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Carcinoids are neuroendocrine malignancies characterized by their over production of various bioactive hormones that lead to the carcinoid syndrome. We have previously shown that AKT serves as a key regulator of growth and phenotypic expression of tumor markers in carcinoids by genetic depletion of AKT expression. However, no small molecule inhibitor of AKT kinase activity has been developed until recently. MK-2206, a novel allosteric inhibitor of AKT, is currently undergoing clinical trials for treatment of solid tumors. In this study, we explored the effect of MK-2206 on carcinoid cell proliferation and bioactive hormone production in vitro in two carcinoid cell lines-pancreatic carcinoid BON and bronchopulmonary H727. Treatment with MK-2206 effectively suppressed AKT phosphorylation at serine 473 and significantly reduced cell proliferation in a dose dependent manner. Most importantly, MK-2206 treatment resulted in a significant reduction of ASCL1, CgA, and NSE expression, collectively recognized as markers of neuroendocrine tumor malignancy. Furthermore, MK-2206 treated cells exhibited an increase in levels of cleaved PARP and cleaved caspase-3, with a concomitant reduction in levels of Mcl-1 and XIAP, suggesting that the anti-proliferative effect of MK-2206 occurs through the induction of apoptosis. In conclusion, MK-2206 alters neuroendocrine phenotype and suppresses carcinoid tumor growth, suggesting that this drug may be beneficial for patients with carcinoid syndrome. These studies merit further clinical investigation.
Carcinoid tumors are slow growing neuroendocrine (NE) malignancies with a reported incidence of approximately 5:100,000 [1,2]. They primarily arise from the enterochromaffin cells of the gastrointestinal or pulmonary system, but can originate from a variety of bodily tissues. Because carcinoids follow an indolent course, they often go untreated until later stages, and are therefore frequently metastasize to the liver. This generally results in carcinoid syndrome, a disease which entails the hypersecretion of an array of bioactive hormones, including serotonin, growth hormone, and gastrin [1,2]. However, carcinoid syndrome may occur with bronchopulmonary metastases even in the absence of hepatic involvement. Patients with carcinoid syndrome usually present with debilitating symptoms including flushing, diarrhea, vomiting, abdominal pain, and tricuspid insufficiency. Localized carcinoid tumors are curable with surgery [3,4]. However, systemic therapies for the management of metastatic carcinoids have had limited antitumor efficacy. Current mainstay therapies for carcinoids have focused on symptomatic management and chronic care [5,6]. Therefore, there is much demand for new targeted therapies for patients with metastatic carcinoid disease.
Recent evidence has demonstrated the phosphoinositide 3-kinase (PI3K)/AKT pathway to be hyperactive in human carcinoid tumors and treatment with either inhibitor, such as LY294002, or siRNA against AKT, suppresses growth and alters the malignant phenotype of both pancreatic and bronchopulmonary human derived carcinoid cells [7–9]. Given these findings, carcinoid tumors may be highly susceptible to selective inhibition of AKT. Therefore, AKT poses as a potential pharmacotherapeutic target.
Because competitive ATP inhibitors of AKT often bind to protein sites that are biochemically homologous and nonspecific, they have reported a high incidence of off-target effects. In contrast, allosteric non-competitive ATP inhibitors of AKT kinase activity have displayed a much higher level of target specificity. Recently, Merck reported results of phase I clinical trials characterizing MK-2206, an allosteric small molecule inhibitor of AKT, for the treatment of solid tumors . Similar to other non-competitive ATP inhibitors, MK-2206 binds to a site other than the ATP binding domain, causing conformational changes that prevent AKT localization and subsequent kinase activity [10,11]. Along with MK-2206, several classes of small molecule inhibitors of AKT have been described, each with varying potencies and specificities for AKT isoforms .
Studies on MK-2206 indicate that it is safe with tolerable side effects, including some reports of a grade 1–4 rash. These studies have also established pharmacologic parameters including dose limiting toxicities, pharmacokinetics, pharmacodynamics, and alternative day dosing regimens for phase II clinical trials . This drug has also has been purported to possess exceptional antitumor efficacy in both in vitro and in vivo as a single agent, in addition to being a potent sensitizer to a number of other chemotherapeutic agents [10,13–15].
In this present study, we investigated the effect of MK-2206 on NE tumor cell growth, biomarker expression, and apoptosis in both pancreatic and bronchopulmonary carcinoid cell lines.
Human pancreatic carcinoid cancer cells (BON), were provided by Drs. B. Mark Evers and Courtney M. Townsend, Jr. (University of Texas Medical Branch, Galveston, TX, USA), and bronchopulmonary carcinoid (H727) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). BON cells were maintained in DMEM/F-12 (Life Technologies, Grand Island, NY, USA) and H727 cells were maintained in RPMI1640 (Life Technologies, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Life Technologies, Grand Island, NY, USA). Both cell lines were grown in a humidified atmosphere of 5% CO2 at 37°C. MK-2206 was dissolved in dimethyl-sulfoxide (DMSO, Fischer Scientific, Pittsburgh, PA, USA) and cells were treated with varying doses of MK-2206.
Carcinoid tumor cell viability was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) by a standard rapid colorimetric assay. BON and H727 cells were plated in 24-well plates and allowed to adhere overnight. Cells were then treated with MK-2206 in quadruplicate at each dose. Treatments lasted for up to 6 days, and were replenished after 48 hours. On the day of cell viability determination, media was replaced with 250 μL of serum-free medium containing 0.5 mg/mL MTT. Plates were then incubated at 37°C for 3.5 hours, followed by the addition of 750 μL of DMSO, and measured at 540 nm using a spectrophotometer (μQuant, Bio-Tek Instruments, Winooski, VT, USA).
Following treatment, cells were washed in 1X PBS and lysed in lysis buffer (50 mM Tris, 0.15 M NaCl, 0.5% Na/deoxycholate, 0.1% SDS, 1% Nonidet P-40, 0.1% protease inhibitor cocktail and 0.6 mM phenylmethanesulfonyl fluoride), and the lysates were then prepared as described . A bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA) was used to determine the concentration of total cellular protein. Subsequently, these samples were denatured, resolved on 7%, 10%, or 12% SDS-PAGE gels (Invitrogen, Grand Island, NY, USA). Proteins were then transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) and the protein bound membranes were blocked for at least 30 minutes in a PBS-T containing milk (1x PBS, 5% dry milk, 0.05% Tween-20), and then incubated overnight at 4°C in their respective primary antibodies. Each antibody was diluted as follows: 1:2000 for mammalian achaete scute complex-like1 (BD PharMingen, San Diego, CA, USA), 1:3000 for chromogranin A (Zymed Laboratories, San Francisco, CA, USA), 1:10,000 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD, USA). Phosphorylated AKTser473, phosphorylated AKTthr308, total AKT, total caspase-3, cleaved caspase-3, cleaved caspase-7, total-PARP, cleaved-PARP, X-linked inhibitor of apoptosis (XIAP), Mcl-1, Survivin, and β-Actin (Cell Signaling Technology, Beverly, MA, USA) were all diluted to 1:1000. Following incubation in primary antibody, membranes were washed 3×5 minutes in PBS-T wash buffer (1x Phosphate Buffered Saline, 0.05% Tween 20). Blots were then incubated in either horseradish peroxidase conjugated anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology), depending on the source of the primary antibody. Membranes were washed for 3×5 or 3×10 minutes in PBS-T wash buffer. SuperSignal West Pico, Femto (Pierce, Rockford, IL, USA) or Immunstar (Bio-Rad Laboratories, Hercules, CA, USA) kits were then used to develop the membranes to measure the intensity of the band according to the manufacturers’ instructions.
All results shown represent mean ± SEM, unless specifically noted. The significance between treatments was analyzed using analysis of variance testing (SPSS software ver10.0; SPSS Inc., Chicago, IL). A p value less than 0.05 was considered significant.
We investigated the effects of MK-2206 on cell proliferation in two human carcinoid cell lines, pancreatic carcinoid (BON) and bronchopulmonary (H727) carcinoid cells. Cellular proliferation was assessed by MTT assay up to 6 days after MK-2206 treatment and results indicated that growth was dose dependently decreased in both cell lines compared to the control (DMSO) treatment. There was a modest reduction in H727 cell growth following 2 days of MK-2206 treatment, whereas there was a statistically significant as well as dose-dependent growth reduction in BON cells at following 2 days of treatment. Nevertheless, cellular growth was reduced to 90% in H727 cells treated with 10 μM of MK-2206 treatment (Figure 1A), whereas BON cells required 15 μM of drug treatment in order to achieve 87% growth inhibition (Figure 1B) following 6 days of treatment.
The carcinoid syndrome progression and symptomatology is largely consequent of the expression and co-secretion of a number of bioactive amines among which include chromogranin A (CgA). These biomarkers along with high expression levels of achaete scute complex-like1, (ASCL1) collectively portend a poor prognosis for carcinoid patients and are routinely utilized for diagnostic purposes. Given the prognostic significance of ASCL1 and CgA, we analyzed the impact of MK-2206 on the expression of these neuroendocrine phenotypic markers. The levels of ASCL1 and CgA were significantly reduced by MK-2206 treatment in a dose dependent manner as shown by Western blot analysis of lysate extracts from MK-2206 treated H727 and BON cells (Figure 2A and 2B). Over-expression of ASCL1 is implicated in neuroendocrine tumorigenesis and therefore, suppression of ASCL1 and reduction of CgA may collectively represent a possible mechanism of action of MK-2206 in carcinoid growth reduction. Furthermore, there was a significant reduction in the levels of other bioactive markers such as Neuron Specific Enolase and Synaptophysin in H727 cells treated with MK-2206 (Figure 2A). These findings cumulatively suggest that MK-2206 may have the ability to alleviate symptoms of the carcinoid syndrome.
Based on the reported inhibitory activity of AKT by MK-2206 in other cancer types, we sought to confirm if this agent inhibits the phosphorylation of AKTSer473 and AKTThr308 in carcinoid cell lines. Both H727 and BON cells were treated with MK-2206 for a given period and subsequently underwent Western analysis for the levels of phosphorylated AKT. We observed that MK-2206 inhibited the phosphorylation of AKTSer473. Interestingly, there was no significant effect on levels of phosphorylated AKTThr308 in H727 and BON cells (Figure 3A and 3B) indicating that MK-2206 has no effect on the upstream PI3-K pathway. We have previously reported that silencing AKT expression by AKT-specific siRNA sequences in carcinoid cells resulted in growth suppression, along with a reduction in the expression of neuroendocrine markers [8,9]. Collectively these data suggest that the suppressive effects of MK-2206 on growth and neuroendocrine marker expression in carcinoid cells may be due to inactivation of AKT activity.
Having observed the growth suppressive effects of MK-2206 in H727 and BON cells, we next sought to determine the possible cause of cytotoxicity by determining levels of molecular markers of apoptosis in treated cells. Following treatment with MK-2206 at the appropriate doses,, cells were examined by Western blotting for their expression levels of cleaved poly (ADP)-ribose polymerase (PARP), cleaved caspase-3, and cleaved caspase-7. It is known that during execution of the apoptotic cascade, cleavage of PARP, a caspase-3 substrate, must precede the cleavage of caspase-3, an ICE-like protease activated response to various cytotoxic stimuli. Similarly, cleavage of caspase-7 has also been strongly associated as a precedent to PARP cleavage in a number of stress-induced models. Therefore, the cleavage of PARP and various caspases can be reliable indicators that cells are undergoing apoptosis following external stimuli, such as exposure to MK-2206. Indeed, we observed an elevation in levels of cleaved PARP in both H727 and BON cells after MK-2206 treatment (Figure 4A and 4B). Furthermore, we observed an increase in cleaved caspase-3 and -7 in H727 cells following treatment with MK-2206 (Figure 4A).
In order to further support the mechanism of apoptotic induction following MK-2206 treatment in carcinoids, we measured the activity of various proteins responsible for evasion of apoptosis. Among these molecular components include members of the family regarded as the inhibitors of apoptosis (IAP), which are known for their role in intervening in programmed cell death cascades by binding to caspases to prevent their cleavage and subsequent activation. Among the IAPs, we chose to investigate the activity of XIAP and survivin, in addition to levels of the Bcl-2 family member known as induced myeloid leukemia cell differentiation protein, or Mcl-1. We demonstrated a reduction in expression levels of XIAP, survivin and Mcl-1 in MK-2206 treated H727 and BON cells at doses that demonstrably induced apoptosis as signified by the cleavage of PARP and associated caspases (Figure 5A and 5B). Control cells exhibited high levels of all pro-survival markers, suggesting that these proteins may support cell survival.
The urgency for new therapies for the treatment of carcinoids is underscored by the complicated nature of their management [1,2,6,17]. Carcinoids are generally encountered inadvertently, and are often unresectable at the time of diagnosis. Recent efforts have focused on molecular signaling pathway derangements in carcinoid cell lines in order to develop effective targeted therapies [17–21]. Among these pathways is the phosphoinositide 3-kinase (PI3K)/AKT pathway, which regulates such functions as mitogenesis, cellular motility, and protection from apoptosis in normal tissues [22,23]. Hyperactivation of the PI3K-AKT pathway has been commonly implicated in the growth, progression and phenotypic expression of a number of neoplasms including breast, prostate, colon, ovarian, and neuroendocrine cancers [22,23]. The current course of drug development has attempted to exploit the critical nature of the PI3K-AKT pathway in various cancer pathogeneses with the use of small molecule inhibitors of this pathway .
In this study, we tested the therapeutic potential of the AKT inhibitor MK-2206 in both pancreatic and bronchopulmonary carcinoid tumor cell lines, BON and H727 respectively. We demonstrated that this drug exerts a potent and efficacious inhibitory effect on cell proliferation in both cell lines. This is strongly aligned with reports which describe the capacity of MK-2206 to inhibit cell growth in other cancer cell lines, including thyroid cancer cells that harbor mutations in the PI3K/AKT pathway, in addition to colon cancer, gliomas, non-small cell lung cancers and melanomas [13,15]. Our studies further demonstrated that this observed anti-tumor effect of MK-2206 correlates with a reduction in AKT phosphorylation at the serine 473 residue, in both BON and H727 cells. Furthermore, we determined that the effects of this drug were associated with the inhibition of ASCL1, CgA, SYP, and NSE production, neuroendocrine biomarkers which can be clinically utilized to assess the degree of drug efficacy [19,25,26]. Therefore, these data suggest that MK-2206 may not only inhibit carcinoid tumor cell growth, but may also alleviate symptoms associated with bioactive hormone production. Therefore, MK-2206 may be a therapeutic option for palliation of carcinoid syndrome symptomatology in addition to reducing tumor burden.
The anti-tumor effect of MK-2206 was due to its ability to initiate apoptosis, and reduce the expression of pro-survival protein Mcl-1 along with proteins classified as inhibitors of apoptosis (IAP) such as XIAP and survivin. Recent evidence has supported the oncogenic role of the protein survivin and it is highly expressed in several tumors . The clinicopathological significance of survivin is inextricably linked to its ability to inhibit the apoptotic cascade in various cancerous tissues, and even more importantly, to confer chemo-resistance to cancer cells in the presence of certain anti-cancer drugs. In fact, recent evidence has implicated the presence of high levels of survivin in the metastatic potential of carcinoid tumors, suggesting that this protein, among others, is a reliable indicator of carcinoid tumor burden . Similarly, XIAP has also been described as being frequently elevated in malignant cancerous cells and a culprit for chemoresistance in cancer patients [29–31]. Finally, the role of Mcl-1 as a protector from apoptotic cascades in carcinoid tumor malignancy has been well characterized, as its expression has been found to be significantly elevated in panels of human carcinoid specimens of pancreatic and bronchopulmonary origin, which, not surprisingly, exhibited low levels of apoptosis . The ability of MK-2206 to exert its cytotoxic effects in carcinoid cells, in addition to reducing levels of survivin, XIAP, and Mcl-1, suggests that it may be capable of counteracting cancer cell defense mechanisms against drug induced cytotoxicity. Because carcinoid patients are often subject to extended periods of chemotherapy, they have an increased likelihood of developing resistance or becoming unresponsive to drugs. Therefore, at present, we can only speculate that MK-2206 may have the potential of sensitizing the cells from chemo resistance. Further studies are needed to confirm our hypothesis.
In summary, MK-2206 presents a viable option for reducing carcinoid tumor burden and symptomatology given its ability to reduce carcinoid cell proliferation, neuroendocrine biomarker expression, and pro-survival signals in vitro. Thus, it is important to further explore the use of this compound in further preclinical studies and eventually in clinical trials on carcinoid patients.
Financial Support: This research was supported in part by the Howard Hughes Medical Institute Medical Fellowship Program (Y.S.), NIH grant T-35 (KS), NIH grant R01 CA121115 (H.C.), the American Cancer Society Research Scholars Grant (H.C.), American Cancer Society MEN2 Professorship (H.C.), and NIH grant R03 CA155691 (M.K.).
We would like to acknowledge the Merck® Corporation for graciously providing us with MK-2206 in order to carry out our investigation.
Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.