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Sesterterpenoid and Steroid Metabolites from a Deep-Water Alaska Sponge Inhibit Wnt/β-Catenin Signaling in Colon Cancer Cells

The Wnt/β-catenin signaling pathway is known to play critical roles in a wide range of cellular processes: cell proliferation, differentiation, migration and embryonic development. Importantly, dysregulation of this pathway is tightly associated with pathogenesis in most human cancers. Therefore, the Wnt/β-catenin pathway has emerged as a promising target in anticancer drug screening programs. In the present study, we have isolated three previously unreported metabolites from an undescribed sponge, a species of Monanchora (Order Poecilosclerida, Family Crambidae), closely related to the northeastern Pacific species Monanchora pulchra, collected from deep waters off the Aleutian Islands of Alaska. Through an assortment of NMR, MS, ECD, computational chemical shifts calculation, and DP4, chemical structures of these metabolites have been characterized as spirocyclic ring-containing sesterterpenoid (1) and cholestane-type steroidal analogues (2 and 3). These compounds exhibited the inhibition of β-catenin response transcription (CRT) through the promotion of β-catenin degradation, which was in part implicated in the antiproliferative activity against two CRT-positive colon cancer cell lines.


Introduction
Colorectal cancer is the fourth most prevalent cancer and the third leading cause of cancer-related mortality worldwide. In 2017, approximately 130,000 new cases and 50,000 deaths can be attributed to colorectal cancer in the United States alone [1]. Despite significant advances in surgery, radiotherapy Mar. Drugs 2018, 16, 297; doi:10.3390/md16090297 www.mdpi.com/journal/marinedrugs and chemotherapy, the survival rate of colorectal cancer patients has only slightly increased over the past decade [1]. Besides, emerging side effects and drug resistances are likely unavoidable, which in turn counteract current chemotherapeutic approaches in the cancer treatment. Therefore, extensive investigations of potential chemotypes along with mechanisms of action are significantly required for the development of effective anticancer drug lead structures. The Wnt/β-catenin signaling cascade is one of the well-established cell intrinsic canonical pathways involved in a variety of biological events [2]. A multitude of research has also shown that abnormal activation of the Wnt/β-catenin pathway frequently occurs in various cancer types (e.g., colorectal, leukemia, prostate and breast) [3]. Subsequently, the functional loss of a tumor suppressor known as adenomatous polyposis coli (APC) was found to be highly associated with the aberrant activation of the oncogenic pathway [3]. The APC was initially identified as the main cause of familial adenomatous polyposis (FAP) syndrome; thereafter it was reported that the genes encoding APC are commonly mutated in most sporadic colorectal cancers [4,5]. The interplay between APC and β-catenin, a central transcriptional regulator, has been well elucidated in the pathway. The mutational alteration of the APC gene stimulates the stabilization of β-catenin that abnormally accumulates and moves to the nucleus in order to bind to T-cell factor/lymphocyte enhancer factor (Tcf/LEF) [6]. The β-catenin/Tcf/LEF transcription ultimately regulates the expression of Wnt/β-catenin target genes such as c-Myc and cyclin D1, thereby culminating in tumorigenic processes in the cells [7,8]. A body of evidence has made it apparent that inhibition of the oncogenic β-catenin pathway significantly suppresses the carcinogenesis of cancers [9,10]. Hence, discovering effective inhibitors targeting β-catenin stabilization and downstream signaling could be of high importance.
Previously, we have reported that the flavonoid galangin effectively inhibits β-catenin response transcription (CRT) by promoting β-catenin degradation in colon cancer cell lines [11], and new 4,9-friedodrimane-type sesquiterpenoids, isolated from a marine sponge, suppress β-catenin expression and exhibit cytotoxic activity in colon cancer cells [12]. Emerging evidence indicates that marine sponges are a prolific source of biologically active natural products [13,14]. Extensive chemical studies with marine sponges have identified unique and diverse structural characteristics, many of which are functionally characterized in a wide range of promising pharmaceutical relevance [15][16][17].
In particular, marine sponge species belonging to the genus Monanchora (order Poecilosclerida, family Crambeidae) have yielded a variety of new natural products. Examples include a unique family of polycyclic guanidine alkaloids exhibiting diverse bioactivities, including cytotoxicity, antibiotic, antiviral, and anti-inflammatory [18][19][20][21][22][23]. Cytotoxic sesterterpenoids and anti-inflammatory steroids have been isolated from a Monanchora sp., collected in Korean waters [24,25]. A number of sterols have also been elucidated from this genus [26,27]. Based on these diverse findings, we herein report the investigation of an undescribed species of Monanchora, closely related to the Northeastern Pacific species, Monanchora pulchra [28], collected from deep waters off the Aleutian Islands of Alaska. The details include structural characterization of 1-3 via spectroscopic and spectrometric approaches, and quantum mechanics-based chemical shifts calculation with support of the advanced statistics DP4 [29]. The isolated compounds were also evaluated for their inhibitory activity of β-catenin response transcription (CRT) and antiproliferative activity against CRT-positive colon cancer cells.

Results and Discussion
The fresh sponge (~1 kg) was immediately frozen on site and stored at −30 • C. The material was subsequently thawed, cut into small pieces and extracted with methanol to yield a crude methanol extract (53.2 g). The crude extract (2.5 g) was then subjected to reversed-phase C 18 medium-pressure liquid chromatography (MPLC), followed by HPLC purification, yielding 1 (3 mg), 2 (3 mg) and 3 (2 mg) ( Figure 1).
Assignment of the absolute configuration of 1 was achieved by comparison of its experimental and calculated electronic circular dichroism (ECD) spectra (Supplementary Materials, Figure S8 and Table S1). The experimental ECD spectrum of 1 revealed strong positive Cotton effects at ~212 and 330 nm, and a negative Cotton effect at ~250 nm, which was consistent with that of a simulated ECD The relative configuration of 1 was established by an analysis of the NOESY spectrum ( Figure 2B). A strong NOESY correlation between H-1 (δ H 4.47) and H-6 (δ H 2.54) suggested that these protons were cofacial, establishing the cis-fused ring system. NOESY correlations from H-8 to H-10 and, from H-1 to H-13, supported the presence of the tricyclic moiety possessing a 9S* spiroketal junction ( Figure 2B). NOESY cross-peaks between a vinyl methyl at δ H 1.74 (H 3 -24) and H-2/H-13 established the (Z)-geometry of C14/15. Meanwhile, additional NOESY correlation from H-13 to H 3 -23 was observed, allowing they are on the same face, which is distinct from alotaketal C [32].
Assignment of the absolute configuration of 1 was achieved by comparison of its experimental and calculated electronic circular dichroism (ECD) spectra (Supplementary Materials, Figure S8 and Table S1). The experimental ECD spectrum of 1 revealed strong positive Cotton effects at~212 and 330 nm, and a negative Cotton effect at~250 nm, which was consistent with that of a simulated ECD spectrum of 1 ( Figure 2C).
Only a few tricyclic spiroketal framework-containing sesterterpenoids have been characterized from several marine sponges. To the best of our knowledge, the first documented examples are alotaketals from the marine sponge Hamigera sp. and phorbaketals, isolated from the marine sponge Phorbas sp. [30,31]. Thereafter, the chemical structures of their derivatives have been reported from other sponge species [33][34][35]. These molecules revealed a myriad of biological properties such as inhibition of adipocyte differentiation, mast cell differentiation activity, cytotoxicity, and anti-inflammatory [31,36,37]. Aloketals have also been shown to induce potent activation of the cyclic adenosine monophosphate (cAMP) signaling pathway which is relevant to a number of human diseases [30].
The assignment of relative configuration of C-23 was achieved by the gauge-including atomic orbital (GIAO) NMR chemical shifts calculation. Conformational searches of two possible diastereomers a (23R) and b (23S) of 2 were conducted utilizing Macromodel (Schrödinger LLC) with a relative energy level in the MMFF94 force field, yielding 20 and 22 conformers within 10 kJ/mol, respectively (Supplementary Materials, Table S2). The chemical shifts of those conformers were subsequently computed at the B3LYP/6-31G(d,p) theory level using the Gaussian 09 package (Gaussian Inc.) and the resultant NMR properties were Boltzmann-averaged at 298.15 K (Supplementary Materials, Table S2). The 1 H and 13 C NMR chemical shift values of the diastereomers a and b were compared with those of the experimental NMR data of 2 (Supplementary Materials, Table S3) using the advanced statistics DP4 [29]. The statistical analyses revealed that the probability of diastereomer a (23R) is 99.6% in the consideration of both 1 H and 13 C NMR chemical shift values ( Figure 4A, Supplementary Materials, Figure S16), establishing the relative configuration of C-23 of 2 as 23R*. Thus, the chemical structure of 2 was proposed as 20E,23R*-hydroxy-nor-cholest-4,20-dien-3-one.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 6 of 15 Table S3) using the advanced statistics DP4 [29]. The statistical analyses revealed that the probability of diastereomer a (23R) is 99.6% in the consideration of both 1 H and 13 C NMR chemical shift values ( Figure 4A, Supplementary Materials, Figure S16), establishing the relative configuration of C-23 of 2 as 23R*. Thus, the chemical structure of 2 was proposed as 20E,23R*-hydroxy-nor-cholest-4,20-dien-3-one.  Figure S23). The NOESY correlations of the protons of the cholestane skeleton of 3 were also consistent with those of typical cholestane-based derivatives ( Figure 3B). The NOESY correlation from H-14 to H-16 supported the existence of the 16β-hydroxy group. The relative configuration of C-20 was also conducted by the application of computational NMR chemical shift calculations supported by DP4 analysis ( Figure 4B). Computational data for 3 were obtained from statistical calculations utilizing the aforementioned protocol and level of theory. Conformers (36 for diastereomer a (20R) and 25 for  Figure S21), and the HMBC cross-peaks from the two doublets at δ H 1.03 of the terminal methyl groups (Me-26 and Me-27) and H 2 -28 (δ H 4.75 and 4.68) to C-24 (δ C 156.3) located the exocyclic double bond on C-24. Further HMBC correlation from Me-21 (δ H 1.32) to C-17 (δ C 60.6) established the linkage of this side chain to C-17 and a methyl substitution at an oxygen-bearing quaternary carbon (C-20, δ C 76.5). This overall elucidated the steroidal side chain to be a methylidene group on C-24 ( Figure 3B). The relative configurations of the stereogenic carbons of 3 were established by NOESY correlations ( Figure 3B and Supplementary Materials, Figure S23). The NOESY correlations of the protons of the cholestane skeleton of 3 were also consistent with those of typical cholestane-based derivatives ( Figure 3B). The NOESY correlation from H-14 to H-16 supported the existence of the 16β-hydroxy group. The relative configuration of C-20 was also conducted by the application of computational NMR chemical shift calculations supported by DP4 analysis ( Figure 4B). Computational data for 3 were obtained from statistical calculations utilizing the aforementioned protocol and level of theory. Conformers (36 for diastereomer a (20R) and 25 for b (20S) were subsequently generated (Supplementary Materials, Table S4), and their respective chemical shift values were calculated and averaged for DP4 analysis (Supplementary Materials, Table S5). Comparative DP4 probabilities of the two diastereomers concluded 100.0% for diastereomer a considering both 1 H and 13 C NMR chemical shifts ( Figure 4B and Supplementary Materials, Figure S24). Thus, the structure of 3 was suggested as 16β,20R-dihydroxy-cholest-4,24-dien-3-one.
A large number of studies have reported that abnormal activation of Wnt/β-catenin signaling and overexpression of β-catenin response transcription (CRT) are frequently observed in the development and progression of colorectal cancer [2,3]. To evaluate modulatory activities of 1, 2, and 3 on the CRT in this signaling pathway, a genetically engineered HEK293 cell line (HEK293-FL reporter cells) which stably harbored a synthetic β-catenin/Tcf-dependent firefly luciferase (FL) reporter and hFz-1 expression plasmid was used [38]. Chemical effects on the Wnt/β-catenin pathway in these reporter cells have been normalized by the β-catenin/Tcf-driven FL activity with cell viability. As shown in Figure 5A, FL activity was significantly stimulated upon the treatment with Wnt3a-conditioned medium (Wnt3a-CM). Importantly, the supplementation with 1-3 at three different micromolar concentrations (10, 20, and 40 µM for 1 and 3; and 15, 30, and 60 µM for 2) caused significant inhibition of CRT activity, stimulated by Wnt3a-CM, in a dose-dependent manner ( Figure 5A). Under the same condition, these compounds did not affect cell viability in HEK293-FL reporter cells, which have an intact Wnt/β -catenin pathway (Supplementary Materials, Figure S25).  It has been reported that CRT is primarily dependent on the level of intracellular β-catenin which is controlled by the proteasomal degradation route. Normally, β-catenin is phosphorylated at Ser45 and Ser33/37/Thr41 by casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), respectively, in a complex with APC and axin (called β-catenin destruction complex), leading to the degradation of β-catenin through a ubiquitin-dependent mechanism [39]. Thus we further examined the effects of 1-3 on the protein level of intracellular β-catenin using Western blot analysis. As expected [38], the up-regulation of protein level of β-catenin upon the treatment of Wnt3a-CM ( Figure 5B) and clear It has been reported that CRT is primarily dependent on the level of intracellular β-catenin which is controlled by the proteasomal degradation route. Normally, β-catenin is phosphorylated at Ser45 and Ser33/37/Thr41 by casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), respectively, in a complex with APC and axin (called β-catenin destruction complex), leading to the degradation of β-catenin through a ubiquitin-dependent mechanism [39]. Thus we further examined the effects of 1-3 on the protein level of intracellular β-catenin using Western blot analysis. As expected [38], the up-regulation of protein level of β-catenin upon the treatment of Wnt3a-CM ( Figure 5B) and clear dose-dependent down-regulation in the cytosolic β-catenin level from the treatment with 1-3 were found in the cytosolic proteins prepared from the HEK293-FL reporter cells ( Figure 5B). This implies that 1, 2, and 3 inhibit Wnt/β-catenin signaling through the down-regulation of cytosolic β-catenin levels.
Previous studies have also shown that the suppression of the Wnt/β-catenin pathway inhibits the proliferation of CRT-positive colon cancer cells [39]. Since 1-3 reduced cytosolic β-catenin levels, we ultimately evaluated the inhibitory effect of these compounds on the growth of representative CRT-positive colon cancer cells. As a result, 1, 2, and 3 exhibited cytotoxic activity in HCT116 colon cancer cells, which contain a Ser45 (CK1 phosphorylation site) deletion mutation in β-catenin, with IC 50 values of 43.5, 19.7, and 48.0 µM, respectively ( Figure 6). In addition, the viability of SW480 colon cancer cells, which display elevated β-catenin expression due to mutation in APC, was decreased by treatment with 1 (IC 50 = 54.8 µM), 2 (IC 50 = 24.2 µM), and 3 (IC 50 = 41.3 µM) ( Figure 6). Several lines of evidence in this study indicate that 1, 2, and 3 suppress the Wnt/β-catenin pathway through a mechanism independent of β-catenin destruction complex. These compounds were still able to decrease CRT activity and the cytosolic β-catenin level in the presence of Wnt3a, which inactivates β-catenin destruction complex. Furthermore, 1, 2, and 3 showed cytotoxic activity in HCT116 and SW480 colon cancer cells, which have alteration in destruction complex-mediated β-catenin degradation. The mechanism underlying downregulation of β-catenin induced CRT of 1-3 needs to be further investigated.

Sponge Collection
The specimen used for compound extraction and subsequent chemical analyses (2010-AK-49) was collected with a bottom trawl during a fisheries stock assessment survey aboard the RV Sea Storm.

Taxonomic Notes
Monanchora pulchra was first described by Lambe [28], from beach-thrown specimens collected from Gull and Unalaska Islands, in the Aleutian Islands Archipelago. The species has since been collected from other island ridges and passes in the Aleutian Islands [40]. In a review of trawl-caught specimens from the Aleutian Island region in 2010 a new, undescribed species, M. n. sp. 1 (yellow fan) was reported with a listing of key differences between the two species [13]. Subsequent careful study of numerous specimens collected from the region prior to 2010 with submersibles [41] revealed yet a third group referred to as M. cf. pulchra. Study of the gross morphology and bathymetric distribution of the three groups of sponges enhances our ability to target future collections and determine if the active metabolites are species-specific; the shape and thickness of the sponge lamella and the colour in life, appear to be reasonably specific to each taxon, and there is some bathymetric differentiation evident as well.
The first taxon is M. pulchra, described by Lambe (Supplementary Materials, Figure S26) [28], as a thin, yellowish fan with deeply incised margins and narrow surface aquiferous canals visible in life, large style megascleres (1100 µm), small robust microscleres (unguiferous arcuate chelae) with longish teeth-like alae, and sigma microscleres. The second taxon is referred to as M. cf. pulchra (Supplementary Materials, Figure S27) because the specimens generally resemble the original species, but form thin, leafy, pumpkin-to dark orange-colored fans, with oscules along the margins and on one side. The spiculation differs in key areas as well: the style megascleres are smaller than those of M. pulchra sensu stricto (900 µm long) and the microsclere chelae are smaller, thinner and predominantly anchorate in form; they are only occasionally unguiferous arcuate in form (as in M. pulchra sensu stricto) with numerous very short alae. These sponges were collected consistently in relatively shallow water (< 100 m). The third group, identified here as Monanchora n. sp. 1 (large isochelae and styles), differs considerably from M. pulchra sensu stricto (Supplementary Materials, Figure S28) in that the specimens may be digitate or more commonly form stalked, thick fans with a deeply cracked surface. The surface appears to be more densely siliceous and opaque in life, than in the other groups. The style megascleres are large and very similar to those in M. pulchra sensu stricto, but the key difference is the possession of large, robust, anchorate/arcuate chelae with numerous very short alae. This third group of specimens, and the single specimen of M. pulchra sensu stricto collected, were all captured in deeper waters around 150 m. One of our future objectives is to determine whether the active compounds are found in all three closely-related taxa, and in what proportions. As the compounds of interest are not abundant, it may be more expedient to pool all specimens of Monanchora for extraction rather than embarking on the detailed determination of each individual's identification.

Ecological Observations
Review of video footage of the seafloor collected throughout the central Aleutian Islands with submersibles in 2002-2004, coupled with voucher specimen collections, indicated that M. pulchra was widely distributed, locally abundant, and found at depths between 79 and 330 m [41]. Taxonomic analyses of collected specimens, all from depths < 100 m, indicated that all were M. pulchra. Our new analysis of additional collected specimens indicates that there are three distinct taxa or groups and that they may occupy different depths and habitats. Monanchora cf. pulchra appears to occupy rougher habitats including bedrock outcrops at depths principally shallower than 100 m. The other two taxa, M. pulchra and Monanchora n. sp. 1, appear to occupy lower-profile habitat consisting of sand, pebbles, and small cobbles and at depths > 100 m. The latter habitat type is accessible to trawl sampling, hence the collection of those specimens by trawl gear. All species occupy habitats that are subjected to moderate to high water currents.

Organic Extraction and Compound Isolation
Monanchora sp. (~1 kg) was cut into small pieces and extracted with MeOH to yield a dark colored-solid extract (53.2 g). The MeOH extract (2.5 g) was subjected to C 18

Computational Details for ECD Simulation
A truncated structure of 1 (1a, Supplementary Materials, Figure S8) was proposed to minimize computational complexity and expense, and conformational searches with 1a were performed using MacroModel with the MMFF force field (gas phase), a 10 kcal/mol upper energy limit and 0.001 kJ (mol Å) −1 convergence threshold on the rms gradient. Redundant conformers were eliminated utilizing a 0.5 Å root-mean-square deviation (RMSD) cut-off for the atoms indicated in red (Supplementary Materials, Figure S8), ultimately leading to identify eight unique conformers. The geometries of these conformers were optimized at B3LYP/6-31G(d) basis set in the polarizable continuum solvation model (PCM) with a dielectric constant representing MeOH. The optimized conformers were then proceeded to ECD calculations at B3LYP/6-31G(d,p) (PCM, MeOH), and the generated excitation energies and rotational strengths were Boltzmann-averaged on the basis of calculated Gibbs free energy (Supplementary Materials, Table S1) and visualized utilizing SpecDis.

Computational NMR Chemical Shifts Calculations for DP4 Analysis
Conformational searches were performed using the MacroModel (Version 9.9, Schrödinger LLC, New York, NY, USA) program interfaced in Maestro (Version 9.9, Schrödinger LLC) with a mixed torsional/low-mode sampling method. Advanced conformational searches were carried out in the MMFF force field, in the gas phase with a 50 kJ/mol energy window and 10,000 maximum iterations based on the original authors' recommendations [29]. NMR chemical shift calculations of all conformers within 10 kJ/mol of the relative energy were implemented at the Gaussian 09 package (Gaussian Inc., Wallingford, CT, USA) without geometry optimization in the B3LYP/6-31G(d,p) theory level. Chemical shifts values were calculated via an equation below where δ x calc is the calculated NMR chemical shift for nucleus x, σ o is the shielding tensor for the proton and carbon nuclei in tetramethylsilane calculated at the B3LYP/6-31G(d,p) basis set. For the consistency of computational outcomes, the geometry of tetramethylsilane was optimized at the aforementioned theory level.

Western Blotting
The cytosolic fraction was prepared as previously described [42]. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in a 4 to 12% gradient gel (Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk and probed with anti-β-catenin (BD Transduction Laboratories, Lexington, KY, USA) and anti-actin antibodies (Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with horseradish-peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA) and visualized using the ECL system (Santa Cruz Biotechnology, Dallas, TX, USA).

Cell Viability Assay
Cells were dispensed into 96-well plates followed by the treatment with compounds 1-3 and further incubated for 48 h. The cell viability from compounds treatment was measured using CellTiter-Glo assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. All experiments were carried out in triplicate.

Conclusions
In summary, we have isolated a new spirocyclic ring-containing sesterterpenoid (1) and two new cholestane-type steroids (2 and 3) from a deep-water Alaska sponge. The sponge was taxonomically characterized to be an undescribed species of Monanchora. The chemical structures of these metabolites were elucidated by the interpretation of NMR and MS spectral data, and their absolute configurations were determined by comparison of the experimental and calculated ECD spectra, and computational chemical shifts calculation followed by DP4 analysis. We also demonstrated that the isolated compounds exhibit an antiproliferative effect on two β-catenin response transcription (CRT)-positive colon cancer cell lines, HCT116 and SW480. We further showed that these metabolites inhibit CRT activity by stimulating the degradation of β-catenin. To the best of our knowledge, this is the first report that sesterterpenoid shows inhibitory effects on the Wnt/β-catenin pathway. Among steroidal metabolites, corticosteroids such as glucocorticoid are known to inhibit the Wnt/β-catenin pathway [43]. Collectively, our studies provide further evidence that marine sponges are an untapped natural resource for the discovery of new bioactive metabolites that expand our view on the structural diversity for the development of the anti-cancer lead structures.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/16/9/297/s1, Figure S1-S25: HRESIMS, 1D ( 1 H and 13 C) and 2D NMR spectra, computational studies of compounds 1-3, Figure S26-S28: sponge specimen in this study, Table S1: Gibbs Free Energy and Boltzmann Population of 1a for ECD computation, Table S2: The major conformers of diastereomers of compound 2, Table S3: Experimental and calculated NMR chemical shift values (ppm) of compound 2 with diastereomers (diastereomers a and b are described in the main text), Table S4: The major conformers of diastereomers of compound 3, Table S5: Experimental and calculated NMR chemical shift values (ppm) of compound 3 with diastereomers (diastereomers a and b are described in the main text).