Induction of cellular senescence in cancerous cells is an important strategy which is used in the treatment of cancer. However, cancer cells are capable of exhibiting resistance to cellular ...senescence through inactivation of tumor suppressors. Because of this, establishment of a route to cellular senescence induction in cancer cells is a crucial direction for developing future cancer therapies. In this study, we demonstrate the involvement of TSC-22 homologous gene-1 (THG-1, also called TSC22D4) in the suppression of cellular senescence. CRISPR/Cas9 gene editing was used to establish THG-1 knockout (KO) cells in a THG-1 positive esophageal tumor cell line. It was found that THG-1 KO cells exhibited delayed cell proliferation as well as cellular senescence. The elevated expression of the CDK inhibitor P21(CDKN1A) was also identified in senescent cells. Through the investigation of the upstream pathway for induction of P21(CDKN1A), the JUNB pathway was identified to play a critical role in P21(CDKN1A) transcription; in fact, the siRNA-mediated knockdown of JUNB reduced the abundance of P21(CDKN1A) mRNA and cellular senescence in THG-1 KO cells. These findings provide a novel insight into the induction of cellular senescence in THG-1 positive cancer cells.
•Senescence is one of the important tumor suppressor mechanisms that limit cancer initiation and progression.•The establishment of a route to cellular senescence in cancer cells is thought to be crucial for future cancer therapy.•THG-1 (TSC-22 homologous gene-1, also called TSC22D4) involves in suppression of cellular senescence.•THG-1 knockout cells exhibited the retard cell proliferation and cellular senescence.•Activation of JUNB-P21(CDKN1A) axis plays a crucial role on cellular senescence in THG-1 knockout cells.
Prostate transmembrane protein androgen-induced 1 (PMEPA1)/transmembrane prostate androgen-induced protein (TMEPAI), a direct target and a negative regulator of transforming growth factor beta ...signalling, has an oncogenic role in many cancers. We observed that knockout (KO) of PMEPA1 in human breast cancer cell line MDA-MB-231 using a CRISPR-Cas9 system resulted in reduction of in vivo tumour growth and lung metastasis but not of in vitro monolayer growth capacity of these KO cell lines. This phenomenon was associated with PMEPA1 KO-mediated downregulation of the key proangiogenic factors vascular endothelial growth factor alpha (VEGFA) and interleukin-8 (IL8) that are essential for in vivo but not in vitro growing cells and are also substantial for initiation of lung metastasis.
Laryngeal squamous cell carcinoma (LSCC), although one of the most common head and neck cancers, has a static or slightly decreased survival rate because of difficulties in early diagnosis, lack of ...effective molecular targeting therapy, and severe dysfunction after radical surgical treatments. Therefore, a novel therapeutic target is crucial to increase treatment efficacy and survival rates in these patients. Glycoprotein NMB (GPNMB), whose role in LSCC remains elusive, is a type 1 transmembrane protein involved in malignant progression of various cancers, and its high expression is thought to be a poor prognostic factor. In this study, we showed that GPNMB expression levels in LSCC samples are significantly higher than those in normal tissues, and GPNMB expression is observed mostly in growth‐arrested cancer cells. Furthermore, knockdown of GPNMB reduces monolayer cellular proliferation, cellular migration, and tumorigenic growth, while GPNMB protein displays an inverse relationship with Ki‐67 levels. Therefore, we conclude that GPNMB may be an attractive target for future LSCC therapy.
In the present study, we showed higher expression levels of GPNMB mRNA in all clinical stages and grades of head and neck squamous cell carcinoma (HNSCC) patients compared with normal tissues and the remarkably higher expression of GPNMB in malignant lesions compared with normal squamous epithelium in laryngeal squamous cell carcinoma (LSCC) clinical samples. Furthermore, we demonstrated the critical roles of GPNMB in 2D monolayer proliferation, cellular migration, 3D sphere growth in vitro, and xenograft tumor formation in vivo in LSCC cell lines. Additionally, we revealed the mutually exclusive expression of GPNMB and Ki‐67 within LSCC cells in the xenografted tumors and clinical samples, suggesting that GPNMB works in Ki‐67–negative, dormant cancer cells.
Three‐dimensional (3D) culturing mimics the heterogeneous cellular conditions of the in vivo tumor microenvironment compared to 2D monolayer‐cultured cells and 3D cultures of established cancer cell ...lines (sphere culture) or patient‐derived cancer cells (organoid culture) are frequently used for cancer research or drug screening and evaluation. To establish more cost and time‐efficient 3D culture methods for cancer cell lines, we supplemented sphere culture medium with polyvinyl alcohol (PVA) and found that 3D sphere cultures of breast and pancreatic cancer cell lines were significantly increased. Mechanistically, we found that PVA prevented cell death and promoted cellular proliferation while maintaining levels of stemness‐related gene expression. Furthermore, we showed that polyvinyl formal resin (PVF) 3D scaffolds made by cross‐linked PVA can function in serum‐free, long‐term 3D cultures to support maintenance of sphere‐ or tumor‐like cell masses for diverse cancer cell types. Taken together, we demonstrate the effectiveness of PVA and PVF in human cancer cell line culture protocols.
In this study, we evaluated the effect of polyvinyl alcohol (PVA) on multiple human cancer cell lines and found that sphere growth was supported by reducing apoptosis and promoting cellular proliferation. We also found that polyvinyl formal resin (PVF) 3D scaffolds made by cross‐linked PVA are useful for long‐term 3D culturing and allow for mass formation even in cell lines that cannot be easily sphere cultured.
c‐MYC stimulates cell proliferation through the suppression of cyclin‐dependent kinase (CDK) inhibitors including P15 (CDKN2B) and P21 (CDKN1A). It also activates E‐box‐mediated transcription of ...various target genes including telomerase reverse transcriptase (TERT) that is involved in cellular immortality and tumorigenesis. Transforming growth factor‐beta 1 (TGF‐β1)‐stimulated clone 22 (TSC‐22/TSC22D1) encodes a highly conserved leucine zipper protein that is induced by various stimuli, including TGF‐β. TSC‐22 inhibits cell growth in mammalian cells and in Xenopus embryos. However, underlying mechanisms of growth inhibition by TSC‐22 remain unclear. Here, we show that TSC‐22 physically interacts with c‐MYC to inhibit the recruitment of c‐MYC on the P15 (CDKN2B) and P21 (CDKN1A) promoters, effectively inhibiting c‐MYC‐mediated suppression of P15 (CDKN2B) and also P21 (CDKN1A) promoter activities. In contrast, TSC‐22 enhances c‐MYC‐mediated activation of the TERT promoter. Additionally, the expression of TSC‐22 in embryonic stem cells inhibits cell growth without affecting its pluripotency‐related gene expression. These results indicate that TSC‐22 differentially regulates c‐MYC‐mediated transcriptional activity to regulate cell proliferation.
Transforming growth factor‐β1‐stimulated clone 22 differentially regulates c‐MYC recruitment to the promoter regions of the CDK inhibitors (P15, P21) and TERT genes.
TMEPAI/PMEPA1 is a transmembrane protein that was originally identified as a prostatic RNA, the synthesis of which is induced by testosterone or its derivatives. We have recently identified TMEPAI as ...a direct target gene of transforming growth factor‐β (TGF‐β)/Smad signaling that participates in negative feedback control of the duration and intensity of TGF‐β/Smad signaling. TMEPAI is constitutively and highly expressed in many types of cancer and is associated with poor prognosis. Here, we report that TMEPAI is highly expressed in the lung adenocarcinoma cell lines Calu3, NCI‐H23, and RERF‐LC‐KJ. Expression of TMEPAI in these cancer cells was significantly suppressed by a TGF‐β receptor kinase antagonist, SB208, and by TGF‐β neutralizing antibodies. These results suggest that constitutive expression of TMEPAI in these cancer cells depends on autocrine TGF‐β stimulation. Knockdown of TMEPAI in Calu3 and NCI‐H23 cells enhanced levels of Smad2 phosphorylation and significantly suppressed cell proliferation in the presence of TGF‐β, indicating that highly expressed TMEPAI suppresses levels of Smad phosphorylation in these cancer cells and reduces the growth inhibitory effects of TGF‐β/Smad signaling. Furthermore, knockdown of TMEPAI in Calu3 and NCI‐H23 cells suppressed sphere formation in vitro and tumor formation in s.c. tissues and in lungs after tail vein injection in NOD‐SCID mice in vivo. Together, these experiments indicate that TMEPAI promotes tumorigenic activities in lung cancer cells.
TMEPAI is constitutively expressed in lung adenocarcinoma cell lines. Knockdown of TMEPAI significantly reduced tumorigenic activities of these cells.
Triple-negative breast cancer (TNBC) is particularly aggressive and difficult to treat. For example, the transforming growth factor-β (TGF-β) pathway is implicated in TNBC progression and metastasis, ...but its opposing role in tumor suppression in healthy tissues and early-stage lesions makes it a challenging target. Therefore, additional molecular characterization of TNBC may lead to improved patient prognosis by informing the development and optimum use of targeted therapies. We found that musculoaponeurotic fibrosarcoma (MAF) oncogene family protein K (MAFK), a member of the small MAF family of transcription factors that are induced by the TGF-β pathway, was abundant in human TNBC and aggressive mouse mammary tumor cell lines. MAFK promoted tumorigenic growth and metastasis by 4T1 cells when implanted subcutaneously in mice. Overexpression of MAFK in mouse breast epithelial NMuMG cells induced epithelial-mesenchymal transition (EMT) phenotypes and promoted tumor formation and invasion in mice. MAFK induced the expression of the gene encoding the transmembrane glycoprotein nmb (GPNMB). Similar to MAFK, GPNMB overexpression in NMuMG cells induced EMT, tumor formation, and invasion, in mice, whereas knockdown of MAFK in tumor cells before implantation suppressed tumor growth and progression.
and
expression correlated with poor prognosis in TNBC patients. These findings suggest that MAFK and its target gene
play important roles in the malignant progression of TNBC cells, offering potentially new therapeutic targets for TNBC patients.
Transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4(+) T helper cells (TH17); yet their signalling network remains largely unknown. ...Here we show that the highly homologous TGF-β receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.
Glycoprotein NMB (GPNMB) is highly expressed in many types of malignant tumors and thought to be a poor prognostic factor in those cancers, including breast cancer. Glycoprotein NMB is a type IA ...transmembrane protein that has a long extracellular domain (ECD) and a short intracellular domain (ICD). In general, the ECD of a protein is involved in protein‐protein or protein‐carbohydrate interactions, whereas the ICD is important for intracellular signaling. We previously reported that GPNMB contributes to the initiation and malignant progression of breast cancer through the hemi‐immunoreceptor tyrosine‐based activation motif (hemITAM) in its ICD. Furthermore, we showed that the tyrosine residue in hemITAM is involved in induction of the stem‐like properties of breast cancer cells. However, the contribution of the ECD to its tumorigenic function has yet to be fully elucidated. In this study, we focused on the region, the so‐called kringle‐like domain (KLD), that is conserved among species, and made a deletion mutant, GPNMB(ΔKLD). Enhanced expression of WT GPNMB induced sphere and tumor formation in breast epithelial cells; in contrast, GPNMB(ΔKLD) lacked these activities without affecting its molecular properties, such as subcellular localization, Src‐induced tyrosine phosphorylation at least in overexpression experiments, and homo‐oligomerization. Additionally, GPNMB(ΔKLD) lost its cell migration promoting activity, even though it reduced E‐cadherin expression. Although the interaction partner binding to KLD has not yet been identified, we found that the KLD of GPNMB plays an important role in its tumorigenic potential.
Glycoprotein NMB (GPNMB) is a poor prognostic factor in many types of cancers. In the present study, we clarified the importance of the kringle‐like domain of GPNMB for its tumorigenic potential.
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