TMEPAI genome editing in triple negative breast cancer cells

Wardhani, Bantari Wisynu Kusuma; Puteri, Meidi Utami; Watanabe, Yuji; Louisa, Melva; Setiabudy, Rianto; Kato, Mitsuyasu

doi:10.13181/mji.v26i1.1871

Cited by 6 publications

(5 citation statements)

References 14 publications

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“…Construction of TMEPAI guide RNA expression vectors and establishment of TMEPAI KO cell lines were described previously (Amalia et al, 2019; Wardhani et al, 2017). Knockout (KO) cell lines which are negative for all isoforms of TMEPAI were established from Hs578T and BT‐549 cells using a CRISPR/Cas9 system.…”

Section: Methodsmentioning

confidence: 99%

PMEPA1/TMEPAI isoforms function via its PY and Smad‐interaction motifs for tumorigenic activities of breast cancer cells

et al. 2020

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PMEPA1 (prostate transmembrane protein, androgen-induced 1)/TMEPAI (transmembrane prostate androgen-induced protein) is highly expressed in diverse cancers, including breast, lung and prostate cancers. It consists of four isoforms with distinct extracellular regions (isoforms a-d). The expression and function of these isoforms are still poorly understood. Hence, we aimed to identify the preferentially expressed isoforms in breast cancer cells and analyze possible differences in tumorigenic functions. In this study, we used 5′ Rapid Amplification of cDNA Ends (RACE) and Western blot analyses to identify the mRNA variants and protein isoforms of TMEPAI and found that TMEPAI isoform d as the major isoform expressed by TGF-β stimulation in breast cancer cells. We then generated CRISPR/ Cas9-mediated TMEPAI knockout (KO) breast cancer cell lines and used a lentiviral expression system to complement each isoform individually. Although there were no clear functional differences between isoforms, double PPxY (PY) motifs and a Smad-interaction motif (SIM) of TMEPAI were both essential for colony and sphere formation. Collectively, our results provide a novel insight into TMEPAI isoforms in breast cancer cells and showed that coordination between double PY motifs and a SIM of TMEPAI are essential for colony and sphere formation but not for monolayer cell proliferation. K E Y W O R D Sbreast cancer, isoform, PY motifs, SIM, TMEPAI |Genes to Cells PUTERI ET al.

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Section: Methodsmentioning

confidence: 99%

PMEPA1/TMEPAI isoforms function via its PY and Smad‐interaction motifs for tumorigenic activities of breast cancer cells

et al. 2020

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“…SDS-PAGE was done on cell lysates using the method as mentioned previously. 25 The primary antibodies were rabbit anti-TMEPAI antibodies (homemade, 1:250), 4 rabbit anti-GFP antibodies (Santa Cruz Biotechnology, 1:2000), and mouse anti-β-actin antibody (Sigma, 1:5000). Primary antibodies were bounded with horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare, 1:10,000) and anti-mouse IgG (GE Healthcare, 1:10,000).…”

Section: Sds-page and Western Blottingmentioning

confidence: 99%

<p>TGF-β-Induced TMEPAI Attenuates the Response of Triple-Negative Breast Cancer Cells to Doxorubicin and Paclitaxel</p>

Wardhani¹,

Puteri²,

Watanabe³

et al. 2020

JEP

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Purpose: Triple-negative breast cancer (TNBC) is a refractory type of breast cancer with poor prognosis and limited choice for treatment. Previous studies had shown that TNBC has high expressions of transmembrane prostate androgen-induced protein (TMEPAI). TMEPAI was known to be induced by TGF-β/Smad signaling and have tumorigenic functions that converting TGF-β from tumor suppressor to tumor promoter and inducing epithelialmesenchymal transition (EMT). Therefore, we aimed to define the role of TMEPAI in triple-negative breast cancer cells treatment using several anti-cancers in the presence of TGF-β. Methods: TMEPAI-knock out (KO) was carried out in a triple-negative breast cancer cell, BT549. TMEPAI editing was developed using the CRISPR-Cas9 system using two combinations of sgRNA to remove exon 4 of the TMEPAI gene entirely. Genotyping and proteomic analysis were performed to check the establishment of the TMEPAI-KO cells. Wild type (WT) and KO cells were used to determine inhibitory concentration 50% (IC 50) of several anti-cancers: doxorubicin, cisplatin, paclitaxel, and bicalutamide in the presence of TGF-β treatment. Results: KO cells were successfully established by completely removing the TMEPAI gene, which was proven in genomic and proteomic analysis. Further, in TMEPAI-KO cells, we found a significant reduction of IC 50 for doxorubicin and paclitaxel, and minimal effects were seen for cisplatin and bicalutamide. Our findings suggest that TGF-β-induced TMEPAI attenuates the response of TNBC to doxorubicin and paclitaxel, but not to cisplatin and bicalutamide. Conclusion: TGF-β induced TMEPAI contributes to the reduced response of TNBC treatment to doxorubicin and paclitaxel, but minimal on cisplatin and bicalutamide. Further study is needed to confirm our findings in other growth factor-induced cells, as well as in in vivo model.

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“…Hs578T TMEPAI knockout (KO) cells were previously established from Hs578T wild-type (WT) using a CRISPR-Cas9 system. 16 Both WT and KO Hs578T were cultured in Dulbecco's modified essential medium (Invitrogen) supplemented with 10% fetal bovine serum (Gibco), 10 µg/ml insulin, 100 units/ml penicillin G, and 0.1 mg/ml streptomycin sulfate (Wako). Cells were maintained in a 5% CO 2 incubator at 37°C.…”

Section: Cell Culturementioning

confidence: 99%

Decreased sensitivity of several anticancer drugs in TMEPAI knockout triple-negative breast cancer cells

et al. 2019

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BACKGROUND Transmembrane prostate androgen-induced protein (TMEPAI) was reported to be highly amplified in the majority of patients with triple-negative breast cancer (TNBC). TMEPAI is related to poorer prognosis, limited treatment options, and prone to drug resistance compared with other proteins. One of the established markers to determine cancer resistance to drugs is the increased expression levels of drug efflux transporters. However, the role of TMEPAI in cancer resistance to drugs has not been elucidated. This study was aimed to investigate whether TMEPAI participates in cancer resistance to drugs by regulating drug efflux transporters. METHODS TMEPAI knockout (KO) cells were previously developed from a TNBC cell line, Hs578T (wild-type/WT), using a CRISPR-Cas9 system. The expression levels of drug efflux transporters were determined in Hs578T-KO and Hs578-WT by quantitative reverse transcriptase polymerase chain reaction. Cytotoxic concentration 50% (CC50) of several anticancer drugs (doxorubicin, cisplatin, and paclitaxel) were determined in the two cell lines via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. RESULTS The results showed that the mRNA expression of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) was significantly increased in Hs578T-KO compared with that in Hs578T-WT cells. CC50 of several anticancer drugs investigated (doxorubicin, paclitaxel, and cisplatin) in Hs578T-KO cells was higher than that in Hs678-WT. CONCLUSIONS TMEPAI participated in the regulation of mRNA expression levels in drug efflux transporters (P-gp, BCRP, and multidrug resistance-associated protein 1). Further studies are necessary to confirm whether this finding might be dependent on the development of cancer cell sensitivity to anticancer agents.

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TMEPAI genome editing in triple negative breast cancer cells

Cited by 6 publications

References 14 publications

PMEPA1/TMEPAI isoforms function via its PY and Smad‐interaction motifs for tumorigenic activities of breast cancer cells

PMEPA1/TMEPAI isoforms function via its PY and Smad‐interaction motifs for tumorigenic activities of breast cancer cells

<p>TGF-β-Induced TMEPAI Attenuates the Response of Triple-Negative Breast Cancer Cells to Doxorubicin and Paclitaxel</p>

Decreased sensitivity of several anticancer drugs in TMEPAI knockout triple-negative breast cancer cells

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