Response Rate Is Associated with Prolonged Survival in Patients with Advanced Non-small Cell Lung Cancer Treated with Gefitinib or Erlotinib

Introduction:Gaining a higher response rate (RR) has usually been determined as a primary end point in phase II trials evaluating the efficacy of new molecular targeted drugs. However, a relationship between clinical response and survival benefit has not been well studied in the patients treated with molecular targeted agents.Methods:Prospective trials of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) monotherapy in non-small cell lung cancer were extracted from MEDLINE, EMBASE, and the annual meetings in 2007 of the American Society of Clinical Oncology, European Cancer Conference, and World Conference on Lung Cancer.Correlation between clinical response and survival was examined using linear regression analysis. We also tried to compare the significance of RR as surrogate markers for survival with that of disease control rate (DCR) by calculating the area under their receiver operating characteristic (ROC) curves.Results:We identified 24 phase II trials and 4 phase III trials with a total of 6171 patients and 30 treatment arms, including 22 arms for the gefitinib group and 8 arms for the erlotinib group. Both RR and DCR strongly correlated with median survival time (MST; p < 0.0001 and p = 0.003, respectively). In an ROC analysis, the area under the ROC curve predicting MST prolongation by RR was 0.918, which was higher than the area under the ROC curve by DCR.Conclusions:We found a significant relationship between RR and MST in clinical trials with EGFR-TKIs. RR could be an independent surrogate marker for MST in the current response criteria in the clinical trials of EGFR-TKIs

ΔNp63 silencing, DNA methylation shifts, and epithelial-mesenchymal transition resulted from TAp63 genome editing in squamous cell carcinoma

TP63 (p63) is strongly expressed in lower-grade carcinomas of the head and neck, skin, breast, and urothelium to maintain a well-differentiated phenotype. TP63 has two transcription start sites at exons 1 and 3′ that produce TAp63 and ΔNp63 isoforms, respectively. The major protein, ΔNp63α, epigenetically activates genes essential for epidermal/craniofacial differentiation, including ΔNp63 itself. To examine the specific role of weakly expressed TAp63, we disrupted exon 1 using CRISPR-Cas9 homology-directed repair in a head and neck squamous cell carcinoma (SCC) line. Surprisingly, TAp63 knockout cells having either monoallelic GFP cassette insertion paired with a frameshift deletion allele or biallelic GFP cassette insertion exhibited ΔNp63 silencing. Loss of keratinocyte-specific gene expression, switching of intermediate filament genes from KRT(s) to VIM, and suppression of cell-cell and cell-matrix adhesion components indicated the core events of epithelial-mesenchymal transition. Many of the positively and negatively affected genes, including ΔNp63, displayed local DNA methylation changes. Furthermore, ΔNp63 expression was partially rescued by transfection of the TAp63 knockout cells with TAp63α and application of DNA methyltransferase inhibitor zebularine. These results suggest that TAp63, a minor part of the TP63 gene, may be involved in the auto-activation mechanism of ΔNp63 by which the keratinocyte-specific epigenome is maintained in SCC

C-terminal α Domain of p63 Binds to p300 to Coactivate β-Catenin

TP63 (p63), a member of the tumor suppressor TP53 (p53) gene family, is essential for ectodermal tissue development and suppresses malignant progression of carcinomas. The most abundant isoform, ΔNp63α (referred to as p63), lacks the N-terminal transactivation (TA) domain, and was originally characterized as a dominant-negative type suppressor against p53 family proteins. It also binds to TCF/LEF to inhibit β-catenin. Nevertheless, transcriptional activation by p63 has also been observed in varied systems. To understand the puzzling results, we analyzed the structure–function relationship of p63 in the control of β-catenin-dependent transcription. p63 acted as a suppressor of moderately induced β-catenin. However, when nuclear targeted S33Y β-catenin was applied to cause the maximum enhancer activation, p63 displayed a β-catenin-coactivating function. The DNA-binding domain of p63 and the target sequence facilitated it. Importantly, we newly found that, despite the absence of TA domain, p63 was associated with p300, a general adaptor protein and chromatin modifier causing transcriptional activation. C-terminal α domain of p63 was essential for p300-binding and for the coactivator function. These results were related to endogenous p63-p300 complex formation and Wnt/β-catenin-responsive gene regulation by p63 in squamous cell carcinoma lines. The novel p63-p300 interaction may be involved in positive regulation of gene expression in tissue development and carcinogenesis

Induction of ΔNp63 by the Newly Identified Keratinocyte-Specific Transforming Growth Factor β Signaling Pathway with Smad2 and IκB Kinase α in Squamous Cell Carcinoma12

The expression of p63 (TP63/p51) occurs in the basal cells of stratified epithelia and is strongly enhanced at the early stages of squamous cell carcinomas (SCCs) of the head and neck, skin, cervix, and others. We analyzed a promoter/enhancer region (2kΔN) that drives the predominant expression of ΔNp63 for sensitivity to Smad signaling pathways. Reporter assays in HepG2 cells showed a moderate activation of 2kΔN by Smad2 and IκB kinase α (IKKα), partners of the newly identified keratinocyte-specific transforming growth factor β (TGF-β) signaling, but not by other Smad molecules. In A431 cells, 2kΔN was activated by Smad2 and IKKα, for which a Smad binding element (SMD2) at -204 was essential. Binding of Smad2 to the chromosomal SMD2 site was detectable. The association of Smad2 with IKKα was evident in the nucleus of A431, accounting for the enhancement of ΔNp63 expression by TGF-β. Moreover, both ΔNp63 and IKKα were necessary to maintain the noninvasive phenotype of this cell line. FaDu, an invasive, Smad4-deficient SCC, also allowed 2kΔN transactivation by transfected Smad2 in the presence of endogenous IKKα. Reflecting the lack of chromosomal SMD2-Smad2 association and the absence of nuclear IKKα, however, endogenous ΔNp63 was not controlled by TGF-β or IKKα in FaDu. SCC tissue arrays showed nuclear accumulation of IKKα and p63 intensification in well-differentiated noninvasive lesions. This study indicates that p63 is a target gene of the proposed keratinocyte-specific TGF-β signal pathway for suppression of the malignant conversion of SCC

Repression of Wnt/β-catenin response elements by p63 (TP63)

<p>Submitted: TP63 (p63), a member of the tumor suppressor TP53 (p53) gene family, is expressed in keratinocyte stem cells and well-differentiated squamous cell carcinomas to maintain cellular potential for growth and differentiation. Controversially, activation of the Wnt/β-catenin signaling by p63 (Patturajan M. et al., 2002, Cancer Cells) and inhibition of the target gene expression (Drewelus I. et al., 2010, Cell Cycle) have been reported. Upon p63 RNA-silencing in squamous cell carcinoma (SCC) lines, a few Wnt target gene expression substantially increased, while several target genes moderately decreased. Although ΔNp63α, the most abundant isoform of p63, appeared to interact with protein phosphatase PP2A, neither GSK-3β phosphorylation nor β-catenin nuclear localization was altered by the loss of p63. As reported earlier, ΔNp63α enhanced β-catenin-dependent <i>luc</i> gene expression from pGL3-OT having 3 artificial Wnt response elements (WREs). However, this activation was detectable only in HEK293 cells examined so far, and involved a p53 family-related sequence 5′ to the WREs. In Wnt3-expressing SAOS-2 cells, ΔNp63α rather strongly inhibited transcription of pGL3-OT. Importantly, ΔNp63α repressed WREs isolated from the regulatory regions of <i>MMP7</i>. ΔNp63α-TCF4 association occurred in their soluble forms in the nucleus. Furthermore, p63 and TCF4 coexisted at a WRE of <i>MMP7</i> on the chromatin, where β-catenin recruitment was attenuated. The combined results indicate that ΔNp63α serves as a repressor that regulates β-catenin-mediated gene expression.</p

Activation of the Long Terminal Repeat of Human Endogenous Retrovirus K by Melanoma-Specific Transcription Factor MITF-M12

The human and Old World primate genomes possess conserved endogenous retrovirus sequences that have been implicated in evolution, reproduction, and carcinogenesis. Human endogenous retrovirus (HERV)-K with 5′LTR-gag-pro-pol-env-rec/np9-3′LTR sequences represents the newest retrovirus family that integrated into the human genome 1 to 5 million years ago. Although a high-level expression of HERV-K in melanomas, breast cancers, and teratocarcinomas has been demonstrated, the mechanism of the lineage-specific activation of the long terminal repeat (LTR) remains obscure. We studied chromosomal HERV-K expression in MeWo melanoma cells in comparison with the basal expression in human embryonic kidney 293 (HEK293) cells. Cloned LTR of HERV-K (HML-2.HOM) was also characterized by mutation and transactivation experiments. We detected multiple transcriptional initiator (Inr) sites in the LTR by rapid amplification of complementary DNA ends (5′ RACE). HEK293 and MeWo showed different Inr usage. The most potent Inr was associated with a TATA box and three binding motifs of microphthalmia-associated transcription factor (MITF). Both chromosomal HERV-K expression and the cloned LTR function were strongly activated in HEK293 by transfection with MITF-M, a melanocyte/melanoma-specific isoform of MITF. Coexpression of MITF and the HERV-K core antigen was detected in retinal pigmented epithelium by an immunofluorescence analysis. Although malignant melanoma lines MeWo, G361, and SK-MEL-28 showed enhanced HERV-K transcription compared with normal melanocytes, the level of MITF-M messenger RNA persisted from normal to transformed melanocytes. Thus, MITF-M may be a prerequisite for the pigmented cell lineage-specific function of HERV-K LTR, leading to the high-level expression in malignant melanomas