Small Molecule Cjoc42 Improves Chemo-Sensitivity and Increases Levels of Tumor Suppressor Proteins in Hepatoblastoma Cells and in Mice by Inhibiting Oncogene Gankyrin

Abstract

Objective: Relapsed hepatoblastoma (HBL) and upfront hepatocellular carcinoma (HCC) are notoriously chemoresistant tumors associated with poor outcomes. Gankyrin (Gank) is a known oncogene that is overexpressed in pediatric liver cancer and implicated in chemo-resistance. The goal of this study was to evaluate if the Gank-tumor suppressor axis is activated in chemoresistant hepatoblastoma patients and examine if an inhibitor of Gank, Cjoc42, might improve the chemosensitivity of cancer cells.Methods: Expression of Gank and its downstream targets were examined in fresh human HBL samples using immunostaining, QRT-PCR, and Western Blot. Cancer cells, Huh6 (human HBL) and Hepa1c1c7 (mouse HCC) were treated with Cjoc42 and with Cjoc42 in combination with cisplatin or doxorubicin. Cell proliferation, apoptosis, and chemoresistance were examined. To examine activities of Cjoc42 in vivo, mice were treated with different doses of Cjoc42, and biological activities of Gank and cytotoxicity of Cjoc42 were tested.Results: Elevation of Gank and Gank-mediated elimination of TSPs are observed in patients with minimal necrosis after chemotherapy and relapsed disease. The treatment of Huh6 and Hepa1c1c7 with Cjoc42 was not cytotoxic; however, in combination with cisplatin or doxorubicin, Cjoc42 caused a significant increase in cytotoxicity compared to chemotherapy alone with increased apoptosis. Examination of Cjoc42 in WT mice showed that Cjoc42 is well tolerated without systemic toxicity, and levels of tumor suppressors CUGBP1, Rb, p53, C/EBPα, and HNF4α are increased by blocking their Gank-dependent degradation.Conclusions: Our work shows that Cjoc42 might be a promising adjunct to chemotherapy for the treatment of severe pediatric liver cancer and presents mechanisms by which Cjoc42 increases chemo-sensitivity.

Small-Molecule Gankyrin Inhibition as a Therapeutic Strategy for Breast and Lung Cancer

“…To determine that compounds 51b and 51d manifest their anti-proliferative activity against MDA-MB-231 cells through gankyrin inhibition, western blot analyses were determined (Figure B,D). Treatment with both compounds resulted in an increase in Rb levels, while gankyrin levels remained relatively unchanged, which agrees with the previous reports of cjoc42-treated liver cancer cells . This suggests that compounds 51b and 51d inhibit gankyrin and disrupt the proteasomal degradation pathway in a similar manner to that observed for cjoc42. , Additionally, treatment with compounds 51b and 51d resulted in relatively unchanged levels of p53.…”

“…Treatment with both compounds resulted in an increase in Rb levels, while gankyrin levels remained relatively unchanged, which agrees with the previous reports of cjoc42-treated liver cancer cells . This suggests that compounds 51b and 51d inhibit gankyrin and disrupt the proteasomal degradation pathway in a similar manner to that observed for cjoc42. , Additionally, treatment with compounds 51b and 51d resulted in relatively unchanged levels of p53. This result was most likely due to the high expression levels of mutant p53 in MDA-MB-231 cells, which does not possess the typical tumor suppressor activity of wild-type p53. , Additionally, compound 51d treatment of HuH6 cells (Figure S1) resulted in increased levels of both p53 and Rb, further suggesting its ability to disrupt the proteasomal degradation pathway.…”

“…Specifically, Rb controls the expression of various genes ( e.g. , E2F transcription factor family, topoisomerase IIα, and cyclin E) required for progression into the S phase of the cell cycle, thereby gatekeeping cell proliferation and growth. , Additionally, p53 plays a critical role in maintaining the G1 to S phase checkpoint, and degradation of p53 inhibits the DNA damage response, causing an increase in cell proliferation. , Gankyrin’s regulation of multiple TSPs lends itself as an important regulator of the G0/G1 and S phases of the cell cycle. , Studies have shown that decreased gankyrin expression levels cause an increase in intracellular TSP levels, which has been associated with reduced cancer cell growth, , illustrating gankyrin’s promise as a therapeutic target. Therefore, inhibiting gankyrin’s ability to facilitate the degradation of various TSPs will arrest the cell cycle and decrease cancer cell proliferation.…”

“…16,17 Gankyrin's regulation of multiple TSPs lends itself as an important regulator of the G0/G1 and S phases of the cell cycle. 7,18 Studies have shown that decreased gankyrin expression levels cause an increase in intracellular TSP levels, which has been associated with reduced cancer cell growth, 19,20 illustrating gankyrin's promise as a therapeutic target. Therefore, inhibiting gankyrin's ability to facilitate the degradation of various TSPs will arrest the cell cycle and decrease cancer cell proliferation.…”

CELF Family Proteins in Cancer: Highlights on the RNA-Binding Protein/Noncoding RNA Regulatory Axis

“…Dephosphorylated CELF1 was shown to be degraded by Gank (Gankyrin oncogene). The Cjoc42, a tiny Gank-inhibiting compound, stimulates CELF1 expression, improves chemo-sensitivity, and reduces liver cancer cell proliferation in vitro [70,159]. Another natural product, Fraxinellone, notably inhibits the mRNA expression of CELF1 in models of kidney and liver fibrosis [160].…”