No abstract

Profiling of p53-responsive genes has been carried out in different cellular models, most of which involved genetic modifications or cytotoxic stimulation. We report on the utilization of IPH-926 human lobular breast cancer cells for the profiling of p53-responsive genes using a novel approach without such modifications. We discovered that IPH-926 cells harbor a homozygous TP53 missense mutation encoding for a rare p53 mutant (E285K) with temperature-sensitive (ts) loss of function characteristics. This mutation had evolved as a late, secondary genetic event during the natural clonal evolution of the corresponding lobular carcinoma. In vitro temperature shifts reconstituted endogenous wild-type p53 activity in IPH-926, as evidenced by induction of p21Waf1 . Transcriptional alterations associated with restored p53 function were profiled using Affymetrix microarrays and a new strategy to gate out non-specific temperature effects. At the P ¼ 0.0005 significance level, 60 genes were differentially expressed following reconstitution of p53 activity. These genes included CDKN1A, MDM2 and PHLDA3, a recently described p53-inducible inhibitor of AKT. Similar transcriptional alterations were observed upon reconstitution of p53 activity in BT-474 cells, which also harbor ts-p53 E285K, and in ASPC1 cells transduced with ts-p53 A138V. Consistent with these models, low PHLDA3 expression was associated with nuclear p53 accumulation, indicative of deleterious TP53 mutations, in primary breast cancers. From a molecular point of view, IPH-926 thus provides a new tool to study transcriptional programs controlled by p53. From a tumor pathology perspective, IPH-926 also provides the first direct evidence of a p53-related clonal evolutionary pathway in lobular breast cancer progression. Mutational inactivation of the TP53 tumor suppressor gene encoding for p53 is frequent in human breast cancer, especially in estrogen receptor (ER)-negative, medullary, basallike and BRCA1-related cases. 1-4 The p53 protein functions as a stress-induced transcription factor that regulates various cellular processes, such as growth arrest, apoptosis and energy metabolism. 5 External stresses that stabilize p53, and thus induce p53 signaling, include DNA-damaging agents and hypoxia. 6 Internal stresses that induce p53 signaling include activation of oncogenes, such as PIK3CA and PTEN. 7 Characterization of p53 transcriptional targets is of importance for understanding how mutational inactivation of TP53 promotes tumor initiation and progression. Profiling of p53-responsive genes has been carried out in different cellular models. Some of these models relied on cytotoxic cell stimulation for induction of a p53 response. 8,9 Other models utilized genetically engineered cells, which overexpressed wild-type (wt) p53, TP53 targeting shRNAs for p53 knockdown, or p53 inactivating polypeptides for functional neutralization of p53. [10][11][12][13][14][15][16] Another useful approach has been ectopic overexpression of the murine, temperature-sensitive (ts) p53 mi...

Infiltrating lobular breast cancer (ILBC) is a tumor-biologically distinct breast cancer subtype. A high frequency of oncogenic PIK3CA mutations has been reported in ILBC, which may allow for targeted therapy with newly developed PI3K inhibitors. This is of particular clinical relevance for ILBC patients, who have failed to respond to current treatment regimes and suffer from tumor recurrence or dissemination. In anticipation of this therapeutic strategy, we investigated PIK3CA mutations in ILBC with special reference to late stage tumor progression. A total of 88 ILBCs from 73 patients, including primary tumors (PTs, n = 43), ipsilateral locally recurrent tumors (LRTs, n = 15), and distant organ metastases (DOMs, n = 30), were compiled on tissue microarrays. Established ILBC marker proteins were evaluated by immunohistochemistry and PIK3CA hot spot mutations in exons 9 and 20 by direct sequencing. Matched PT/LRT, PT/DOM, and DOM/DOM cases were characterized on a patient-by-patient basis. Following correction for redundant patient representations, mutation frequencies were compared in PTs versus LRTs or DOMs. Nearly all specimens were E-cadherin-negative (99%), estrogen receptor (ER)-positive (91%), and lacked basal epithelial markers (100%), demonstrating correct ILBC classification. PIK3CA mutations were detected in 32/88 (36%) specimens. The mutation rate was similar in PTs (33%) and DOMs (26%, P = 0.769), but approximately two-fold increased in LRTs (69%, P = 0.022). Consistently, matched PT/LRT and LRT/DOM cases showed additional PIK3CA mutations in LRTs. Intriguingly, these findings imply that PIK3CA mutations are positively selected for during ILBC progression to local recurrence but not distant metastasis, which may have clinical implications for PI3K inhibitor-based therapy.

Inherited mutations in PALB2 are known to be associated with increased breast cancer risk. We aimed to investigate the prevalence and risk association of a recurrent PALB2 mutation, c.509_510delGA, among 3,924 unselected breast cancer patients from Belarus, Russia or Germany. High-resolution melting analyses and direct sequencing identified the c.509_510delGA allele in 3/1,008 (0.3 %) German breast cancer patients, 2/994 (0.2 %) Russian breast cancer patients and 5/1,922 (0.3 %) Byelorussian breast cancer patients. Breast tumours were mainly estrogen receptor positive and included both ductal and lobular histology. Only one of the ten patients had a first-degree family history of breast cancer. The mutation was not detected in 2,827 healthy females from the same populations, confirming the association of PALB2*c.509_510delGA with breast cancer risk (p = 0.007). These data indicate that the PALB2*c.509_510delGA mutation is prevalent in about 1 in 400 breast cancer patients from Central and Eastern Europe, and the low occurrence of familial clustering is consistent with a moderate penetrance of this mutation.

BackgroundMutations in NBN, the gene for Nijmegen Breakage Syndrome (NBS), are thought to predispose women to developing breast cancer, but a breast cancer cell line containing mutations in NBN has not yet been described. The p.R215W missense mutation occurs at sub-polymorphic frequencies in several populations. We aimed to investigate its functional impact in breast cancer cells from a carrier of this NBN mutation.MethodsBreast cancer cell lines were screened by immunoblotting for NBN protein levels, and the NBN coding region was sequenced for mutation analysis. Radiosensitivity assays and functional studies were performed through immunocytochemistry and immunoblotting, and flow cytometry was employed to assess cell cycle progression. Impedance measurements were used to study the consequences of PARP1 inhibition. Statistical comparisons between cell lines were performed using t-tests.ResultsHCC1395 breast cancer cells exhibited reduced NBN protein levels. Direct sequencing identified the NBN p.R215W mutation in the hemizygous state, in addition to a truncation in BRCA1. Mutations in both genes were already present in the heterozygous state in the patient’s germline. HCC1395 cells were highly radiosensitive, susceptible to apoptosis and were deficient in the formation of NBN foci. There was also evidence for some impairment in the formation of γH2AX, MDC1, and 53BP1 foci after irradiation; these foci appeared smaller and irregular compared with repair foci in wild-type cells, although ATM signalling was largely unaffected. In line with their deficiency in NBN and BRCA1, HCC1395 cells were particularly sensitive to PARP1 inhibition.ConclusionOur results indicate that the p.R215W mutation in the HCC1395 breast cancer cell line impairs NBN function, making this cell line a potentially useful cellular model for studying defective NBN protein within a mutant BRCA1 background.