Doxorubicin‐induced senescence promotes stemness and tumorigenicity in EpCAM−/CD133− nonstem cell population in hepatocellular carcinoma cell line, HuH‐7

Abstract

The therapeutic induction of senescence is a potential means to treat cancer, primarily acting through the induction of a persistent growth‐arrested state in tumors. However, recent studies have indicated that therapy‐induced senescence (TIS) in tumor cells allows for the prolonged survival of a subgroup of cells in a dormant state, with the potential to re‐enter the cell cycle along with an increased stemness gene expression. Residual cells after TIS with increased cancer stem cell phenotype may have profound implications for tumor aggressiveness and disease recurrence. Herein, we investigated senescence‐associated stemness in EpCAM+/CD133+ liver cancer stem cell and EpCAM−/CD133− nonstem cell populations in HuH7 cell line. We demonstrated that treatment with doxorubicin induces senescence in both cell populations, accompanied by a significant increase in the expression of reprogramming genes SOX2, KLF4, and c‐MYC as well as liver stemness‐related genes EpCAM, CK19, and ANXA3 and the multidrug resistance‐related gene ABCG2. Moreover, doxorubicin treatment significantly increased EpCAM + population in nonstem cells indicating senescence‐associated reprogramming of nonstem cell population. Also, Wnt/β‐catenin target genes were increased in these cells, while inhibition of this signaling pathway decreased stem cell gene expression. Importantly, Dox‐treated EpCAM−/CD133− nonstem cells had increased in vivo tumor‐forming ability. In addition, when SASP‐CM from Dox‐treated cells were applied onto hİPSC‐derived hepatocytes, senescence was induced in hepatocytes along with an increased expression of TGF‐β, KLF4, and AXIN2. Importantly, SASP‐CM was not able to induce senescence in Hep3B‐TR cells, a derivative line rendered resistant to TGF‐β signaling. Furthermore, ELISA experiments revealed that the SASP‐CM of Dox‐treated cells contain inflammatory cytokines IL8 and IP10. In summary, our findings further emphasize the importance of carefully dissecting the beneficial and detrimental aspects of prosenescence therapy in HCC and support the potential use of senolytic drugs in HCC treatment in order to eliminate adverse effects of TIS.

Changes in Wnt and TGF-β Signaling Mediate the Development of Regorafenib Resistance in Hepatocellular Carcinoma Cell Line HuH7

“…The plasmids used in TCF/LEF reporter assay were a gift from Dr. Hans Clevers at Hubrecht Institute. Transfections were performed as previously described ( Karabicici et al, 2021 ) using Lipofectamine 2000. For TCF/LEF reporter assays, 24 h after transfection, cells were treated with Regorafenib (5 or 10 μM), Wnt3a/R-Spo1, IWR-1(10 μM) or a combination of these molecules.…”

“…Immunofluorescence stainings were performed as previously described ( Karabicici et al, 2021 ), Beta-catenin (BD, 610153)(1:50), p-Beta-catenin (S675)(4176)(1:50), Cytokeratin 19 (sc-6278)(1:50), E-Cadherin (sc8426)(1:50), EpCAM (2929S)(1:50), Vimentin (sc-373717)(1:250), alpha-SMA (ab21027)(1:50), p-Smad2 (S255)(ab188334)(1:50), t-Smad2 (ab40855)(1:50) and Phalloidin-iFluor 488 (ab176753) antibodies were used for the stainings. Cells were visualized using the Confocal LSM 800 microscope.…”

“…Real-time q-PCR experiments were performed as previously described ( Karabicici et al, 2021 ). using7500 Fast RT PCR System (Applied Biosystems).…”

“…Flow cytometry experiments were performed as previously described ( Karabicici et al, 2021 ). For all cell lines, 2 × 10 6 cells were seeded into a 10 cm dish 1 day before the experiment.…”

“…β-galactosidase assay was performed as previously described ( Karabicici et al, 2021 ). Briefly, cells were seeded on the six well plates (7 × 10 4 ) or 12 well plates (2 × 10 4 ) 1 day before treatment.…”