ANXA7 GTPase; AMPK/mTORC1/STAT3

SEC (20 µM) prevents PC3 prostate cancer cells and HEK 293T RKIP−/− cells from migrating [1]. The ANXA7 GTPase-specific inhibitor ABO reverses the considerable increase in AMPK phosphorylation in PC3 cells caused by SEC (20 µM), suggesting that activated ANXA7 with increased phosphorylation levels promotes AMPK phosphorylation (cell-selective) [1].
SEC specifically induces apoptosis in tumor cells with high expression of ITGB4 by promoting ITGB4 nuclear translocation. [2]

In the PC-3M-Luc orthotopic implantation nude mouse model, SEC (3 mg/kg/day or 18 mg/kg/day) prevents metastases [1].
SEC suppressed metastasis in PC-3M-Luc orthotopic implantation nude mice model[1]
Next, we evaluated in vivo efficacy of SEC by monitoring orthotopic prostate cancer metastasis in mice. Mice receiving orthotopic inoculation of PC-3M-Luc were injected with SEC (3 mg/kg/day or 18 mg/kg/day) for 3 weeks. Importantly, SEC decreased the bioluminescent signals expressed as photon counts (Fig. 7, A and B). The lymph node is a frequent metastatic site for prostate cancer. Then, aortic lymph nodes were isolated and followed by bioluminescence in vitro imaging. PC-3M-Luc cells were able to spread to distant organs, as indicated by the presence of lymph node metastasis. Notably, SEC compromised bioluminescent signals of metastatic lymph nodes and reduced the number of aortic lymph node with prostate cancer metastasis (Fig. 7, C to E). Meanwhile, SEC treatment did not affect the body weight of the mice (Fig. 7F). Consistently, SEC injection indeed decreased the mRNA level of CCL2, APLN and IL6ST in implanted tumors (Fig. 8). These in vivo observations indicate that SEC suppresses the lymph node metastatic capacities of PC-3M-Luc cells in the nude mice model.[1]

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SEC inhibited the growth of human tumor xenografts in an avian embryo model[2]
Next, we determined whether SEC had apoptosis-promoting effects in vivo. Because of the immune-deficient environment and the dense capillary network, the chick embryo chorioallantoic membrane (CAM) is widely used for tumor engraftment to evaluate the efficacy of anticancer drugs and for the action of proangiogenic or antiangiogenic factors. Therefore, we used the CAM model to study the action of SEC in tumor growth as well as normal angiogenesis. A549 cells were deposited on the CAM and formed a solid tumor within 2 days. The solid adenocarcinoma was locally treated from day 2 to 8 with PBS or PBS containing SEC every 2 days. Treatment with PBS plus SEC substantially inhibited tumor growth as compared with PBS alone (Figure 6A). 5FU treatment was beneficial in reducing the size of xenografts (Supplementary Figure 6). TUNEL assay of frozen sections of tumors revealed that SEC effectively promoted apoptosis in vivo, and an even stronger signal was detected in the peritumoral edge (Figure 6B). To determine whether SEC disturbed normal angiogenesis accompanied by suppressing tumor growth, we directly measured the angiogenic action of SEC on CAM. SEC had no effect on CAM normal angiogenesis (Figure 6C). Fully consistent with the in vivo angiogenesis studies, SEC did not interfere with the formation of capillary-like tube structures with serum and FGF-2 in vitro Matrigel assay (Figure 6D). Therefore, SEC effectively inhibited tumor growth in vivo by inducing apoptosis with no adverse effects on normal CAM angiogenesis.

In vitro ANXA7 activity assay[2]
The coding region of human wide type ANXA7 and ANXA7-mt1 (T275A) and -mt2 (T286A) mutants were subcloned into the mCherry-N1 expression vector with a 6*His tag. When the density of HEK293 plated onto 10-cm dishes reached 70% to 80% confluence, cells were transfected with the indicated expression vector. At 24 h after transection, total proteins were harvested, and expressed recombinant ANXA7 was extracted and purified by column chromatography by use of the His GraviTrap Ni-NTA protein purification assay kit. Purified ANXA7 was incubated at 37°C with or without SEC for indicated concentrations and times. The GTPase activity of ANXA7 was measured with use of the ATPase/GTPase Activity Assay Kit.

Cell viability assay[1]
Cell Types: HEK293T RKIP−/− cells. [1]
Tested Concentrations: 20 µM.
Incubation Duration: 24 hrs (hours).
Experimental Results: Inhibited the migration of HEK293T RKIP−/− cells, but had no effect on HEK293T RKIP+/+ cells.

Animal/Disease Models: Luciferase-labeled PC-3M-Luc cells (2 × 106 cells per 50 µL sterile HBSS−/−) were in situ inoculated into the prostate of 8weeks old nude mice [1].
Doses: 3 mg/kg/day or 18 mg/kg/day.
Doses: Daily IP for 3 weeks.
Experimental Results: Inhibited the lymph node metastasis ability of PC-3M-Luc cells in nude mouse model. Decreases the mRNA levels of CCL2, APLN and IL6ST in implanted tumors. Does not affect mouse body weight.

[1]. SEC-induced Activation of ANXA7 GTPase Suppresses Prostate Cancer Metastasis. Cancer Lett. 2018 Mar 1;416:11-23.

[2]. A small molecule induces integrin β4 nuclear translocation and apoptosis selectively in cancer cells with high expression of integrin β4. Oncotarget. 2016 Mar 29; 7(13): 16282–16296.

Annexin A7 (ANXA7) is an inhibitory factor in prostate cancer tumorigenesis and metastasis. Activated ANXA7 GTPase promotes apoptosis in prostate cancer cells. However, the role and potential mechanisms of ANXA7 GTPase in prostate cancer metastasis remain unclear. RKIP is a metastasis inhibitor that is downregulated in prostate cancer metastases. The binding of RKIP to its target proteins may inhibit the activation of their interacting proteins. However, the effect of RKIP on ANXA7 GTPase activation is still unknown. This study found that the small molecule SEC ((S)-1-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3-(4-methoxyphenyl)-1H-pyrazole-5-carboxylic acid ethyl ester) can effectively activate ANXA7 GTPase, thereby inhibiting prostate cancer metastasis. Mechanistically, activated ANXA7 promotes AMPK phosphorylation, leading to decreased mTORC1 activity, inhibition of STAT3 nuclear translocation, and downregulation of pro-metastatic genes, including CCL2, APLN, and IL6ST. Conversely, RKIP interacts with ANXA7 and inhibits the activation of ANXA7 GTPase by SEC and its downstream signaling pathways. Notably, in in situ in vivo analysis, SEC treatment inhibited the metastasis of prostate cancer cells. In summary, our findings provide new insights into how prostate cancer metastasis with low RKIP expression is inhibited by SEC-induced activation of ANXA7 GTPase via the AMPK/mTORC1/STAT3 signaling pathway. [1] Elevated integrin β4 (ITGB4) levels are associated with malignant progression in various cancers. However, there are currently no reported selective therapeutic strategies for cancer cells that highly express ITGB4. This paper reports for the first time a chiral small molecule SEC that selectively promotes apoptosis in cancer cells that highly express ITGB4 by inducing nuclear translocation of ITGB4. Intranuclear ITGB4 can bind to the ATF3 promoter region, activating ATF3 expression and thereby upregulating the expression of downstream pro-apoptotic genes. In addition, SEC promotes the binding of annexin A7 (ANXA7) to ITGB4 and enhances the GTPase activity of ANXA7. Activated ANXA7 promotes the nuclear translocation of ITGB4 by triggering phosphorylation of ITGB4 at the Y1494 site. SEC can also inhibit the growth of xenograft tumors in avian embryonic models. We have discovered a small molecule SEC that selectively promotes apoptosis in cancer cells that highly express ITGB4, both in vitro and in vivo, by triggering the binding of ITGB4 to ANXA7, the nuclear translocation of ITGB4, and the expression of pro-apoptotic genes. [2]

C22H23CLN2O5

430.881425142288

430.1295

C, 61.33; H, 5.38; Cl, 8.23; N, 6.50; O, 18.57

1802997-81-4

1802997-81-4;918879-70-6 (recemate);

146681186

Light yellow to yellow ointment

4

1

6

10

30

524

1

O=C(C1=CC(C2=CC=C(OC)C=C2)=NN1C[C@H](O)COC3=CC=C(Cl)C=C3)OCC

OFKGGSVHXHMDDN-KRWDZBQOSA-N

InChI=1S/C22H23ClN2O5/c1-3-29-22(27)21-12-20(15-4-8-18(28-2)9-5-15)24-25(21)13-17(26)14-30-19-10-6-16(23)7-11-19/h4-12,17,26H,3,13-14H2,1-2H3/t17-/m0/s1

ethyl 2-[(2S)-3-(4-chlorophenoxy)-2-hydroxypropyl]-5-(4-methoxyphenyl)pyrazole-3-carboxylate

Sec; 1802997-81-4; ethyl 2-[(2S)-3-(4-chlorophenoxy)-2-hydroxypropyl]-5-(4-methoxyphenyl)pyrazole-3-carboxylate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~232.08 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.80 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.80 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.80 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)