Joshua A. Desotelle

Therapy-induced senescence in cancer.

Cellular senescence is a response to nonlethal stress that results in persistent cytostasis with a distinct morphological and biochemical phenotype. The senescence phenotype, detected in tumors through the expression of mRNA and protein markers, can be generated in cancer cells lacking functional p53 and retinoblastoma protein. Current research suggests that therapy-induced senescence (TIS) represents a novel functional target that may improve cancer therapy. TIS can be induced in immortal and transformed cancer cells by selected anticancer compounds or radiation, and accumulating data indicate that TIS may produce reduced toxicity-related side effects and increased tumor-specific immune activity. This review examines the current status of TIS-regulated mechanisms, agents, and senescence biomarkers with the goal of encouraging further development of this approach to cancer therapy. Remaining hurdles include the lack of efficient senescence-inducing agents and incomplete biological data on tumor response. The identification of additional compounds and other targeted approaches to senescence induction will further the development of TIS in the clinical treatment of cancer.

A methyl-deficient diet modifies histone methylation and alters Igf2 and H19 repression in the prostate.

Folate and methyl‐group deficiency has been linked to prostate cancer susceptibility, yet the mechanisms underlying these observations are incompletely understood. The region of the genome containing the imprinted genes insulin‐like growth factor 2 (Igf2) and H19, both of which display oncogenic functions, may be particularly sensitive to environmental influences.

Aging and Cancer-Related Loss of Insulin-like Growth Factor 2 Imprinting in the Mouse and Human Prostate

Loss of imprinting (LOI) is an epigenetic alteration involving loss of parental origin-specific expression at normally imprinted genes. A LOI for Igf2, a paracrine growth factor, is important in cancer progression. Epigenetic modifications may be altered by environmental factors. However, is not known whether changes in imprinting occur with aging in prostate and other tissues susceptible to cancer development. We found a LOI for Igf2 occurs specifically in the mouse prostate associated with increased Igf2 expression during aging. In older animals, expression of the chromatin insulator protein CTCF and its binding to the Igf2-H19 imprint control region was reduced. Forced down-regulation of CTCF leads to Igf2 LOI. We further show that Igf2 LOI occurs with aging in histologically normal human prostate tissues and that this epigenetic alteration was more extensive in men with associated cancer. This finding may contribute to a postulated field of cancer susceptibility that occurs with aging. Moreover, Igf2 LOI may serve as a marker for the presence of prostate cancer.

Drug-induced senescence bystander proliferation in prostate cancer cells in vitro and in vivo.

Senescence is a distinct cellular response induced by DNA-damaging agents and other sublethal stressors and may provide novel benefits in cancer therapy. However, in an ageing model, senescent fibroblasts were found to stimulate the proliferation of cocultured cells. To address whether senescence induction in cancer cells using chemotherapy induces similar effects, we used GFP-labelled prostate cancer cell lines and monitored their proliferation in the presence of proliferating or doxorubicin-induced senescent cancer cells in vitro and in vivo. Here, we show that the presence of senescent cancer cells increased the proliferation of cocultured cells in vitro through paracrine signalling factors, but this proliferative effect was significantly less than that seen with senescent fibroblasts. In vivo, senescent cancer cells failed to increase the establishment, growth or proliferation of LNCaP and DU145 xenografts in nude mice. Senescent cells persisted as long as 5 weeks in tumours. Our results demonstrate that although drug-induced senescent cancer cells stimulate the proliferation of bystander cells in vitro, this does not significantly alter the growth of tumours in vivo. Coupled with clinical observations, these data suggest that the proliferative bystander effects of senescent cancer cells are negligible and support the further development of senescence induction as therapy.

The Circadian Control of Skin and Cutaneous Photodamage

Biologically, light including ultraviolet (UV) radiation is vital for life. However, UV exposure does not come without risk, as it is a major factor in the development of skin cancer. Natural protections against UV damage may have been affected by lifestyle changes over the past century, including changes in our sun exposure due to working environments, and the use of sunscreens. In addition, extended “day time” through the use of artificial light may contribute to the disruption of our circadian rhythms; the daily cycles of changes in critical bio‐factors including gene expression. Circadian disruption has been implicated in many health conditions, including cardiovascular, metabolic and psychiatric diseases, as well as many cancers. Interestingly, the pineal hormone melatonin plays a role in both circadian regulation as well as protection from UV skin damage, and is therefore an important factor to consider when studying the impact of UV light. This review discusses the beneficial and deleterious effects of solar exposure, including UV skin damage, Vitamin D production, circadian rhythm disruption and the impact of melatonin. Understanding these benefits and risks is critical for the development of protective strategies against solar radiation.

A High-Throughput Method to Identify Novel Senescence-Inducing Compounds

Cellular senescence is a persistently growth-arrested phenotype in normal and transformed cells induced by noncytotoxic stress. Cytostasis as a method of cancer treatment has recently generated significant interest. Research into the induction of cellular senescence as cancer therapy has been hindered by a lack of compounds that efficiently induce this response. The authors describe a semiautomated high-throughput method to identify library compounds that induce senescence using prostate cancer cells cultured in 96-well plates. Primary hits are identified by low cell numbers after 3 days in culture, measured by Hoechst 33342 fluorescence. A secondary visual assessment of senescence-associated β-galactosidase staining and cellular morphology in the same wells distinguishes senescence from quiescence, apoptosis, and other false positives. This method was used to screen a 4160-compound library of known bioactive compounds and natural products at a 10-µM dose. Candidate compounds were further selected based on persistent growth arrest after drug removal and increased expression of previously described senescence marker genes. Four lead compounds not previously associated with senescence were identified for further investigation. This is the first successful assay to identify novel agents from compound libraries based on senescence induction in cancer cells. (Journal of Biomolecular Screening 2009:853-858)

Androgen deprivation induces senescence characteristics in prostate cancer cells in vitro and in vivo

The treatment of non‐localized prostate cancer involves androgen deprivation (AD) therapy which results in tumor regression. Apoptosis has been implicated in the tumor response to AD, but constitutes a small fraction of the total tumor at any time. Cellular senescence is a response to sub‐lethal stress in which cells are persistently growth arrested and develop distinct morphological and biochemical characteristics. The occurrence of senescence in prostate tumor tissue after AD therapy has not previously been investigated.

Analysis of Promoter Non-CG Methylation in Prostate Cancer

BACKGROUND In vertebrates, DNA methylation occurs primarily at CG dinucleotides but recently, non-CG methylation has been found at appreciable levels in embryonic stem cells. MATERIALS & METHODS To assess non-CG methylation in cancer, we compared the extent of non-CG methylation at several biologically important CG islands in prostate cancer and normal cell lines. An assessment of the promoter CG islands EVX1 and FILIP1L demonstrates a fourfold higher rate of non-CG methylation at EVX1 compared with FILIP1L across all cell lines. These loci are densely methylated at CG sites in cancer. RESULTS No significant difference in non-CG methylation was demonstrated between cancer and normal. Treatment of cancer cell lines with 5-azacytidine significantly reduced methylation within EVX1 at CG and CC sites, preferentially. CONCLUSION Non-CG methylation does not correlate with CG methylation at hypermethylated promoter regions in cancer. Furthermore, global inhibition of DNA methyltransferases does not affect all methylated cytosines uniformly.

Abstract 1920: Small molecule inhibitor of polo-like kinase 1 (Plk1) causes significant melanoma growth delay and regression in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Melanoma is an aggressive skin cancer that rapidly metastasizes to become lethal, if not diagnosed early. An analysis of the Surveillance, Epidemiology, and End Results (SEER) program has suggested that the age-adjusted annual incidence of melanoma, both for young men and women, has increased over the past 20 years. In 2011, it is projected that over 70,000 new cases of melanoma will be diagnosed with nearly 9,000 cases resulting in death. Current preventive and treatment approaches have not been able to effectively manage this neoplasm. Therefore, new mechanism- and target- based approaches are urgently needed. Better understanding of the genetic control of cellular proliferation and cell division may provide the basis for the rational design of specific therapeutics for the management of cancers, including melanoma. We have previously demonstrated that the mitotic regulator Polo-like kinase 1 (Plk1) is over-expressed in clinical melanoma and a targeted depletion of Plk1 through lentiviral shRNA or a small-molecule inhibitor causes mitotic catastrophe and induction of apoptosis in human melanoma cells (J Invest Dermatol 129: 2843-53, 2009). In this study, we assessed the effect of BI6727, a second generation Plk1 specific small molecule inhibitor, in vivo in athymic nu/nu nude mice implanted with human melanoma cells. A375 cells (1x106 cells) were implanted in athymic nude by subcutaneous injection on the right flanks. The tumors were allowed to grow and the mice bearing established tumors (>100 mm3) were randomized into 3 groups (containing 12 mice each). The mice were treated with BI6727 at two doses (10 mg/kg or 25 mg/kg) or a vehicle control via tail vein injections twice weekly. Our data demonstrates that in BI6727 treated animals, the rate of tumor growth (to reach to a 1000 mm3 target volume) was significantly delayed. We also found that BI6727 at 25 mg/kg resulted in a near complete tumor regression. The observed tumor regression was stable for several days following discontinuation of treatment, after which, the tumors started growing again. Additionally, we found that BI6727 caused i) a significant inhibition of cell growth and viability, and ii) induction of apoptosis in multiple melanoma cell lines. Based on this study, we suggest that Plk1 is a druggable target for melanoma and pharmacological inhibitors of Plk1 could be clinically useful in melanoma therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1920. doi:1538-7445.AM2012-1920

Abstract 3159: Identification of circulating tumor cells from renal cell carcinoma patients by a multi-parameter flow cytometry assay

BACKGROUND: Renal cell carcinoma (RCC) is one of the top ten causes of cancer death in the United States. The last ten years have shown a dramatic increase in the number of available treatment options, however metastatic RCC remains largely incurable. The classes of drugs that have been developed can be divided into agents that target the Vascular Endothelial Growth Factor (VEGF) pathway (Sunitinib, Sorafenib, Pazopanib, Axitinib, Bevacizumab), those that target the mammalian Target of Rapamycin (mTOR) pathway (Everolimus, Temsirolimus) and immune based therapies (IL-2 and PD-1 inhibition). There are currently limited biomarkers to guide clinical decisions, mainly due to the lack of tumor cells for longitudinal molecular analysis. Circulating tumor cells (CTCs) are potential a source of tumor cells that can be identified from a blood draw for serial analysis. CTCs have not been reliably detected in RCC due to the significant heterogeneity and high rate of false positive events in this disease when EpCAM has been used. We sought to identify RCC CTCs with alternative markers, such as carbonic anhydrase IX (CAIX) which is found in greater than 90% of clear cell RCC tumors. METHODS AND RESULTS: To evaluate for the presence and subtypes of CTCs from patients with RCC, we utilized a multi-parametric flow cytometry assay. We evaluated heterogeneity across subpopulations of putative CTCs with Epithelial Cell Adhesion Molecule (EpCAM), Carbonic Anhydrase IX (CAIX), Carbonic Anhydrase XII (CAXII), PAX8, and Cytokeratin (CK). Negative controls for immune and endothelial events were performed with markers for CD45, CD14, CD34, CD11b and CD61. We tested twenty blood samples from patients with RCC at the Dana Farber Cancer Institute and University of Wisconsin Carbone Cancer Center. CTC frequency in RCC ranges from 0-5410 CAIX+/CK+ events with a median of 24.5 putative CTCs/7.5mL of blood. A subset of patients with radiographic progression had a higher number of CTCs with a median of 295.2 CTCs/7.5 mL. A range of 1-231 EpCAM+/CK+ events were identified with a median of 5.5 CTCs/7.5mL. There was low frequency of events being CAIX+/EpCAM+/CK+, with a median of 1 CTC/7.5mL of blood. Assay specificity was dramatically improved through the combination of multiple positive markers with stains for immune and endothelial cells given frequent non-specific staining for cytokeratin in RCC blood samples. CONCLUSIONS: CTCs can be identified in patients with RCC using non-traditional markers. CAIX is a more sensitive marker than EpCAM to identify putative CTCs from patients with RCC. Specificity in the assay is critical given the high frequency of false positive events identified if only CD45 is used as a marker for immune cells. Our investigation is ongoing for further molecular characterization of orthogonal endpoints in identified CTCs. Future directions include longitudinal monitoring of CTCs during treatment with VEGF inhibitors. Citation Format: Joshua A. Desotelle, Chorom Pak, Erika Heninger, Jennifer L. Schehr, Rana R. McKay, Benjamin K. Gibbs, Craig Norton, Toni K. Choueiri, Joshua M. Lang. Identification of circulating tumor cells from renal cell carcinoma patients by a multi-parameter flow cytometry assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3159.