Purpose: We developed a method to monitor copy number variations (CNV) in plasma cell-free DNA (cfDNA) from patients with metastatic squamous non-small cell lung cancer (NSCLC). We aimed to explore the association between tumor-derived cfDNA and clinical outcomes, and sought CNVs that may suggest potential resistance mechanisms. Experimental Design: Sensitivity and specificity of low-pass whole-genome sequencing (LP-WGS) were first determined using cell line DNA and cfDNA. LP-WGS was performed on baseline and longitudinal cfDNA of 152 patients with squamous NSCLC treated with chemotherapy, or in combination with pictilisib, a pan-PI3K inhibitor. cfDNA tumor fraction and detected CNVs were analyzed in association with clinical outcomes. Results: LP-WGS successfully detected CNVs in cfDNA with tumor fraction !10%, which represented approximately 30% of the first-line NSCLC patients in this study. The most frequent CNVs were gains in chromosome 3q, which harbors the PIK3CA and SOX2 oncogenes. The CNV landscape in cfDNA with a high tumor fraction generally matched that of corresponding tumor tissue. Tumor fraction in cfDNA was dynamic during treatment, and increases in tumor fraction and corresponding CNVs could be detected before radiographic progression in 7 of 12 patients. Recurrent CNVs, such as MYC amplification, were enriched in cfDNA from posttreatment samples compared with the baseline, suggesting a potential resistance mechanism to pictilisib. Conclusions: LP-WGS offers an unbiased and highthroughput way to investigate CNVs and tumor fraction in cfDNA of patients with cancer. It may also be valuable for monitoring treatment response, detecting disease progression early, and identifying emergent clones associated with therapeutic resistance.
The p53-related transcription factor p63 is required for maintenance of epithelial cell differentiation. We found that activated forms of the Harvey Rat Sarcoma Virus GTPase (H-RAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) oncogenes strongly repress expression of ΔNp63α, the predominant p63 isoform in basal mammary epithelial cells. This regulation occurs at the transcriptional level, and a short region of the ΔNp63 promoter is sufficient for repression induced by H-RasV12. The suppression of ΔNp63α expression by these oncogenes concomitantly leads to an epithelial-tomesenchymal transition (EMT). In addition, the depletion of ΔNp63α alone is sufficient to induce EMT. Both H-RasV12 expression and ΔNp63α depletion induce individual cell invasion in a 3D collagen gel in vitro system, thereby demonstrating how Ras can drive the mammary epithelial cell state toward greater invasive ability. Together, these results suggest a pathway by which RAS and PIK3CA oncogenes induce EMT through regulation of ΔNp63α.p63 | H-Ras | epithelial mesenchymal transition | transcriptional repression | breast cancer H -Ras, a member of the Ras family of GTPases, was originally identified as the transforming protein encoded by the Harvey rat sarcoma retrovirus (1). Activating mutations in H-Ras and its family members, K-Ras and N-Ras, were identified in a variety of cancers, and nearly 30% of all cancers have mutations in one of the Ras genes (2, 3). The EGFR2 receptor, also known as HER2/ neu, is an important upstream activator of Ras and is amplified in about 20-30% of breast cancers (4, 5). Although many HER2 + cancers are initially responsive to treatment with the monoclonal antibody Herceptin (trastuzumab), resistance usually develops (6, 7). Therefore, it is of great importance to understand signaling pathways that are downstream of HER2 and Ras, to identify key factors responsible for their tumor-promoting effects. It may also be possible to target such factors, in combination with HER2 or Ras inhibitors, to achieve greater clinical efficacy.A major effector downstream of Ras is the phosphatidylinositol 3-kinase (PI3K) pathway. The catalytic subunit of PI3K, PIK3CA, is frequently found mutated in cancers, especially those cancers originating in the breast (8, 9). Like mutant Ras, expression of mutant PIK3CA can cause nontumorigenic cells to undergo transformation and gain invasive abilities (10, 11). Carcinoma cells, in particular, may gain increased invasive abilities by undergoing an epithelial-to-mesenchymal transition (EMT), where adherens junctions formed by E-cadherin are disrupted (12, 13). Canonical transcription factors that repress E-cadherin include Twist, Snail, Slug, and Zeb1, although this network has grown more complex in recent years (14,15).The transcription factor p63 not only induces transcription of canonical p53 targets but is also a master regulator of epithelial cells (16,17). Mice losing both alleles of p63 display complications due to the loss of epithelial stratif...
The p53-related gene p63 is required for epithelial cell establishment and its expression is often altered in tumor cells. Great strides have been made in understanding the pathways and mechanisms that regulate p63 levels, such as the Wnt, Hedgehog, Notch, and EGFR pathways. We discuss here the multiple signaling pathways that control p63 expression as well as transcription factors and post-transcriptional mechanisms that regulate p63 levels. While a unified picture has not emerged, it is clear that the fine-tuning of p63 has evolved to carefully control epithelial cell differentiation and fate.
Functional in a tetrameric state, the protein product of the p53 tumor suppressor gene confers its tumorsuppressive activity by transactivating genes which promote cell-cycle arrest, senescence, or programmed cell death. How p53 distinguishes between these divergent outcomes is still a matter of considerable interest. Here we discuss the impact of 2 mutations in the tetramerization domain that confer unique properties onto p53. By changing lysines 351 and 357 to arginine, thereby blocking all post-translational modifications of these residues, DNA binding and transcriptional regulation by p53 remain virtually unchanged. On the other hand, by changing these lysines to glutamine (2KQ-p53), thereby neutralizing their positive charge and potentially mimicking acetylation, p53 is impaired in the induction of cell cycle arrest and yet can still effectively induce cell death. Surprisingly, when 2KQ-p53 is expressed at high levels in H1299 cells, it can bind to and transactivate numerous p53 target genes including p21, but not others such as miR-34a and cyclin G1 to the same extent as wild-type p53. Our findings show that strong induction of p21 is not sufficient to block H1299 cells in G1, and imply that modification of one or both of the lysines within the tetramerization domain may serve as a mechanism to shunt p53 from inducing cell cycle arrest.
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