Identification of CDK10 as an important determinant of resistance to endocrine therapy for breast cancer.

Therapies that target estrogen signaling have transformed the treatment of breast cancer. However, the effectiveness of these agents is limited by the development of resistance. Here, an RNAi screen was used to identify modifiers of tamoxifen sensitivity. We demonstrate that CDK10 is an important determinant of resistance to endocrine therapies and show that CDK10 silencing increases ETS2-driven transcription of c-RAF, resulting in MAPK pathway activation and loss of tumor cell reliance upon estrogen signaling. Patients with ER alpha-positive tumors that express low levels of CDK10 relapse early on tamoxifen, demonstrating the clinical significance of these observations. The association of low levels of CDK10 with methylation of the CDK10 promoter suggests a mechanism by which CDK10 expression is reduced in tumors.

iASPP preferentially binds p53 proline-rich region and modulates apoptotic function of codon 72-polymorphic p53.

iASPP is one of the most evolutionarily conserved inhibitors of p53, whereas ASPP1 and ASPP2 are activators of p53. We show here that, in addition to the DNA-binding domain, the ASPP family members also bind to the proline-rich region of p53, which contains the most common p53 polymorphism at codon 72. Furthermore, the ASPP family members, particularly iASPP, bind to and regulate the activity of p53Pro72 more efficiently than that of p53Arg72. Hence, escape from negative regulation by iASPP is a newly identified mechanism by which p53Arg72 activates apoptosis more efficiently than p53Pro72.

Tip60 is a haplo-insufficient tumour suppressor required for an oncogene-induced DNA damage response.

The acetyl-transferase Tip60 might influence tumorigenesis in multiple ways. First, Tip60 is a co-regulator of transcription factors that either promote or suppress tumorigenesis, such as Myc and p53. Second, Tip60 modulates DNA-damage response (DDR) signalling, and a DDR triggered by oncogenes can counteract tumour progression. Using E(mu)-myc transgenic mice that are heterozygous for a Tip60 gene (Htatip) knockout allele (hereafter denoted as Tip60+/- mice), we show that Tip60 counteracts Myc-induced lymphomagenesis in a haplo-insufficient manner and in a time window that is restricted to a pre- or early-tumoral stage. Tip60 heterozygosity severely impaired the Myc-induced DDR but caused no general DDR defect in B cells. Myc- and p53-dependent transcription were not affected, and neither were Myc-induced proliferation, activation of the ARF-p53 tumour suppressor pathway or the resulting apoptotic response. We found that the human TIP60 gene (HTATIP) is a frequent target for mono-allelic loss in human lymphomas and head-and-neck and mammary carcinomas, with concomitant reduction in mRNA levels. Immunohistochemical analysis also demonstrated loss of nuclear TIP60 staining in mammary carcinomas. These events correlated with disease grade and frequently concurred with mutation of p53. Thus, in both mouse and human, Tip60 has a haplo-insufficient tumour suppressor activity that is independent from-but not contradictory with-its role within the ARF-p53 pathway. We suggest that this is because critical levels of Tip60 are required for mounting an oncogene-induced DDR in incipient tumour cells, the failure of which might synergize with p53 mutation towards tumour progression.

The prolyl isomerase Pin1 orchestrates p53 acetylation and dissociation from the apoptosis inhibitor iASPP.

The tumor-suppressor function of p53 relies on its transcriptional activity, which is modulated by post-translational modifications and interactions with regulatory proteins. The prolyl isomerase Pin1 has a central role in transducing phosphorylation of p53 into conformational changes that affect p53 stability and function. We found that Pin1 is required for efficient loading of p53 on target promoters upon stress. In addition, Pin1 is recruited to chromatin by p53 and stimulates binding of the p300 acetyltransferase and consequent p53 acetylation. Accordingly, tumor-associated mutations at Pin1-binding residues within the p53 proline-rich domain hamper acetylation of p53 by p300. After phosphorylation of p53 at Ser46 triggered by cytotoxic stimuli, Pin1 also mediates p53's dissociation from the apoptosis inhibitor iASPP, promoting cell death. In tumors bearing wild-type p53, expression of Pin1 and iASPP are inversely correlated, supporting the clinical relevance of these interactions.

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.

Survival prediction and frequency of anticancer treatment in cancer patients hospitalized due to acute conditions. Role of clinical parameters and PaP score.

PURPOSE: Survival prediction is useful in selecting patients for palliative care or active anticancer therapy. The palliative and prognostic (PaP) score was shown to predict 1-month survival in terminally ill patients. Its application to patients with less advanced disease is a subject of debate. We assessed the value of the PaP score and of other clinical parameters in predicting survival in patients admitted in an oncological ward due to acute conditions. We also evaluated the frequency of active anticancer treatment in the last weeks of life. METHODS: All the 208 patients, consecutively admitted in a department of medical oncology and radiotherapy in a 9-month period, were included. Patients and disease features together with the PaP score were assessed and included in a multivariable model for survival prediction. RESULTS: Overall, median survival was 19 weeks and 12-week survival was 59.6%. The PaP score accurately predicted 4-week survival. Among the 39 patients who died within 4 weeks, 36% were on active treatment. The reason of admission, disease control, treatment, and PaP score were independently related to 12-week survival in the multivariate analysis; however patients with a 12-week survival lower than 30% were a minority. CONCLUSIONS: Although the PaP score accurately predicts life expectancy, its use in the setting of acute conditions seems not straightforward, due to the overall good prognosis of these patients. Active treatment in the last period of life is common. The potential reversibility of acute conditions makes prognostic measures inadequate for the purpose of treatment choices.

Functional regulation of p73 and p63: development and cancer.

The transcription factor and tumour suppressor p53 and its two homologues p63 and p73 form a family of proteins. p63 and p73 show much greater molecular complexity than p53 because they are expressed both as multiple alternatively spliced C-terminal isoforms, and as N-terminally deleted, dominant-negative proteins that show reciprocal functional regulation. In addition, several other factors, such as post-translational modifications and specific and common family regulatory proteins, result overall in subtle modulation of their biological effects. Although all p53, p63 and p73 family members are regulators of the cell cycle and apoptosis, the developmental abnormalities of p73- and p63-null mice do not show enhanced tumour susceptibility of p53 knockouts, suggesting that complex regulatory processes modulate the functional effects of this family of proteins.

Epigenetic inactivation implies independent functions for insulin-like growth factor binding protein (IGFBP)-related protein 1 and the related IGFBPL1 in inhibiting breast cancer phenotypes.

PURPOSE: To analyze epigenetic regulation of two related genes, insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) and IGFBPL1, and its significance as a determinant of clinical phenotypes in human breast cancer. EXPERIMENTAL DESIGN: We have investigated the expression and epigenetic regulation of IGFBP-rP1 and IGFBPL1 in human breast cancer cell lines and primary and metastatic carcinomas. RESULTS: Expression of IGFBP-rP1 and IGFBPL1 is down-regulated in breast cancer cell lines. Aberrant methylation in the CpG islands of each gene correlates well with loss of expression at the mRNA level. Analysis of methylation in DNA isolated from human primary breast tumors showed that methylation in either gene was associated with a worse overall survival (OS; P=0.008) and disease-free survival (DFS) following surgery (P=0.04) and worse DFS following adjuvant chemotherapy (P=0.01). Methylation of IGFBP-rP1 alone was associated with a trend toward decreased OS (P=0.10) and decreased DFS (P=0.25). Methylation in IGFBPL1 was clearly associated with worse OS (P=0.001) and DFS (P<0.0001). Methylation in either IGFBP-rP1 or IGFBPL1 was significantly associated with nodal disease (P<0.001). CONCLUSIONS: Expression of IGFBP-rP1 and IGFBPL1 is regulated by aberrant hypermethylation in breast cancer, implying that inactivation of these genes is involved in the pathogenesis of this malignancy. Analysis of methylation of these genes may have utility in prediction of clinical phenotypes, such as nodal disease and response to chemotherapy.

A phase I-II preoperative biomarker trial of fenretinide in ascitic ovarian cancer.

PURPOSE: To evaluate study feasibility, toxicity, drug concentrations, and activity of escalating doses of the synthetic retinoid fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] in ovarian cancer by measuring serum CA125 and cytomorphometric biomarkers in cancer cells collected from ascitic fluid before and after treatment. METHODS: Twenty-two naive patients with ascitic ovarian cancer were treated with escalating doses of 4-HPR at 0, 400, 600, and 800 mg/d for 1 to 4 weeks before surgery. Changes in the proportion of proliferating cells expressed by Ki67 and computer-assisted cytomorphometric variables (nuclear area, DNA index, and chromatin texture) were determined in ascitic cells. Drug levels were measured by high-performance liquid chromatography. RESULTS: Doses up to 800 mg/d were well tolerated, and no adverse reactions occurred. There was no effect of 4-HPR on changes in serum CA125, Ki67 expression, which were assessed in 75% of subjects, and cytomorphometric variables, which were assessed in 80% of subjects. Plasma retinol levels were significantly lower in affected women than healthy donors. 4-HPR plasma concentrations increased slightly with increasing doses and attained a 1.4 micromol/L concentration with 800 mg/d. Drug levels in malignant ascitic cells and tumor tissue were higher than in plasma but were 50 and 5 times lower, respectively, than in carcinoma cells treated in vitro with 1 micromol/L 4-HPR. CONCLUSIONS: Cell biomarkers can be measured in ascitic cells to assess drug activity. Under our experimental conditions, 4-HPR did not show activity in advanced ovarian cancer cells. However, clinical evidence supports further investigation of fenretinide for ovarian cancer prevention.

Gemcitabine and cisplatin in a concomitant alternating chemoradiotherapy program for locally advanced head-and-neck cancer: a pharmacology-guided schedule.

PURPOSE: Administration of gemcitabine together with cisplatin at cytotoxic doses in a chemoradiotherapy regimen is hampered by a high degree of local toxicity. Using the pharmacologic properties of the drug we designed a modified schedule aimed at reducing toxicity while preserving activity. METHODS AND MATERIALS: Patients with squamous cell carcinomas of the oral cavity, pharynx and larynx, bulky T4, and/or N2 to N3 were eligible. Gemcitabine was administered at a dose of 800 mg/m2 on Days 1 and 12 and cisplatin at a dose of 20 mg/m2 on Days 2 to 5, every 21 days for 3 courses. Radiotherapy, delivered with standard fractionation, was given on Days 8 to 12 and 15 to 19 and was repeated 3 times up to a total dose of > or = 60 Gy. RESULTS: A total of 28 patients were selected. Grade 3 to 4 stomatitis was recorded in 25 patients (89%). Thirteen patients (46%) experienced Grade 3 to 4 neutropenia. Febrile neutropenia occurred in 8 patients (29%) and in 2 was complicated by infection and death. The overall complete response rate was 79%. At a median follow up of 71 months, 11 patients had a locoregional relapse (3-year locoregional control, 64%); 6 patients had distant metastases, among whom only 2 were without locoregional recurrence. The 3-year progression-free survival is 39% and 3-year overall survival has been 43%. CONCLUSION: The schedule modification did not attenuate local toxicity. Moreover, infections and especially pneumonia, were a major problem. The high activity of gemcitabine when combined with radiotherapy would most likely be better exploited in the context of modified radiation schemes.

Epigenetic inactivation of 14-3-3 sigma in oral carcinoma: association with p16(INK4a) silencing and human papillomavirus negativity.

In vitro studies have identified 14-3-3sigma as a regulator of senescence in human keratinocytes. To assess its contribution to squamous neoplasia, we have analyzed genetic and epigenetic changes in this gene in squamous cell carcinomas (SCCs) and dysplastic lesions of the oral cavity. No mutations were detected in the coding sequence of 14-3-3sigma in 20 oral carcinomas, and there was loss of heterozygosity in only 7 of 40 informative cases. In contrast to the absence of genetic change, aberrant methylation within 14-3-3sigma was detected in 32 of 92 squamous cell carcinomas and in 3 of 6 oral dysplasias and was associated with reduced or absent expression at both mRNA and protein levels. Methylation was not detected in matched, normal epithelial tissue controls. Carcinomas in which 14-3-3sigma was methylated were significantly more likely to lack DNA sequences from human papillomavirus and to have coincident methylation of p16(INK4a) than cases that expressed 14-3-3sigma. Methylation was detected in SCC, both wild-type and mutant for p53, but was more commonly detected in cancers with wild-type p53. These results implicate coincident epigenetic abrogation of function in both sigma and p16(INK4a) in a subset of SCCs of the oral cavity.

Polymorphism in wild-type p53 modulates response to chemotherapy in vitro and in vivo.

A single-nucleotide polymorphism (SNP) in exon 4 results in expression of either arginine (72R) or proline (72P) at codon 72 of p53. We demonstrate that the in vitro response of cells exposed to anticancer agents is strongly influenced by this SNP in wild-type p53. In inducible systems and in cells expressing the endogenous protein, expression of 72P wild-type p53 results in a predominant G1 arrest, with only a minor apoptosis, at drug concentrations causing extensive apoptosis in cells expressing the 72R wild-type variant. The superior apoptosis-inducing activity of the 72R form correlates with more efficient induction of specific apoptosis-associated genes, and is maximal in the presence of serine 46 (S46). In vivo, the outcome of chemo-radiotherapy of squamous carcinomas is more favourable in cancers retaining a wild-type 72R allele, such cases having higher response rates and longer survival than those with wild-type 72P. Together, these results reveal that this SNP is an important determinant of response to anticancer agents in cells expressing wild-type p53. Analysis of complete p53 genotype (mutation and SNP) merits detailed investigation as a simple means for prediction of treatment response and survival in clinical oncology.

Coincident inactivation of 14-3-3sigma and p16INK4a is an early event in vulval squamous neoplasia.

The structure and expression of 14-3-3 sigma(sigma) was analysed in squamous carcinomas (SCC) of the vulva and in the vulval pre-malignant lesion vulval intraepithelial neoplasia (VIN). Sequence analysis of the sigma coding region did not detect mutations in any case of SCC or VIN III and loss of heterozygosity (LOH) occurred in only 2 out of 27 informative cases. In contrast to the absence of genetic change, methylation-specific PCR (MSP) analysis revealed dense CpG methylation within the sigma gene in approximately 60% of cases of vulval SCC, but methylation was not detected in matched, normal epithelial tissue. Methylation was associated in all cases with reduced or absent expression of sigma mRNA. There was no correlation between sigma methylation and HPV or p53 status. Analysis of pre-malignant vulval intraepithelial neoplasia (VIN) revealed that sigma methylation was detectable early in neoplastic development. Co-incident methylation, accompanied by loss of expression, of sigma and p16INK4a was commonly detected in both SCC and VIN III, suggesting that epigenetic silencing of these two genes is an early and important event in vulval neoplasia.

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.

SERMs in chemoprevention of breast cancer.

Selective estrogen receptor modulators (SERMs) play a key role in breast cancer chemoprevention. Tamoxifen has been shown to reduce breast cancer incidence by 30-40% in at-risk subjects in large phase III trials. However, toxicity may be a limiting factor. Thus, different strategies are being pursued to improve the risk: benefit ratio of using these compounds in chemoprevention. Firstly, the second generation SERM raloxifene is currently undergoing evaluation in comparison with tamoxifen in a large phase III trial. Also, lower doses of tamoxifen are being assessed in phase II-III trials. In addition, the combination of hormone replacement therapy (HRT) or aromatase inhibitors and tamoxifen at low doses may reduce the risks while retaining the benefits of either agents. Finally, new agents that interfere with the onset of ER-negative breast cancer are being sought for combination chemoprevention since almost a third of breast cancers will not be sensitive to hormonal modulation.

Paclitaxel, cisplatin, 5-fluorouracil and radiotherapy in the management of advanced squamous cell carcinoma of the head and neck: a phase II trial.

Aim of the present study was to test, activity and toxicity of a rapidly alternating chemoradiation (paclitaxel based) in 31 patients with unresectable, locally advanced or recurrent after surgery, head and neck cancer. Three-year overall survival and progression-free survival were 61.4 and 73.7%, respectively. Main side effects remain a major problem.

Carboplatin and weekly paclitaxel in non-small cell lung cancer patients unfit for or pretreated with chemotherapy.

BACKGROUND: Carboplatin-Paclitaxel is one of the most active regimens in non-small cell lung cancer (NSCLC). We assessed the administration of weekly Paclitaxel as second-line chemotherapy, or as first-line chemotherapy in unfit patients. PATIENTS AND METHODS: Forty-eight patients received Carboplatin at the dose of 6 x area under the concentration-time curve (AUC) on day 1 and Paclitaxel 100 mg/m2 on days 1, 8, 15 every 28. Thirty-two had received a prior platinum-based treatment, while 16 were chemotherapy-naive, unfit patients. RESULTS: Grade 3-4 neutropenia occurred in 16 patients (33%); grade 3-4 thrombocytopenia in 7 (15%); grade 1-3 peripheral sensory neuropathy in 35 (73%). Nineteen patients (39.6%; 95% C.I.: 25.8% - 53.4%) achieved an objective response without any difference between the first-line and second-line group. One-year survival was 39.5% (95% CI: 25.4% - 53.6%). CONCLUSION: The impressive activity of this regimen makes it suitable for further investigation in the second-line setting. Toxicity seen in the unfit population mandates some modification of the regimen.

TP53 mutations in familial breast cancer: functional aspects.

Mutation in p53 (TP53) remains one of the most commonly described genetic events in human neoplasia. The occurrence of mutations is somewhat less common in sporadic breast carcinomas than in other cancers, with an overall frequency of about 20%. There is, however, evidence that p53 is mutated at a significantly higher frequency in breast carcinomas arising in carriers of germ-line BRCA1 and BRCA2 mutations. Some of the p53 mutants identified in BRCA1 and BRCA2 mutation carriers are either previously undescribed or infrequently reported in sporadic human cancers. Functional characterization of such mutants in various systems has revealed that they frequently possess properties not commonly associated with those occurring in sporadic cases: they retain apoptosis-inducing, transactivating, and growth-inhibitory activities similar to the wild-type protein, yet are compromised for transformation suppression and also possess an independent transforming phenotype. The occurrence of such mutants in familial breast cancer implies the operation of distinct selective pressures during tumorigenesis in BRCA-associated breast cancers.

Single-agent pegylated liposomal doxorubicin (Caelix) in chemotherapy pretreated non-small cell lung cancer patients: a pilot trial.

Polyethylene glycol-coated (pegylated) liposomal doxorubicin (PLD) is a new formulation of doxorubicin with peculiar pharmacokinetic and pharmacodinamic properties, a favorable toxic profile and a demonstrated activity in solid tumors. We tested PLD in locally advanced or metastatic NSCLC patients, progressed after a platinum-based first-line chemotherapy. PLD was administered at the dose of 35 mg/m(2) every 21 days. After the first six patients had been accrued, due to the low toxicity shown in the first six patients, the dose was escalated to 45 mg/m(2). Seventeen patients were enrolled in the study and were considered eligible for evaluation of toxicity and response. Stomatitis, palmar-plantar erythrodysaesthesia (PPE) and asthenia were the most common toxicities and affected approximately half of the treated patients. Stomatitis occurred in 8/17 patients and was grade 3-4 in three. PPE was seen in 9/17 and was grade 3 in one. In the group treated at the dose of 45 mg/m(2) PPE was more frequent and severe and required treatment delay in some cases. Other toxicities were equally distributed among the two groups. Hematological toxicity was not common and never reached grade 3-4. However, one patient with grade 2 leucopenia had pneumonia and died. Clinically evident heart failure was never recorded. Left ventricular ejection fraction was assessed in three patients after PLD treatment (in one case after the first course, due to the occurrence of atrial fibrillation, and in two cases after six courses) and was unchanged compared to pre-treatment assessment. One confirmed partial response was observed (5.8%); five patients (29.4%) had stable disease (including one minor response) and nine (52.9%) had disease progression. Median time to progression was 9.5 weeks, median survival 18.6 weeks. PLD at the doses employed in this study can be safely administered and has shown activity in platinum pretreated NSCLC patients.

The p53 network in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and, despite advances in treatment, still represents a clinical challenge. Inactivation of one or more components of the p53 network is an extremely common event in human neoplasia. In HNSCC, disabling of p53 occurs in a high proportion of cases by mutation in the p53 gene, but other mechanisms of inactivation, such as the presence of human papillomavirus (HPV) and molecular abnormalities in other components of the pathway, are also recognised. The frequent changes occurring in the p53 pathway in HNSCC imply that molecular genetic and immunocytochemical analysis of this critical tumour suppressor network may be of diagnostic and prognostic utility in the clinical management of HNSCC. Further, these changes also provide targets for the development of novel therapeutic approaches to this increasingly common cancer, in which clinical cure for advanced disease remains an elusive goal.