Numerous epidemiological and experimental studies provided evidence linking obesity to an increased risk of developing different malignancies, including breast, colorectal, prostate and endometrial cancers [76
]. In addition, calorie-rich diet has been shown to induce inflammatory responses in microglia cells, which potentially can promote development of brain neoplasms [80
In obese individuals, especially in those with high visceral fat content, adiponectin levels are low [11
]. According to epidemiological studies, low adiponectin levels are associated with elevated cancer risk and development of more aggressive neoplasms [4
]. How exactly adiponectin might prevent or restrict cancer is yet not clear. The relevant mechanisms could involve activation of intracellular metabolic changes similar to those produced by calorie restriction, i.e., stimulation of intracellular signals, such as AMPK, and inhibition of abnormal growth and survival pathways [11
]. Thus, pharmacological activation of adiponectin signaling in obese individuals that are refractory to lifestyle modifications could help to restore beneficial pathways normally controlled by this adipokine.
However, development of the whole adiponectin protein as a drug is difficult because of the extreme insolubility of the C-terminal globular domain and its larger peptide fragments. In addition, until now, the adiponectin active site has not been mapped. Consequently, we attempted to generate small peptides that would produce biological effects similar or superior to that of gAd, but would be suitable for pharmaceutical modifications.
First, using peptide arrays and biological screening assays, we mapped the adiponectin active site to amino acids 149-166 within the globular domain of the whole adipokine (Figure ). In parallel experiments, we found that peptides covering the active site displayed high affinity to an extended version of the AdipoR1 loop 1 (sequence: Arg-Pro-Asn-Met-Tyr-Fen-Met-Ale-Pro-Leu-Gln-Glu-Lys-Val-Val) that shares 86% homology with the loop 1 in AdipoR2. Further modifications of the active site, followed by structure-function screening resulted in the development of the lead peptidomimetic, ADP 355, as optimal AdipoR agonist.
The identified active site of adiponectin can be characterized as a turn region followed by a β-pleated sheet fragment (Figure ). When removed from the protein environment, MD studies indicated that the isolated native peptide 25 loses the β-pleated sheet character and forms a series of turns (Figure ). During MD simulations, the initial turn- β-sheet structures of both peptide 25 and ADP 355 peptides were substantially changed and showed high flexibility. The backbone RMSD values fluctuated with high frequency between 0.1 and 0.7 mm. However, in the case of ADP 355, from 80 ns to 250 ns, the RMSD remained around 0.6 nm, indicting that the peptidomimetic folded into a more stable conformation characterized by a hairpin incorporating almost the entire peptide. In the cluster analysis, the most populated cluster of the peptidomimetic contained more than twice as many structures as the native fragment (31.6% vs 12.4%). If the dominant β-hairpin structure is indeed the active conformation, the significantly increased population of this conformer can explain the improved in vitro activity of ADP 355 relative to that of its precursor peptide 25.
ADP 355 energy analysis. Representative energy minimized structures of peptide 25 (red) and ADP 355 (purple) overlaid to the conformation of the 153-162 sequence found in adiponectin protein (grey).
Functional assays with ADP 355 demonstrated that the peptide restricts cancer cell proliferation in a dose-dependent manner at 100 nM-10 μM concentrations. In all studied cell lines, this growth inhibition was superior to that obtained with gAd (Figure ). Cytostatic activity of ADP 355 is in agreement with several other reports showing similar effects of adiponectin or gAd in cancer models [26
]. However, some studies failed to demonstrate any anti-neoplastic activity of this adipokine [54
]. These discrepancies likely reflect differences in experimental design as well as cell context, including differential levels of AdipoR1/2 and signaling proteins. Indeed, our work clearly suggests that the levels of AdipoR1 and AdipoR2 vary among cell lines. Some previous reports suggested that cytostatic effects of adiponectin in breast cancer cells are primarily mediated through AdipoR1 [51
], and our results with AdipoR2-negative cells and AdipoR2-knockdown cells confirm this notion.
Our signaling studies further confirmed that cell response to adiponectin or its derivatives may be cell-specific. We demonstrated that cytostatic effects ADP 355 coincided with the modulation of specific adiponectin signals that have been associated with growth or survival control, i.e., AMPK, Akt, ERK1/2, and STAT3. Interestingly, the major metabolic adiponectin pathway--AMPK was transiently induced only in MCF-7 cells, while in MDA-MB-231 and LN18 cells, the peptide or gAd did not have any effects (Figure ).
In MCF-7 cells, ADP 355, but not gAd, decreased ERK1/2 signaling. STAT3 was activated in this cell line by both ADP 355 and gAd. In MDA-MB-231 cells, like in MCF-7 cells, ADP 355 decreased ERK1/2 activation and transiently increased STAT3 signaling. In both breast cancer cell lines, ADP 355 did not affect the major growth/survival Akt pathway. In contrast, ADP 355 and gAd significantly inhibited Akt and STAT3 signals in LN18 cells. Interestingly, the effects on Akt concerned total levels of the enzyme, suggesting that ADP 355 might affect its turnover.
Published data on adiponectin signaling in cancer cells seem to support the notion that the cytokine might induce different signaling pathways in different cell lines. For instance, in many cancer cell lines (breast MCF-7, MDA-MB-231, T47D; colorectal HT-29, CaCO2, SW480; prostate PC3) adiponectin activated AMPK [26
]. On the other hand, adiponectin either reduced or did not affect ERK1/2 in MCF-7 or MDA-MB-231 cells, but stimulated the pathway in some colorectal cancer cell lines [1
]. Akt was inhibited by adiponectin in MDA-MB-231 breast cancer cells, but activated in prostate cancer cells LNCaP [82
]. The upregulation of AMPK and reduction of Akt in response to adiponectin in MDA-MB-231 cells [82
] is in contrast with our study and might be related to significantly lower gAd and ADP 355 concentrations used in our experiments, while high doses used by Kim et al. were toxic in our system. Consistent with our results, moderate STAT3 stimulation by adiponectin was noted in MDA-MB-231 cells, while the transcription factor was inhibited in DU145 prostate cancer cells [1
]. These differences, in part, could reflect variable experimental settings, such as baseline growth conditions, adiponectin reagents used as well as treatment timing and dosage.
To further assess the efficacy of ADP 355, we carried out a preliminary in vivo study. In scid mice carrying MCF-7 orthotopic xenografts, ADP 355 treatment reduced the growth of established tumors by ~31%, validating AdipoR as a target for breast cancer therapy.