This study demonstrates that AC upregulation is a conserved response to radiation therapy across multiple tumor types and AC inhibition can directly improve the clinical response to radiotherapy in vitro and in vivo. IR exposure induced AC upregulation at the mRNA, protein, and enzyme activity level. Dependence of this process on ceramide expression was confirmed by exogenously or endogenously manipulating ceramide levels as well as suppression of IR-stimulated ceramide biogenesis. This observation led to our hypothesis that a critical threshold of intracellular ceramide exists beyond which a mechanism of AC gene transactivation is evoked in order to temper ceramide-mediated cell death. A transcription factor activity array and subsequent RNAi validation revealed that the c-Jun/AP-1 transcription factor significantly induces upregulation of AC following IR through de novo ceramide generation. In our hands, 90 percent of surviving clonal cell populations after 80-Gy IR overexpressed AC, and the level of AC expression correlated positively with proliferation and subsequent radiation insensitivity. Human prostate tissues, in addition to exhibiting AC upregulation in the tumor compared with benign tissues or PIN lesions, had significantly higher levels of AC following radiation therapy failure. In murine xenograft models, interference of AC gene transcription by the TAM67 dominant-negative mutant of c-Jun, RNAi against the AC gene, or lysosomotropic small molecule inhibition of the AC enzyme improved the therapeutic response to IR. Importantly, experiments with combination IR and LCL521 therapy provided a durable cure following completion of therapy. Finally, we demonstrated by in vitro clonogenic studies that rescue of AC overexpression in cells with endogenous AC suppression restored radioresistance and relapse following IR therapy proving the importance of this enzyme in the resistance pathway.
Curiously, the AC upregulation induced by radiation exposure in numerous adenocarcinoma and squamous cell carcinoma cell lines was not recapitulated in 3 noncancer prostate cell lines we evaluated: primary prostate epithelium (PrEC), immortalized prostate epithelium (PWR-1E), and immortalized prostate stroma cells (WPMY-1) (as seen in Supplemental Figure 8). Profiling the ceramide species of irradiated (5 Gy) and mock-irradiated cells revealed differences in total ceramide expression and relative species levels between the noncancer and cancer cell lines. These results suggest associations between a 2-fold increase of the overall intracellular ceramide burden, and/or specifically among the C16
-ceramide species, and AC induction. Investigations to distinguish differences in sphingolipid profile expression between “normal” versus malignant cells at baseline or in vitro growth conditions have identified higher levels of ceramide expression in cancer tissues and cells as well as species-specific variations (63
). At the level of enzymatic function, elucidating the control of sphingolipids upon chemotherapeutic or radiotherapeutic challenge may yield novel differences between cell types (65
). This offers the tantalizing possibility that a distinct difference in sphingolipid regulation at clinically relevant IR dosage presents targets for therapeutic enhancement. Work in this laboratory is currently invested in identifying the underpinnings of such differential responses.
The mechanisms and pathways of irradiation-induced cell death in PCa cells are critical avenues of investigation. Canonical pathways of cell death would include the well-characterized pathway of de novo ceramide accumulation subsequent to irradiation supported by the findings of the Kolesnick group and others (23
). The Kolesnick group characterized this de novo ceramide generation pathway in HeLa cells as dependent on CerS5/6 activation (70
). Initial observations indicating that similar ceramide generation pathways were functioning in the PCa cells included the delayed and sustained onset of ceramide accumulation consistent with de novo ceramide synthesis rather than induction of ceramide accumulation through the action of sphingomyelinase. Ceramide accumulation was positively correlated with radiation dose and sensitivity to radiation, as evaluated by clonogenic assay. Furthermore, inhibition of both ceramide synthases with FB1 (51
) and the de novo pathway with myriocin (52
) prevented IR-induced AC upregulation, ceramide generation, and IR-induced caspase activity, whereas inhibition of acid sphingomyelinase had no effect. Thus, we appear to be observing the diminution of proapoptotic ceramide signaling by ceramide-mediated induction of AC, an enzyme with demonstrated oncogenic activity in PCa (42
), which in turn directly catabolizes ceramide itself.
We sought to further characterize the pathways of cell death in response to irradiation in PCa cells. Radiation treatment of cells induces marked G2
/M arrest, with increased apoptotic cells in a dose- and time-dependent manner, as determined by annexin V staining. Caspase-3 activity was increased following irradiation, with activity augmented by siRNA inhibition of AC. Apoptotic cell death mediated by de novo ceramide accumulation critically functions in mediating cell death following irradiation. Inhibition of necrotic cell death did not rescue IR-induced cell death in PPC-1 cells, suggesting that necrosis, unlike apoptosis, does not appear to be the major cell death pathway in this setting. Although we identified the cell death pathway following IR through de novo ceramide generation and apoptosis augmented by the inhibition of AC, we have not yet ruled out a contribution of mitotic catastrophe mediated by factors such as caspase-2. This mechanism of IR-induced cell damage/death is of great interest to us, as ceramide may be of importance to this process (73
Although the onset of ceramide accumulation would predict a favorable outcome to therapy, due to ceramide’s roles in apoptosis and growth arrest, we also observed substantial increases in the ceramide catabolite sphingosine and its anabolite S1P. As free sphingosine is mainly produced by deacylation of ceramide, this observation was highly suggestive of increased flux through 1 or more of the 5 human ceramidases. Interestingly, we found that AC was increased specifically, with no significant alteration in activity or mRNA expression of neutral or alkaline ceramidases. This may be due, in part, to the increase of C16
-ceramide — a preferred substrate of AC (44
) — relative to other species under conditions radiation exposure or exogenous ceramide treatment. We might also posit that the wave of autophagic activity engendered by a dose of IR that is not immediately lethal accounts for a conserved induction of lysosomal components (74
), of which AC is a significant member. How this process may be connected to IR-stimulated c-Jun/AP-1 activity (76
) remains underappreciated (78
) and an important matter for investigation. In addition, no significant change of sphingosine kinase activity in irradiated PPC-1/PC-3 cells (49
) coincides with IR-induced AC, which led to our hypothesis that AC represents a rate-limiting step for IR-induced ceramide catabolism.
Ceramide is a pleiotropic signaling lipid and IR induces myriad alterations to cellular signaling (as reviewed in ref. 80
). Mechanistically, we were interested to find that inhibition of ceramide generation following IR by pretreatment with FB1 or myriocin prevented not only ceramide accumulation, but also upregulation of AC transcription and protein expression. This observation lead to the hypothesis that ceramide generation induces AC upregulation as a feedback mechanism to dampen the apoptotic ceramide signal. Moreover, the rapid catabolism of stress-induced ceramide into mitogenic/oncogenic S1P counteracts apoptosis signaling propagated by ceramide accumulation (17
). Intriguingly, we observed that nonmalignant prostate epithelial and stromal cell lines do not upregulate AC with IR exposure, which may reveal a survival mechanism that contributes to evasion of apoptosis in malignant cells, as opposed to the orderly apoptotic program maintained by normal cells.
A role for ceramide in regulating gene expression has long been appreciated (81
). In order to seek out the transcriptional mechanism for IR-induced AC upregulation, we identified transcription factor alterations that were common to ceramide treatment and IR individually. Both ceramide and IR activated AP-1, and siRNA knockdown of the various Jun/Fos components indicated c-Jun involvement in these processes. c-Fos involvement was implicated as well, although results were shy of significance and, therefore, equivocal. Interestingly, JUNB binding was confirmed at the ASAH1 promoter as well, although the level of interaction between this protein and the promoter was unaffected by IR or C6
-ceramide induction. Also of note was the failure of myriocin to inhibit c-Jun or c-Fos binding stimulated by C6
-ceramide. These observations may result from the bypass of de novo ceramide generation achieved with the exogenous administration of a ceramide source, which may feed the recycling pathway of ceramide biosynthesis, mediated by FB1-sensitive ceramide synthases (88
The c-Jun proto-oncogene has long been appreciated for mediating cellular transformation, growth, and angiogenesis (89
) and may have more labile activity in malignant cells (96
). The Kester group demonstrated that AP-1 transactivates neutral ceramidase in human coronary artery smooth muscle cells with stimulation by fetal bovine serum (97
). Here, through a combination of luciferase reporter truncations/mutations and ChIP–RT-PCR, we deduced the node of c-Jun/AP-1 interaction with the ASAH1
promoter to the –475/–468–bp site. It was suggested that AP-1 control of neutral ceramidase transcription is tightly regulated by predicted overlapping binding of NF-Y or interaction with proximate cis-
binding factors, considerations that may direct context-dependent transcription of either neutral or AC under disparate stimuli. Investigation of other transcription factors regulating AC expression, such as CREB (54
) and KLF6 (98
), via RNAi studies have not yet revealed involvement of those particular factors in IR-induced AC upregulation (data not shown). However, it should be noted that in silico detection by TFBind of CREB, Stat-1, and Elk-1 consensus binding sequences on the same promoter region as AP-1 may suggest competition for or interaction of binding on the AC gene promoter under various conditions of stimulation.
Approximately 50% of patients with cancer undergo radiation therapy (99
), typically administered in fractionated doses up to 2 Gy every weekday for 7 to 8 weeks in order to avoid toxicity to adjacent structures (101
). However, experimental models of fractionated radiation to clinically achievable doses remain underreported. In the present study, we attempted to mimic clinical irradiation of cancer cells, irradiating PCa cells in vitro at 2-Gy doses for 40 times for the cumulative IR dose of 80 Gy. In our analysis of the surviving clones, we were surprised to find that fractionated irradiation did not completely ablate the reproductive capacity of cancer cells, and, interestingly, 90% of these IR subclones continued to overexpress AC. Although neither the average radiation resistance nor proliferation rate of these IR subclones was as robust as therapy-naive cells, their levels of AC expression showed remarkable correlation with these parameters, harkening to the eventual biochemical failure and relapse of a proportion of radiotherapy patients. As seen in our animal studies using PPC-1 xenografts bearing a doxycycline-inducible AC expression cassette, irradiated mice with forced AC overexpression demonstrated a poorer response to IR compared with mice without AC induction. The irradiated mice overexpressing AC also exhibited earlier relapse compared with IR-treated mice with no AC induction, suggesting a significant contribution to therapy resistance. Taken together, these results demonstrate that AC supports a radioresistance phenotype and inform the hypothesis that enhanced AC expression would be found in prostate tumors that fail definitive courses of radiation therapy.
This was confirmed by evaluation of human prostate tissues by immunohistochemistry. These data suggest that AC is upregulated following IR and support the notion that AC may contribute to resistance and relapse. Retrospective analysis of tissues from difficult to obtain relapsed malignancies showed that radiotherapy patient samples have elevated AC expression. These data provide an indication that our in vitro interrogation of PCa cell responses to IR revealed valid findings. Future studies could use quantification of AC protein in human plasma, urine, or expressed prostatic secretions, which are amenable to high-throughput platforms and allow noninvasive evaluation of tumor response to IR and potential for relapse. This is an attractive approach to studying AC in cancer cells, as their enhanced autophagy and secretory activity enriches the extracellular concentration of lysosomal contents (102
) and because AC can be detected and isolated from human urine (105
Clinically achievable radiation doses are limited by systemic and local toxicity to adjacent structures. Thus, adjuvant therapy that selectively sensitizes tumors to IR might enable lower doses with lower adverse events. The efficacy of IR on xenografts with tightly controlled AC expression supports the notion of inhibiting AC as a strategy for improved cancer therapy. Indeed, our experiments indicate that targeting AC upregulation by interfering with c-Jun activation and by small molecule inhibition augments the efficacy of IR without an increase in total dose of radiation (Figures and ). Moreover, inhibition of AC with the novel lysosomotropic prodrug AC inhibitor, LCL521, prevented relapse (resumption of tumor growth after apparent eradication) in the mice (n = 10), whereas approximately 70% of mice receiving only IR experienced relapse. This situation recapitulates relapse after definitive radiation therapy in human patients with locally advanced disease, 50% of who ultimately experience recurrence of disease. These promising in vivo studies suggest that AC may be an ideal clinical target in the setting of solid tumor irradiation.
An important concern with radiosensitizing agents is the potential for sensitization of normal tissue or augmentation of systemic toxicity, effectively defeating the purpose of adjuvant therapy. Our studies indicate no increased toxicity in mice receiving IR plus LCL521 in comparison with that in the group receiving only LCL521. Although no benefit was observed for LCL521 as a single agent against tumor growth at the dose administrated, synergistic cell killing in combination with IR characterizes LCL521 as an efficacious radiosensitizer. Using our rationally designed AC inhibitors, a previous study demonstrated that susceptibility to AC inhibition corresponds to the level of basal AC expression in treated cells (106
). This suggests that AC inhibition can produce greater therapeutic benefit in treated tumors, due to specific AC induction via focal IR delivery. Additional evaluation of radiosensitization of nontargeted tissue is necessary as we move forward with evaluation of this therapeutic modality; however, the specificity and lack of apparent unintended sensitization is encouraging. Additionally, while LCL521 is at this point highly experimental and its clinical feasibility is not well known (but currently under evaluation), our study clearly outlines a role for AC inhibition in radiosensitization of prostate tumors.
In summary, our data strongly support AC as a major factor in the development of radiation resistance in PCa therapy. Multiple methods clearly demonstrate that interference of AC induction and activity sensitizes cancer cells to IR in vitro and in vivo. Upregulation of AC in radiotherapy-relapsed human tumors confirms the described phenomenon and justifies further study into the feasibility and efficacy of targeting AC in patients receiving IR for PCa. Therefore, AC inhibition presents a rational approach for improving radiotherapy in the preclinical environment with the potential for advancement into patients.