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 a suppressor of tumorigenesis and metastasis in prostate cancer. Activated ANXA7 GTPase promotes prostate cancer cell apoptosis. However, the role and underlying mechanism of ANXA7 GTPase in prostate cancer metastasis have not been established. RKIP is a metastatic suppressor and downregulated in prostate cancer metastases. The binding of RKIP and its target proteins could inhibit the activation of its interactive partners. However, the effect of RKIP on ANXA7 GTPase activation is not clear. Here, we report that activation of ANXA7 GTPase by a small molecule SEC ((S)-ethyl 1-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3- (4-methoxyphenyl)-1H-pyrazole-5-carboxylate) effectively inhibited prostate cancer metastasis. Mechanistically, activated ANXA7 promoted AMPK phosphorylation, leading to decreased mTORC1 activity, suppressed STAT3 nuclear translocation, and downregulation of pro-metastatic genes, including CCL2, APLN, and IL6ST. Conversely, RKIP interacted with ANXA7 and impaired activation of ANXA7 GTPase by SEC and its downstream signaling pathway. Notably, SEC treatment suppressed metastasis of prostate cancer cells in in vivo orthotopic analysis. Together, our findings provide a novel insight into how metastasis of prostate cancer with low RKIP expression is suppressed by SEC-induced activation of ANXA7 GTPase via the AMPK/mTORC1/STAT3 signaling pathway.[1]

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Increased integrin β4 (ITGB4) level is accompanied by malignant progression of multiple carcinomas. However, selective therapeutic strategies against cancer cells expressing a high level of ITGB4 have not been reported. Here, for the first time, we report that a chiral small molecule, SEC, selectively promotes apoptosis in cancer cells expressing a high level of ITGB4 by inducing ITGB4 nuclear translocation. Nuclear ITGB4 can bind to the ATF3 promoter region and activate the expression of ATF3, then upregulate the downstream pro-apoptosis genes. Furthermore, SEC promoted the binding of annexin A7 (ANXA7) to ITGB4 and increased ANXA7 GTPase activity. Activated ANXA7 promoted ITGB4 nuclear translocation by triggering ITGB4 phosphorylation at Y1494. SEC also inhibited the growth of xenograft tumors in the avian embryo model. We identified a small molecule, SEC, with selective pro-apoptosis effects on cancer cells with high expression of ITGB4, both in vitro and in vivo, by triggering the binding of ITGB4 and ANXA7, ITGB4 nuclear trafficking, and pro-apoptosis gene expression.[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.)