessed by immunostaining (P=0.015). Our results show that: (1) p16INK4a and p19ARF expression are altered in almost half of resectable NSCLC; (2) methylation within exon 1α is a frequent, but not the only mechanism of p16INK4a downregulation; and that (3) the inverse association of p19ARF and p53 alteration is consistent with a linked pathway.

Absence of mutations in the ATM gene in breast cancer patients with severe responses to radiotherapy

The effectiveness of cancer radiotherapy is compromised by the small proportion (approximately 5%) of patients who sustain severe normal tissue damage after standard radiotherapy treatments. Predictive tests are required to identify these highly radiosensitive cases. Patients with the rare, recessively inherited, cancer-prone syndrome ataxia-telangiectasia (A-T) sustain extremely severe normal tissue necrosis after radiotherapy and their cultured cells are also highly radiosensitive. Clinically normal carriers (heterozygotes) of the A-T gene have an increased risk of breast cancer, account for approximately 4% of all breast cancer cases and show a modest increase in cellular radiosensitivity in vitro. It has been suggested that a substantial proportion of highly radiosensitive (HR) breast cancer patients may be A-T heterozygotes, and that screening for mutations in the A-T gene could be used as a predictive test. We have tested this hypothesis in a group of cancer patients who showed adverse reactions to radiotherapy. Sixteen HR breast cancer patients showing mainly acute reactions (and seven HR patients with other cancers) were tested for ATM mutations using the restriction endonuclease fingerprinting assay. No mutations typical of those found in obligate A-T heterozygotes were detected. If the estimate that 4% of breast cancer cases are A-T gene carriers is correct, then ATM mutations do not confer clinical radiosensitivity. These early results suggest that screening for ATM mutations in cancer patients may not be of value in predicting adverse reactions.

Maspin - the most commonly-expressed gene of the 18q21.3 serpin cluster in lung cancer - is strongly expressed in preneoplastic bronchial lesions.

Maspin, SCCA1/2 and hurpin were identified by cDNA microarray analyses as dramatically differentially expressed transcripts in primary non-small cell lung cancer (NSCLC). These sequences are located within a 10-gene serpin cluster on 18q21.3. Using comparative RT–PCR, we have investigated the expression of each of these serpins, including their flanking loci, in an independent NSCLC series. Whereas six of the genes (maspin, SCCA1, SCCA2, hurpin, megsin and pAI-2) were commonly differentially expressed in primary lesions, each significantly more often in squamous cell tumours, maspin was identified as the most frequently over-represented sequence in both squamous cell carcinoma and adenocarcinoma. Using a well-characterized monoclonal antibody, we have shown strong maspin expression in tumour protein extracts, detected multiple isoforms of the 42 kDa protein and shown that maspin is localized specifically to the tumour cells within neoplastic lesions. In contrast, most cells in non-neoplastic lung tissue appear not to express the gene, with the exception of the multipotent basal epithelial cells that line the bronchial airway. These reserve cells generally show strong predominantly nuclear localization of maspin. Strong nuclear expression of maspin within primary tumour cells is correlated with increased survival (P=0.05) and a longer remission duration (P=0.02) in resectable-staged patients. However, within the airways of cancer patients and somewhat in contrast to this observation, such expression was more frequently detected in the superficial cells of preneoplastic over non-neoplastic epithelia (P<0.0001), consistent with a role for the protein in early neoplasia.

Xeroderma pigmentosum group D haplotype predicts for response, survival, and toxicity after platinum‐based chemotherapy in advanced nonsmall cell lung cancer

The treatment of lung cancer has reached a therapeutic plateau. Several mechanisms of platinum resistance have been described, including the removal of platinum‐DNA adduct by nucleotide excision repair (NER). Polymorphisms within the Xeroderma pigmentosum Group D protein (XPD), a member of the NER pathway, are associated with alterations in enzyme activity and may change sensitivity to platinum‐based chemotherapy. The authors investigated the relation between XPD polymorphisms and treatment response, toxicity, and overall survival in patients who received platinum‐based chemotherapy for advanced nonsmall cell lung cancer (NSCLC).

Abnormal expression of CCND1 and RB1 in resection margin epithelia of lung cancer patients.

Tumours develop through the accumulation of genetic alterations associated with a progressive increase of the malignant phenotype. In lung cancer, chronic exposure of bronchial epithelium to carcinogens in cigarette smoke may lead to multiple dysplastic and hyperplastic lesions scattered throughout the tracheobronchial tree. Little is known about the genetic alterations in such lesions. This study was carried out to examine cyclin D1 (CCND1) and retinoblastoma (RB1) gene expression in the bronchial epithelium of patients with lung cancer. Lung tumours and their corresponding tumour-free resection margins from 33 patients who underwent resection of non-small-cell lung cancer (NSCLC) were examined by immunostaining with monoclonal antibodies against cyclin D1 (DCS-6; Novocastra) and pRb (NCL Rb-1; Novocastra). Examination of the resection margins revealed four carcinomas in situ, 19 hyperplasias and ten sections showing apparently normal bronchial epithelium. A control group of patients, without lung tumours and who had never smoked, revealed no or weak cyclin D1 and positive pRb staining within bronchial epithelia. Increased cyclin D1 and diminished pRb expression were found in 76% (n = 25) and 27% (n = 9) of the resection margins respectively, and in 12% (n = 4) both cyclin D1 and pRb expression were altered. In the corresponding tumours, 48% (n = 16) were normal, while altered expression was found for cyclin D1 in 33% (n = 11), pRb in 27% (n = 9) and both in 9% (n = 3) of cases. It appears that altered expression of cyclin D1 and pRb is an early event in NSCLC development in almost half of cases analysed. Further investigations are needed to determine the significance of immunostaining of bronchial specimens in individuals at risk of lung cancer, with the possibility that the observations are of importance in the early diagnosis of NSCLC.

METH-2 silencing and promoter hypermethylation in NSCLC.

The antiangiogenic factor METH-2 (ADAMTS-8) was identified in a previous dual-channel cDNA microarray analysis to be at least two-fold under-represented in 85% (28 out of 33) of primary non-small-cell lung carcinomas (NSCLCs). This observation has been validated in an independent series of NSCLCs and adjacent normal tissues by comparative multiplex RT—PCR, and METH-2 mRNA expression was dramatically reduced in all 23 tumour samples analysed. Immunohistochemical analysis of the same sample set demonstrated that METH-2 was strongly expressed in 14 out of 19 normal epithelial sites examined but only one out of 20 NSCLCs. DNA methylation analysis of the proximal promoter region of this gene revealed abnormal hypermethylation in 67% of the adenocarcinomas and 50% of squamous cell carcinomas, indicating that epigenetic mechanisms are involved in silencing this gene in NSCLC. No homozygous deletions of METH-2 were found in lung cancer cell lines. Allelic imbalance in METH-2 was assessed by an intronic single nucleotide polymorphism (SNP) assay and observed in 44% of informative primary samples. In conclusion, the downregulation of METH-2 expression in primary NSCLC, often associated with promoter hypermethylation, is a frequent event, which may be related to the development of the disease.

G1 control gene status is frequently altered in resectable non-small cell lung cancer

Progression through the mammalian cell cycle is controlled by a series of cyclins, cyclin‐dependent kinases (cdks) and cdk inhibitors. Cyclin D1, cdk4 and the tumour suppressors p16 and retinoblastoma protein (pRb) are thought to comprise a linked system governing cell passage through the G1 phase of the cell cycle. Extending an earlier study on cyclin D1 expression, a series of resectable non‐small cell lung carcinomas (NSCLCs) was examined for defects in other elements of this control system. Forty‐six of fifty‐one NSCLC specimens exhibited at least one alteration of these cell‐cycle regulators. Immunohistochemical analysis revealed that 33% and 47% of the tumours failed to express pRb and p16, respectively. Failure to detect pRb did not correlate with loss of heterozygosity at the RB1 locus. Eleven of 12 tumours showing positive (normal) pRb staining over‐expressed nuclear localised cyclin D1, including 8 with amplification of the cyclin D1 gene (CCND1). However, in a number of lesions (n = 5) where cyclin D1 was over‐expressed but localised to the cytoplasm, pRb expression was undetectable. Sequencing of exons 1 and 2 of the p16 gene (CDKN2) revealed 3/51 tumours with somatic mutations (in addition to 1 case with a germ‐line alteration). All of these lesions were positive for p16 protein. No clear homozygous deletions of CDKN2 were observed by multiplex PCR. As assessed by immunostaining using a p16 monoclonal antibody, there was an inverse correlation of pRb and p16 down‐regulation. Whilst patients with tumours over‐expressing cyclin D1 had a significantly lower incidence of local relapse, the group whose tumours failed to express pRb had a significantly greater risk of local relapse and tended to have shortened event‐free survival. Our data show that alteration of at least one cell cycle–regulator gene occurs in the majority of resectable NSCLCs. Int. J. Cancer 74:556–562, 1997.

Linkage studies in a Li-Fraumeni family with increased expression of p53 protein but no germline mutation in p53.

We report a family with the Li-Fraumeni syndrome (LFS) in whom we have been unable to detect a mutation in the coding sequence of the p53 gene. Analysis of linkage to three polymorphic markers within p53 enabled direct involvement of p53 to be excluded. This is the first example of a LFS family in whom exclusion of p53 has been possible. Four affected members of the family with sarcoma or premenopausal breast cancer showed increased expression of p53 protein in their normal tissues as detected by immunohistochemistry. It therefore appears that the LFS phenotype has been conferred by an aberrant gene, showing a dominant pattern of inheritance, which may be acting to compromise normal p53 function rather than by a mutation in p53 itself. In order to try to determine the chromosomal location of this putative gene, we have carried out studies of linkage to candidate loci. By these means we have excluded involvement of Rb1 and BRCA1 on chromosomes 13q and 17q respectively. The MDM2 oncogene on chromosome 12q was considered to be the prime candidate as MDM2 is amplified in sarcomas and the MDM2 product binds to p53. Furthermore, p53 mutation and amplification of MDM2 have been shown to be mutually exclusive events in tumour development. Linkage analysis to two polymorphic markers within MDM2 yielded a three-point LOD score of -5.4 at a recombination fraction theta equal to zero. Therefore MDM2 could be excluded. It is possible that the gene which is responsible for cancer susceptibility in this family, possibly via interaction with p53, will be important in the histogenesis of breast cancer in general. We are now carrying out further studies to locate and identify this gene.