SUMMARY Telomerase, the end-replication enzyme, is reactivated in malignant cancers to drive cellular immortality. While this distinction makes telomerase an attractive target for anti-cancer therapies, most approaches for inhibiting its activity have been clinically ineffective. As opposed to inhibiting telomerase, we use its activity to selectively promote cytotoxicity in cancer cells. We show that several nucleotide analogs, including 5-fluoro-2′-deoxyuridine (5-FdU) triphosphate, are effectively incorporated by telomerase into a telomere DNA product. Administration of 5-FdU results in an increased number of telomere-induced foci, impedes binding of telomere proteins, activates the ATR-related DNA-damage response, and promotes cell death in a telomerase-dependent manner. Collectively, our data indicate that telomerase activity can be exploited as a putative anti-cancer strategy.

SignificanceTelomere length homeostasis is an important mechanism for maintaining genomic stability. Telomere length is regulated by numerous events that include protein–DNA interactions, the length and structure of telomere DNA, and recruitment of telomerase. Here we used hydroxyl radical footprinting to identify environmental changes in the telomere end-binding heterodimer, POT1-TPP1, as a function of telomere length. Our data identified a specific residue (histidine 266) of the POT1 protein that reports differences in solvent accessibility as a function of telomere DNA length. We further show that the chronic lymphocytic leukemia-related H266L POT1 mutation disrupts the ability of POT1-TPP1 to negatively regulate telomerase activity in vitro and in cancer cells.

Since prostaglandins are critically involved in tumorigenesis, COX inhibitors, which reduce prostaglandins, have been investigated for anti‐cancer effects. We and others have shown that the COX‐2 selective inhibitor, celecoxib, induces apoptosis in various cancer cell lines. However, the role of COX inhibition in these anti‐cancer effects remains uncertain. Previously, we reported that celecoxib and another COX‐2 selective inhibitor, etodolac, increased COX‐2 protein levels in several human cancer cell lines (A375 melanoma, CRL‐1620 glioblastoma and HT‐29 colon carcinoma). A non‐selective inhibitor, ibuprofen, failed to increase COX‐2 expression in all but A375 cells. It is well known that COX‐2 is inducible by various stimuli. However, COX‐1 is constitutive and thought to be less dynamic. Here, COX‐1 levels were examined via western blotting to determine whether they were dynamic in response to treatment with various COX inhibitors. Celecoxib treatment for 24 hours induced COX‐1 in A375 cells. In contrast, COX‐1 levels were stable in celecoxib‐treated HT‐29 cells but were decreased in CRL‐1620 cells. COX‐1 levels were unaffected by etodolac treatment in all three cells lines. Ibuprofen treatment for 24 hours induced COX‐1 in A375 and CRL‐1620 cells, but not in HT‐29 cells. Meloxicam, another COX‐2 selective inhibitor, reduced COX‐1 levels but increased COX‐2 levels in CRL‐1620 cells. These results suggest that while COX‐1 is considered a constitutive enzyme, its levels can be modulated by COX inhibitors.

Androgen deprivation therapy persists as first‐line treatment for advanced prostate cancer despite the unescapable progression to castrate‐resistant prostate cancer (CRPCa). Castrate resistance is acquired through numerous mechanisms altering androgenic signaling, further complicating disease progression. Therapeutics have been developed for CRPCa; however, these provide limited benefit, as these agents remain targeted against the altered androgenic signaling environment exhibited by this disease. To address the limited treatment options available, it is essential to interrogate regulatory networks in CRPCa that function independent or downstream of androgenic signaling, ideally identifying fruitful therapeutic targets. Preliminary data suggests SLX4IP, a relatively uncharacterized protein involved in telomere maintenance mechanism (TMM) plasticity, may fulfill this therapeutic prerequisite. TMMs in malignancy are responsible for the elongation of telomeres at chromosomal ends to instill replicative immortality, with the two TMMs available being the telomerase pathway and the alternative lengthening of telomeres (ALT) pathway. It is well established that, if necessary, cancer cells have the ability to utilize the alternate pathway if the primary is disrupted to perpetuate replication. Additionally, the ALT pathway and elevated SLX4IP expression have been shown to correlate with aggressive phenotypes of CRPCa, specifically metastatic and neuroendocrine disease, both exhibiting bleak patient outcomes. To first determine if SLX4IP is capable of mediating TMM plasticity, SLX4IP was overexpressed in a telomerase‐positive cell line followed by TMM characterization. This analysis demonstrated a TMM switch occurred with the cells primarily utilizing the ALT pathway for telomere elongation. Conversely, SLX4IP knockdown in an ALT‐positive cell line initiated a transition to telomerase utilization. After establishing the critical role of SLX4IP in TMM plasticity, SLX4IP was overexpressed in CRPCa cell lines and proliferation rates were evaluated as a surrogate marker of aggressiveness in vitro. Elevated proliferation rates were identified in CRPCa cell lines utilizing ALT however, no significant change was noted in proliferation rates of telomerase‐positive cell lines overexpressing the protein. Taken together these data suggest that SLX4IP may govern the aggressive, ALT‐positive CRPCa phenotype however, additional studies are underway to further confirm this relationship. Ideally, these studies will determine the feasibility of modulating SLX4IP expression in CRPCa to promote telomerase utilization and less aggressive characteristics of the disease. Once the presumed less aggressive, telomerase‐positive phenotype is established, nucleoside analogs that elicit cytotoxicity via telomerase‐dependent misincorporation can then be utilized, providing additional treatment strategies for CRPCa.Support or Funding InformationThis work was supported in part by an NIH T32 (GM008803) grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The COX‐2 selective inhibitor, celecoxib, causes cancer cell death in vitro. However, the mechanism(s) behind the observed cytotoxic effects are still being debated. We have demonstrated that celecoxib treatment induces caspase‐dependent apoptosis in several human tumor cell lines (A375 melanoma, HT‐29 colon carcinoma and CRL‐1620 glioblastoma). Given protective effects of certain prostaglandins, we hypothesized that the apoptotic effects of celecoxib may be in part due to COX‐2 inhibition. However, western blotting for COX‐2 in A375, HT‐29 and CRL‐1620 cells treated with celecoxib for 24 hours revealed a surprising result. Celecoxib induced COX‐2 levels in each of these lines in a dose‐dependent manner. To determine whether COX‐2 induction by celecoxib is a class effect, we included the COX‐2 selective inhibitor etodolac and the non‐selective COX inhibitor ibuprofen. Etodolac treatment for 24 hours induced COX‐2 in a dose‐dependent manner in A375 and CRL‐1620 cells and at the highest dose in HT‐29 cells. Treatment with ibuprofen for 24 hours induced COX‐2 in a dose‐dependent manner in only A375 melanoma cells. These results demonstrate that the COX‐2 inhibitors celecoxib and etodolac induce protein expression of COX‐2 in several human tumor cell lines, whereas induction by ibuprofen was only seen in A375 tumor cells. These data demonstrate that specific and non‐COX specific NSAIDs induce COX‐2 expression.

Telomeres are protective caps found at chromosomal ends that act to prevent attrition of the genome and aberrant activation of DNA repair mechanisms. In somatic cells, telomeric DNA progressively shortens with rounds of replication until a critically short length is reached triggering the cell to undergo senescence to prevent loss of genomic information. However, some cells express telomerase to elongate telomeres and avoid reaching this critical length, thus instilling additional replicative capacity. Notably, 85% of all cancer cells upregulate telomerase and the remaining 15% primarily utilize the alternative lengthening of telomeres (ALT) pathway which relies upon homologous recombination. There is evidence to suggest that cancer cells have the ability to switch to the ALT pathway, should telomerase activity be compromised, but the molecular switch regulating this process is unknown. A recently identified protein, SLX4IP, appears to play a critical role in telomere homeostasis and the telomerase‐to‐‐ALT conversion. In order to identify the basic function of SLX4IP in telomere homeostasis, the interactome of SLX4IP must be interrogated to identify additional proteins involved under specific conditions. For example, identifying differences between the interactomes in telomerase‐positive and ALT‐positive cells will provide insight regarding SLX4IP in telomere maintenance mechanisms. Initially, immunoprecipitation experiments were carried out to identify discrepancies in the proportion of known SLX4IP binding partners among telomerase‐positive and ALT‐positive cell lines. As expected, SLX4IP interacts with TRF2, a telomeric binding protein, and SLX4, an ALT‐associated nuclease scaffold protein, but with varying degrees among the cell types tested. To further dissect the interactome of SLX4IP, wild type SLX4IP, an N‐terminal mutant being interrogated for structural studies, and a C‐terminal mutant which represents a deletion mutant identified in acute lymphoblastic leukemia patients exhibiting poor prognosis were stably overexpressed in telomerase‐positive and ALT‐positive cell lines followed by subsequent immunoprecipitation and proteomic evaluation. This evaluation will provide valuable information as to which regions of SLX4IP are responsible for orchestrating particular interactions in each cell type. With this knowledge, a greater understanding of SLX4IP's role in telomere homeostasis and the telomerase‐to‐ALT conversion can be elucidated.Support or Funding InformationThis work was supported in part by an NIH T32 (GM008803) grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The identification of telomerase-mediated telomeric misincorporation of 5-fluoro-2ʹ-deoxyuridine (5-FdU) uncovered a unique approach to telomeric-based therapeutics. Additionally, identification of such a mechanism supports the utility of telomere maintenance mechanisms in guiding therapeutic decisions. Presented here is a unique perspective of 5-FdU and its clinical implications as a telomeric-based therapeutic.

A small library of chalcone derivatives was synthesized and tested for anti‐proliferative activity in several human cancer cell lines (A375 melanoma, CRL‐1620 glioblastoma, HT‐29 colon carcinoma, and MCF‐7 breast adenocarcinoma). These novel chalcones (RK‐6 and RK‐7) incorporated privileged structures intended to quench glutathione or influence microtubule dynamics. We hypothesized that these compounds would decrease antioxidant capacity or disrupt cell division resulting in anti‐cancer effects. Each of the four cell lines was treated with 50 µM of RK‐6 or RK‐7 for 24 or 48 hours and relative cell numbers were measured using the CyQUANT® NF microplate assay according to the manufacturer's instructions. Treatment with RK‐6 and RK‐7 each resulted in a significant reduction in cell density and visual examination of cells following treatment revealed gross morphology suggestive of apoptosis. Additionally, we determined the GI50 for RK‐6 and RK‐7 in MCF‐7 cells by treating with increasing concentrations (1‐100 µM) of each compound separately for 48 hours, followed by the CyQUANT® NF assay. GI50 concentrations for RK‐6 and RK‐7 in MCF‐7 cells were 36 and 68 µM respectively. To determine whether these compounds were indeed cytotoxic, MCF‐7 cells were treated with RK‐6 or RK‐7 for 24 hours and then subjected to western blotting. RK‐6 but not RK‐7 caused cleavage of effector caspase‐7. Taken together, these results demonstrate that two novel chalcone derivatives possess anti‐cancer effects in multiple human cancer cell lines. Additional studies are underway to determine the mechanisms of anti‐cancer action of these compounds.