PF271 enhanced the lethality of ABT-737.

<p>A-upper panels, Ovarian cancer cell lines were exposed to various concentrations of ABT-737 (⋄: A), sorafenib (△: S), ABT-737 in combination with 1 µM BEZ235 and 5 µM PF271 (: ABP), or sorafenib in combination with 1 µM BEZ235 and 5 µM PF271 (▴: SBP) for 72 hr. Cell viability (%) was calculated and dose response curves were predicted with a three-parameter log-logistic function. A-lower panel, The EC₅₀ of the anti-apoptotic inhibitors alone or in combination. This represents the result of one experiment; two additional studies also exhibited equivalent results. B, The protein expression patterns of the anti-apoptotic proteins and the phosphorylation status of AKT in nine ovarian cancer cell lines. Immunoblots were done to assess the basal expression levels of BCL-2, BCL-XL, MCL1, and phosphorylated AKT. C, The standard mRNA expression levels of BCL-XL and BCL-2 in forty ovarian cancer cell lines (CCLE database).</p

FAK and BCL2/BCL-XL inhibitors induced apoptosis.

<p>A, OCCC cell lines were treated with 1 µM ABT-737 (A), 1 µM BEZ235 (B), or 5 µM PF271 (P), individually or in combinations (AB indicates ABT-737/BEZ235; AP, ABT-737/PF271; BP, BEZ235/PF271; ABP, ABT-737/BEZ235/PF271) at the same drug concentrations for 6 hr. Cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies. Cleavage of caspase-3 (CASP3), caspase-8 (CASP8), PARP, and FAK indicated that apoptosis had occurred. BCL-2 was not expressed in OVMANA and OVISE. B, TOV21G, RMGI and OVISE cells were treated individually or in combination with 1 µM ABT-737, 1 µM BEZ235 and/or 5 µM PF271 for 6 hr. The cells were fixed with 70% ethanol, were stained with propidium iodide (PI) and analyzed using a FACS Calibur flow cytometer.</p

Drug responses of ovarian cancer cell lines to the FAK inhibitors.

<p>A, The chemical structure of the FAK inhibitors PF-573,228 (PF228) and PF-562,271 (PF271). B. OVISE cells were incubated for 24 hr at the indicated concentrations of the FAK inhibitors. Immunoblots were performed to assess inhibition of auto-phosphorylation by the FAK inhibitors. A vehicle control, containing dimethyl sulfoxide (DMSO), was performed. C, The viability of the ovarian cancer cells was determined after exposure to PF228 and PF271 for 72 hr. The results from only one experiment are shown; two additional studies also exhibited equivalent results.</p

FAK overexpression was associated with an increased copy number in OCCCs.

<p>A, Paraffin-embedded tumor sections were immunohistochemically stained using an anti-FAK antibody. The FAK staining results were scored into four categories based on the signal intensity: 0, no detection; 1, weak; 2, moderate; 3, strong. B, Variation in the FAK copy number was assessed with quantitative PCR. Copy number log ratios higher than 0.32 and 1.00 were considered evidence of copy number gain and gene amplification, respectively. The X-axis represents the samples. C, FAK overexpression (IHC score 3) correlated with the gains in copy number (copy number log ratio >0.32). Weak expressions of FAK (IHC score 1) resulted in normal or reduced FAK copy numbers (copy number log ratio <0.32). D, Samples with a mutation in <i>PIK3CA</i> (<i>PIK3CA</i>_Mut) are marked as red diamonds on the plot. The vertical dotted line indicates a copy number log ratio of 0.32.</p

FAK inhibitors down-regulate MCL1.

<p>A, MCL1 was transiently down-regulated by treatment with PF271 or PF228. RMGI and OVISE cells were incubated in the presence of PF271 or PF228 at 5 µM for RMGI and 10 µM for OVISE. The cells were harvested at the indicated time points and lysed. D indicates the dimethyl sulfoxide (DMSO) vehicle control. Immunoblots were performed for p-FAK, FAK, MCL1, AKT, and p-AKT. B, A proposed schematic model by how the combined pharmacological inhibition of FAK/PYK2 and BCL-XL/BCL-2 induces apoptosis.</p

Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples

<div><p>Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.</p></div

CD24 Overexpression Is Associated with Poor Prognosis in Luminal A and Triple-Negative Breast Cancer

<div><p>CD24 is associated with unfavourable prognoses in various cancers, but the prevalence of CD24 expression and its influence on clinical outcome in subtypes of breast cancers remain unclear. CD24 expression was analyzed by immunohistochemistry in 747 breast cancer tissues, and DNA methylation and histone modification status in the promoter region of CD24 were assessed using bisulfite sequencing and chromatin immunoprecipitation assay. 213 (28.5%) samples exhibited high CD24 expression in the membrane and/or cytoplasm of breast cancer cells, and CD24 overexpression was significantly correlated with the presence of lymph node metastasis and more advanced pathological stage. Patients with CD24-high tumours had significantly shorter patient survival than those with CD24-low tumours. Importantly, multivariate analysis that included tumour size, lymph node metastasis and chemotherapy demonstrated that high CD24 expression is independently associated with poorer survival in luminal A and triple-negative breast cancer (TNBC) subtypes. Furthermore, CD24 gene expression was associated with histone acetylation independent of DNA methylation, suggesting its epigenetic regulation in breast cancer. Our results suggest that CD24 overexpression is an independent unfavourable prognostic factor in breast cancer, especially for luminal A and TNBC subtypes, and CD24 may be a promising therapeutic target for specific subtypes of breast cancer.</p></div

Frequency of base transition in formalin-fixed paraffin-embedded (FFPE) samples.

<p><b>A</b>: A strategy for estimation of rates of sequencing errors/background DNA damage and overall base alteration rates. The discrepant bases at homozygous sites in control samples are likely to be sequencing errors or background DNA mutation. Conversely, discrepant bases at homozygous sites in frozen or FFPE tissue samples could be either sequencing errors/background DNA damage or base alteration caused by preservation methods. <b>B</b>: The rate of base alterations caused by formalin fixation. High frequencies of C>T and G>A were observed in FFPE tissue samples only.</p

Distribution of nucleotide quality according to mapping status.

<p>The nucleotide quality scores were analyzed according to mapping status. In the mapped reads, there was no significant difference between the distributions of the 2 types of sample. In the unmapped reads, the blood and frozen samples showed a higher percentage of low-quality bases (≤ 20 on the Phred scale, black arrow) compared to the FFPE samples.</p

Comparison of somatic single nucleotide variant (SNV) calls.

<p>The overall concordance of somatic SNV calls between FFPE-frozen paired samples. A. The overlapping fraction of somatic mutation calls. B. The overlapping fraction of somatic mutation calls in FFPE samples when the matched frozen samples have at least one supporting read at the mutation.</p