Timothy F. Burns

Silencing of the Novel p53 Target Gene Snk/Plk2 Leads to Mitotic Catastrophe in Paclitaxel (Taxol)-Exposed Cells

ABSTRACT Loss of p53 sensitizes to antimicrotubule agents in human tumor cells, but little is known about its role during mitosis. We have identified the Polo-like kinase family member serum inducible kinase (Snk/Plk2) as a novel p53 target gene. Snk/Plk2 mutagenesis demonstrated that its kinase activity is negatively regulated by its C terminus. Small interfering RNA (siRNA)-mediated Snk/Plk2 silencing in the presence of the mitotic poisons paclitaxel (Taxol) or nocodazole significantly increased apoptosis, similar to p53 mutations, which confer paclitaxel sensitivity. Furthermore, we have demonstrated that the apoptosis due to silencing of Snk/Plk2 in the face of spindle damage occurs in mitotic cells and not in cells that have progressed to a G1-like state without dividing. Since siRNA directed against Snk/Plk2 promoted death of paclitaxel-treated cells in mitosis, we envision a mitotic checkpoint wherein p53-dependent activation of Snk/Plk2 prevents mitotic catastrophe following spindle damage. Finally, these studies suggest that disruption of Snk/Plk2 may be of therapeutic value in sensitizing paclitaxel-resistant tumors.

Detection of Cancer DNA in Plasma of Patients with Early-Stage Breast Cancer

Purpose: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection. Experimental Design: In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR. Results: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery. Conclusions: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients. Clin Cancer Res; 20(10); 2643–50. ©2014 AACR.

Tissue specific expression of p53 target genes suggests a key role for KILLER/DR5 in p53-dependent apoptosis in vivo.

The p53 tumor suppressor plays a key role in the cells response to genotoxic stress and loss of this ‘guardian of the genome’ is an important step in carcinogenesis. The ability of p53 to induce apoptosis through transactivation of its target genes is critical for its function as tumor suppressor. We have found that overexpression of p53 in human cancer cell lines resulted in apoptosis as measured by PARP cleavage. Furthermore we observed cleavage of both caspase 9 and caspase 8 after overexpression of p53 and found that p53-dependent apoptosis was inhibited by either cellular (c-Flip-s, Bcl-XL) or pharmacological inhibitors of caspase 8 or caspase 9 respectively. These results indicate that p53 is mediating apoptosis through both the mitochondrial and death receptor pathways. To elucidate the relevant p53 target genes and examine the caspase pathways utilized in vivo, we treated p53+/+ and age matched p53−/− mice with 5 Gy ionizing radiation or 0.5 mg/animal dexamethasone and harvested tissues at 0, 6 and 24 h. We examined the mRNA expression of p21, bax, KILLER/DR5, FAS/APO1 and EI24/PIG8 using TaqMan real time quantitative RT–PCR in the spleen, thymus and small intestine. Although the basal mRNA levels of these genes did not depend on the presence of p53, we observed a p53-dependent induction of all these targets in response to γ-irradiation and a p53-independent regulation for p21 and KILLER/DR5 in response to dexamethasone. Furthermore, we have demonstrated that the relative induction of these p53 target genes is tissue specific. Despite observing otherwise similar levels of death in these tissues, our findings suggest that in some cases apoptosis mediated through p53 occurs by redundant pathways or by a ‘group effect’ while in other tissues one or few targets may play a key role in p53-dependent apoptosis. Surprisingly, KILLER/DR5 is the dominantly induced transcript in both the spleen and small intestine suggesting a potentially important role for this p53 target gene in vivo.

Twist1 Suppresses Senescence Programs and Thereby Accelerates and Maintains Mutant Kras-Induced Lung Tumorigenesis

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with KrasG12D to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

The prognostic and predictive value of KRAS oncogene substitutions in lung adenocarcinoma

The prognostic and therapeutic implications of the spectrum of v‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene substitutions in lung cancer remain poorly understood. The objective of this study was to determine whether KRAS oncogene substitutions differed with regard to prognosis or predictive value in lung adenocarcinoma.

The Twist box domain is required for Twist1-induced prostate cancer metastasis

Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial–mesenchymal transition (EMT) that promotes cancer metastasis. Structure–function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer. Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in prostate cancer cells using in vitro assays, which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extrathoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for prostate cancer cells to colonize metastatic lung lesions and extrathoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in prostate cancer cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and prostate cancer metastasis. Implications: Targeting the Twist box domain of Twist1 may effectively limit prostate cancer metastatic potential. Mol Cancer Res; 11(11); 1387–400. ©2013 AACR.

Inhibition of TWIST1 Leads to Activation of Oncogene-Induced Senescence in Oncogene Driven Non-Small Cell Lung Cancer

A large fraction of non–small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. Although targeted agents exist for EGFR- and EML4-ALK–driven NSCLCs, no therapies target the most frequently found driver mutation, KRAS. Furthermore, acquired resistance to the currently targetable driver mutations is nearly universally observed. Clearly a novel therapeutic approach is needed to target oncogene-driven NSCLCs. We recently showed that the basic helix-loop-helix transcription factor Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in transgenic mouse models and that inhibition of Twist1 in these models led to Kras-induced senescence. In the current study, we examine the role of TWIST1 in oncogene-driven human NSCLCs. Silencing of TWIST1 in KRAS-mutant human NSCLC cell lines resulted in dramatic growth inhibition and either activation of a latent oncogene-induced senescence program or, in some cases, apoptosis. Similar effects were observed in EGFR mutation–driven and c-Met–amplified NSCLC cell lines. Growth inhibition by silencing of TWIST1 was independent of p53 or p16 mutational status and did not require previously defined mediators of senescence, p21 and p27, nor could this phenotype be rescued by overexpression of SKP2. In xenograft models, silencing of TWIST1 resulted in significant growth inhibition of KRAS-mutant, EGFR-mutant, and c-Met–amplified NSCLCs. Remarkably, inducible silencing of TWIST1 resulted in significant growth inhibition of established KRAS-mutant tumors. Together these findings suggest that silencing of TWIST1 in oncogene driver–dependent NSCLCs represents a novel and promising therapeutic strategy. Mol Cancer Res; 11(4); 329–38. ©2013 AACR.

Targeting Heat Shock Proteins in Cancer: A Promising Therapeutic Approach

Heat shock proteins (HSPs) are a large family of chaperones that are involved in protein folding and maturation of a variety of “client” proteins protecting them from degradation, oxidative stress, hypoxia, and thermal stress. Hence, they are significant regulators of cellular proliferation, differentiation and strongly implicated in the molecular orchestration of cancer development and progression as many of their clients are well established oncoproteins in multiple tumor types. Interestingly, tumor cells are more HSP chaperonage-dependent than normal cells for proliferation and survival because the oncoproteins in cancer cells are often misfolded and require augmented chaperonage activity for correction. This led to the development of several inhibitors of HSP90 and other HSPs that have shown promise both preclinically and clinically in the treatment of cancer. In this article, we comprehensively review the roles of some of the important HSPs in cancer, and how targeting them could be efficacious, especially when traditional cancer therapies fail.

Insulin-Like Growth Factor Factor Binding Protein-2 is a Novel Mediator of p53 Inhibition of Insulin-Like Growth Factor Signaling

The p53 tumor suppressor induces cellular growth arrest and apoptosis in response to DNA damage by transcriptionally activating or repressing target genes and also through protein-protein interactions and direct mitochondrial activities. In 1995, insulin-like growth factor binding protein (IGFBP)-3 was identified as one of the genes transcriptionally activated by p53. IGFBP-3 is one of six closely related IGFBP’s, with additional IGFBP-related proteins belonging to the IGFBP superfamily. Here we show that IGFBP-2 is also a p53 target. Like IGFBP-3, IGFBP-2 secretion is reduced when p53+/+ lung cancer cells are transfected with human papillomavirus E6, which targets p53 for degradation. IGFBP-2 mRNA is induced by irradiation in vivo in a p53-dependent manner. p53 protein binds IGFBP-2 intronic sequences in an electrophoretic mobility shift assay, and activates transcription in a luciferase assay. Loss of IGFBP-2 inhibits the ability of p53 to inhibit the activation of extracellular signal-regulated kinase (ERK)1 by IGF-I. Thus, p53 effects on the IGF axis are more complex than previously appreciated, and overall transform the axis from IGF-mediated mitogenesis to growth inhibition and apoptosis. This has significant implications for how growth hormone and IGF-I can induce growth without also inducing cancer.

Lung cancer in 'never-smokers': beyond EGFR mutations and EGFR-TK inhibitors.

The author reports the mean DNA values of interphase nuclei in 25 human tumours of epithelial origin, not previously treated. These values are closely related to the chromosome numbers. The constancy of the observed values for each tumour makes it likely that each tumour represents a clone, derived perhaps from one single cell, in which the genetic changes first occurred. The analysis of tumour cell karyotypes indicates, in agreement with DNA estimations, that the mean chromosome size in tumour cells is larger than normal. The data confirm the view that most of the variations present in tumour cells are not only structural but also represent an increase of the active DNA content per chromosome. The practical implications of these findings are pointed out.