A RAC-GEF network critical for early intestinal tumourigenesis.

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2-/- Vav3-/- Tiam1-/-), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

Germline and sporadic cancers driven by the RAS pathway: parallels and contrasts.

Somatic mutations in RAS and related pathway genes such as NF1 have been strongly implicated in the development of cancer while also being implicated in a diverse group of developmental disorders named the 'RASopathies', including neurofibromatosis type 1 (NF1), Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), Costello syndrome (CS), cardiofaciocutaneous syndrome (CFC), and capillary malformation-arteriovenous syndrome (CM-AVM). It remains unclear why (i) there is little overlap in mutational subtype between Ras-driven malignancies associated with sporadic disease and those associated with the RASopathy syndromes, and (ii) RASopathy-associated cancers are usually of different histological origin to those seen with sporadic mutations of the same genes. For instance, germline variants in KRAS and NRAS are rarely found at codons 12, 13 or 61, the most common sites for somatic mutations in sporadic cancers. An exception is CS, where germline variants in codons 12 and 13 of HRAS occur relatively frequently. Given recent renewed drug interest following early clinical success of RAS G12C and farnesyl transferase inhibitors, an improved understanding of this relationship could help guide targeted therapies for both sporadic and germline cancers associated with the Ras pathway.

The tumour suppressor HACE1 controls cell migration by regulating Rac1 degradation.

The small GTPase Rac1 is a key regulator of cell motility. Multiple mechanisms regulate Rac1 activity including its ubiquitylation and subsequent degradation. Here, we identify the tumour suppressor HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) as an E3 ubiquitin ligase responsible for Rac1 degradation following activation by a migration stimulus. We show that HACE1 and Rac1 interaction is enhanced by hepatocyte growth factor (HGF) signalling, a Rac activator and potent stimulus of cell migration. Furthermore, HACE1 catalyses the poly-ubiquitylation of Rac1 at lysine 147 following its activation by HGF, resulting in its proteasomal degradation. This negative feedback mechanism likely restricts cell motility. Consistent with this, HACE1 depletion is accompanied by increased total Rac1 levels and accumulation of Rac1 in membrane ruffles. Moreover, HACE1-depletion enhances cell migration independently of growth factor stimulation, which may have significance for malignant conversion. A non-ubiquitylatable Rac1 rescues the migration defect of Rac1-null cells to a greater extent than wild-type Rac1. These findings identify HACE1 as an antagonist of cell migration through its ability to degrade active Rac1.

Rho family proteins in cell adhesion and cell migration.

Cell migration and the regulation of cadherin-mediated homotypic cell-cell interactions are critical events during development, morphogenesis and wound healing. Aberrations in signalling pathways involved in the regulation of cell migration and cadherin-mediated cell-cell adhesion contribute to tumour invasion and metastasis. The rho family proteins, including cdc42, rac1 and rhoA, regulate signalling pathways that mediate the distinct actin cytoskeleton changes required for both cellular motility and cell-cell adhesion. Recent studies indicate that rac directly influences rho activity at the GTPase level and that the reciprocal balance between rac and rho activity can determine epithelial or mesenchymal cell morphology and migratory behaviour of epithelial (tumour) cells.

The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells.

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

Sensitivity to transforming growth factor beta 1-induced growth arrest is common in human squamous cell carcinoma cell lines: c-MYC down-regulation and p21waf1 induction are important early events.

Transforming growth factor beta 1 (TGF-beta 1) is a potent inhibitor of keratinocyte proliferation and a potential tumor suppressor of squamous cell carcinomas (SCCs). TGF-beta 1 exerts its antiproliferative effects by inhibiting key transitions required for progression from G1 to the S phase of the cell cycle, exemplified by a rapid reduction of c-MYC and inhibition of the G1 cyclin/cyclin-dependent kinases by induction of their inhibitors p21waf1, p27kip1, and p15INK4B. A significant majority of a new series of human SCC cell lines were found to be as sensitive as primary human epidermal keratinocytes to TGF-beta 1 growth inhibition. Only a minority of cell lines derived from late-stage tumors were resistant. An early and rapid increase in p21waf1 and reduction in c-MYC protein levels were important concomitants for TGF-beta 1 growth inhibition; these changes occurred exclusively in each of the sensitive cell lines. Expression of p15INK4B was found to be neither necessary nor sufficient for TGF-beta 1 growth arrest in the sensitive and resistant cell lines, respectively. TGF-beta 1 induced alterations in other cell cycle regulatory molecules, cyclin-dependent kinase 4, cyclin D1, pRB, and p27Kip1, occurred late and were dispensable in some of the sensitive cell lines. Expression of exogenous mycER fusion protein in one of the sensitive cell lines did not render the cells resistant to TGF-beta 1-induced growth arrest nor prevent p21waf1 induction or down-regulation of both c-MYC and mycER proteins. However, in TGF-beta 1-resistant subclones of sensitive mycER-expressing cells, p21waf1 was not induced, whereas both c-MYC and mycER protein levels decreased following TGF-beta 1 treatment. We conclude that TGF-beta 1 activates multiple cell cycle inhibitory pathways dependent upon p21waf1 induction and c-MYC degradation and that it does not function as a tumor suppressor in the majority of SCCs.

Association of CDKN2A/p16INK4A with human head and neck keratinocyte replicative senescence: relationship of dysfunction to immortality and neoplasia.

We have previously suggested that a gene mapping to chromosome 9p21 could contribute to replicative senescence and suppress cullular immortality in squamous neoplasia. Two candidate genes, the cyclin D1/cyclindependent kinase inhibitors CDKN2A/p16INK4A (p16) and CDKN2B/p15INK4B (p15) have now been identified in this region and we show here that p16 is upregulated when normal human keratinocytes undergo replicative senescence but not when they undergo differentiation. Furthermore, all of 19 immortal neoplastic keratinocyte head and neck lines, including nine showing loss of heterozygosity (LOH) at 9p21, showed undetectable p16 expression, whereas five of six senscent neoplastic cultures showed normal levels of expression. The retinoblastoma protein (pRb) appeared functional in all the cell lines and cultures examined. The mechanism of p16 inactivation appeared to be transcriptional silencing in 10 of 18 lines and homozygous deletions in the rest. Treatment of two of the immortal cell lines which had transcriptionally silent wild type p16 genes with 5aza-2deoxycytidine resulted in the re-expression of p16, thus implicating DNA methylation as one mechanism of transcriptional silencing in the immortal SCC-HN lines. We observed no cases of p16 point mutation. In contrast, the p15 gene was rarely transcriptionally silent and was not deleted in any of the cell lines which showed p16 deletions. Our results show that p16 dysfunction correlates strongly with keratinocyte immortalisation but less strongly with the stage of tumour progression. P16 dysfunction was not related to the neoplastic state or the length of time spent in vitro. The results also suggest that p16 but not p15 is involved in the keratinocyte replicative senescence programme. However, two neoplastic cell cultures which lacked p16 expression were still mortal, suggesting that the loss of p16 is a necessary but insufficient condition for human keratinocyte immortality.

C-jun oncogene expression in nonsmall cell lung-cancer.

Primary lung cancer specimens of the non-small cell (NSC) types were examined for expression of the c-jun gene at the protein level by immunocytochemistry using the polyclonal antibody c-jun 890. The results obtained revealed that the c-jun p39 protein was expressed in 82% (18/22) of squamous cell carcinomas, 77% (10/15) of adenocarcinomas and 40% (2/5) of large cell anaplastic carcinomas, but no staining was seen in the non-cancer cases. Positive immunostaining was also seen in 16.7% (3/18) of the nonneoplastic bronchial cells of positively stained squamous cell carcinomas and in 40% (4/10) of adenocarcinomas. We suggest that elevated expression df c-jun p39 protein which was seen in a total of 75% (30/40) of the NSCLC, plays a role in lung cancer.

P53 expression in end-stage squamous-cell carcinoma of the head and neck prior to chemotherapy treatment - expression correlates with a very poor clinical outcome.

p53 expression was assessed immunohistochemically in 24 'end stage disease' patients with squamous cell carcinoma of the head and neck, prior to selection for a chemotherapy trial. Twelve patients were assigned to each arm of the trial; cisplatinum arm or the cisplatinum and nifedipine arm. p53 expression was assessed using the CM-1 antibody in specimens from biopsies or surgically removed tissue at the time these patients were assessed as end stage disease. Sixty-six per cent had p53 positive nuclear staining but no correlation was found between p53 staining and age, sex, site of primary tumour, tumour stage, or site of the recurrence. Three patients responded to cisplatinum chemotherapy treatment, two of whom had p53 positive staining. p53 survival curves were calculated for these patients from the date they were assessed as 'end stage disease', p53 overexpression was found to correlate with a very poor clinical outcome (P<0.05). Survival curves for 109 head and neck patients calculated from the date the disease first presented showed no correlation with p53 expression.

Detection of hepatitis-B virus-DNA and mutations in k-ras and p53 genes in human hepatocellular carcinomas.

Hepatitis B virus (HBV) infection is considered as one of the major risk factors in the development of human hepatocellular carcinoma (HCC). Recent studies have also suggested the implication of oncogene and onco-suppressor genes in liver carcinogenesis. We studied 41 cases of HCC for the presence of HBV DNA and point mutations in codon 12 of K-ras and codon 249 of p53. We used 'nested' PCR for the amplification of HBV because of the expected low incidence of the virus DNA in the samples. PCR was also used for the amplification of K-ras and p53 regions that contain the codons of interest, followed by RFLP analysis for the detection of point mutations. HBV DNA was amplified in 22 cases (53.7%), while 5 cases (12.2%) appeared to carry mutations in codon 12 of K-ras and 7 cases (17.1%) had mutations in codon 249 of the p53 gene. These results further support the correlation between HBV infection and HCC and also indicate an implication of K-ras and p53 genes in hepatocarcinogenesis.

Expression of ras Rb1 and p53 proteins in human breast cancer.

The ras, Rb and p53 genes have been implicated in the development of human breast cancer. Qualitative or quantitative changes in the expression of the ras p21 may lead to cell transformation, and this has been previously demonstrated in breast cancer. Both the retinoblastoma protein (Rb1) and the p53 gene product appear to function as negative regulators of cell division. We have investigated the expression of ras p21, Rb1 and p53 proteins in human breast cancer patients immunohistochemically, and correlated the results with a range of clinical and pathological parameters. Ras p21 expression was elevated in 65 per cent and p53 in 23 per cent of cases. Rb1 was expressed in 58 per cent of breast cancer tissues and in 75 per cent of normal tissue. Only four patients were found to have loss of Rb1 expression and also overexpression of both p53 and ras gene products. No correlations were found between the expression of these three genes and menopausal status, histological types or tumour grade. However, a correlation was found between Rb1 loss of expression and tumour diameter (greater than 2 cms), and no lymph node metastasis. Also, a significantly higher number of p53 staining specimens were found to be overexpressing the ras gene. These results suggest that all three oncogenes are most likely involved in the development of breast cancer but that their role is complex.

Mutations in the p53 gene at codon 249 are rare in squamous-cell carcinoma of the head and neck.

Mutations in the p53 tumour suppressor gene are common in a wide range of human malignancies. In particular G-T transversions in the p53 gene occur at a high frequency in lung cancer tumours and as the same mutagens maybe responsible for p53 mutations in head and neck squamous cell carcinomas, a preliminary investigation was carried out to establish if this may be the case. Codon 249 of the p53 gene has been found to be a hot spot of G-T transversions in hepatocellular carcinomas, thus it was decided to initially investigate this site using specific primers and restricting the PCR product with the HaeIII restriction endonuclease. Fifty seven squamous cell carcinomas of the head and neck were investigated using this technique and no mutations were found at codon 249 in the p53 gene. In this study twenty six of the specimens were also examined immunohistochemically using a number of antibodies to p53 (PAb 421, PAb 1801 and CM-1), and 19 were found to have positive staining. Moreover as positive p53 staining is considered to be synonymous with p53 mutations, over two thirds of our earlier immunohistochemical study indicated p53 mutations in head and neck tumours. The technique used in this paper has the advantage of scanning a large number of tumours for mutations at suspect sites and maybe of use in the future if specific mutational hot spots are found in the p53 gene in head and neck cancers. In the meantime p53 mutations in these tumours are been investigated by conventional sequencing techniques.

Ras and p53 expression in non-small-cell lung-cancer patients - p53 over-expression correlates with a poor prognosis.

Expression of the tumor suppressor gene p53 and the ras oncogene were examined in 46 tumor and nodal specimens of non-small cell lung cancer (NSCLC) using the antibodies p53 pAb 240 and ras Y13-259 respectively. p53 expression was elevated in 46% and ras p21 was over-expressed in 85% of the tumor specimens analyzed. Fifteen cases of benign lessions were also assessed for both ras p21 and p53 expression; all were found to have negative staining. p53 over-expression was found to correlate with a poor prognosis in both the tumor specimens (p<0.05) and in the nodal tissues (p<0.005). Ras p21 over-expression was found to be associated with survival (p<0.1) in both the tumor and the nodal specimens. Stage of the disease correlated with survival; similarly both p53 and ras p21 over-expression correlated with stage. No correlations were found with the pathological grade of the tumors nor with a history of smoking or duration of smoking. No K-ras mutations at codon 12 were observed in a further 15 NSCLC specimens analyzed. These results indicate that the p53 gene in particular plays a role in the stages of NSCLC.

Elevated P53 expression correlates with a history of heavy smoking in squamous cell carcinoma of the head and neck.

Expression of the tumour suppressor gene p53 was examined in squamous cell carcinoma of the head and neck using two p53 antibodies, PAb 421 and PAb 1801. Elevated p53 expression was found in 67% of the 73 patients investigated. P53 expression was not found to correlate with whether the patient had been previously treated or not, nor any of the clinico-pathological parameters. However a correlation was found between the patients smoking history and positive p53 staining. Six out of seven non-smokers did not express p53 whereas 29 of 37 heavy smokers were found to have elevated p53 expression (P less than 0.005). Also, of a group of ten patients who had given up smoking more than 5 years ago, nine had elevated expression. Epidemiological studies have shown a correlation between heavy smoking and head and neck cancer. The present study indicate a genetic link for this correlation.

Ras p21 expression in brain tumors: elevated expression in malignant astrocytomas and glioblastomas multiforme.

We have employed an immunohistochemical analysis to study the ras p21 oncoprotein in a total of 41 brain tumors and 1 reactive gliosis. The tumors included 33 astrocytomas, 1 oligodendroglioma, 2 ependymomas, 1 neurilemmoma, 1 malignant meningioma, 1 neuroblastoma and 2 medulloblastomas. Our results indicate that elevated ras p21 expression is a common feature in a range of brain tumors. In particular, elevated ras p21 expression has been found in 18 out of 24 high grade astrocytomas (malignant astrocytomas and glioblastomas multiforme) compared to 3 out of 9 low grade (well differentiated astrocytomas) (P less than 0.05). These results suggest that ras p21 expression may be an important molecular marker of the malignant astrocytomas and glioblastomas multiforme.

p53 expression in cytologic specimens from benign and malignant breast lesions.

This study was undertaken to determine the expression of p53 gene in cytologic specimens from benign and malignant breast lesions. To detect p53 an immunocytochemical assay with p53 (pAb421) monoclonal antibody was used. Abnormalities in p53 expression were found in 19 out of 40 Fine Needle Aspiration (FNA) smears with infiltrating ductal breast carcinomas. Benign epithelial breast cells obtained from fibroadenomas, fibrocystic disease and smears from nipple discharge reacted negatively for p53 in 38 out of 39 cases. Moderate positive reaction, confined to a few clusters of epithelial cells, was observed in one smear of fibroadenoma with cellularity. The results recorded in this study show that no significant association was found between p53 staining and stage of disease, tumor size or nodal status and that the immunocytochemical assay represents a simple method for the detection of p53 associated proteins in breast lesions.

Implication of the ras and myc oncoproteins in the pathogenesis of mycosis fungoides.

In the present work, we studied the expression of the c-myc oncoprotein p-62 and the ras oncoprotein p-21 in the dermal cellular infiltrate of paraffin embedded skin specimens, obtained from patients suffering from Mycosis Fungoides and Sezary syndrome. Nineteen specimens from early stage Mycosis Fungoides, nineteen from advanced stage Mycosis Fungoides and four from Sezary syndrome were included in the study. The oncoprotein detection was achieved immunohistochemically, using the mouse monoclonal antibody myc 1-9E10 and the rat monoclonal antibody Y13-259 for p-62 and p-21 respectively. Increased detection of both p-62 and p-21 in atypic lymphoid cells was shown in advanced stages of Mycosis Fungoides (third stage plaques and tumors) as compared to early stages (premycotic erythema, second stage plaques). In advanced stages, however, the percentage of P-62+ atypic cells proved to be higher than that of p-21+ atypic lymphoid cells. The implication of increased p-62 and p-21 oncoprotein expression in the process of lymphomagenesis in cutaneous T-cell lymphomas is discussed.

Elevated expression of AP-1 activity in human breast tumors as compared to normal adjacent tissue.

The levels of AP-1 activity in human breast lesions and in adjacent normal tissue were studied by a gel retardation assay. A thirty nucleotide long consensus oligonucleotide to the adenovirus E3 gene AP-1 sequence (E3AP-1) was end labelled and reacted with nuclear extracts from breast lesions and adjacent normal tissue. A total of 20 tissue extracts (8 pairs of tumor and normal tissue from the same patient and 4 tumors) were examined. All 12 tumor tissues showed elevated levels of AP-1 as compared to the 8 normal tissues. These results suggest that the AP-1 transcription factor may play a role in breast neoplasia.