E2F8 exerts cancer-promoting effects by transcriptionally activating RRM2 and E2F8 knockdown synergizes with WEE1 inhibition in suppressing lung adenocarcinoma.

Ribonucleotide reductase (RR) is a rate-limiting enzyme that facilitates DNA replication and repair by reducing nucleotide diphosphates (NDPs) to deoxyribonucleotide diphosphates (dNDPs) and is thereby crucial for cell proliferation and cancer development. The E2F family of transcription factors includes key regulators of gene expression involved in cell cycle control. In this study, E2F8 expression was significantly increased in most cancer tissues of lung adenocarcinoma (LUAD) patients and was correlated with the expression of RRM2 through database and clinical samples analysis. The protein expression of E2F8 and RRM2 were positively correlated with tumor-node-metastasis (TNM) pathological stage, and high expression of E2F8 and RRM2 predicted a low 5-year overall survival rate in LUAD patients. Overexpression and knockdown experiments showed that E2F8 was essential for LUAD cell proliferation, DNA synthesis, and cell cycle progression, which were RRM2-dependent. Reporter gene, ChIP-qPCR, and DNA pulldown-Western blot assays indicated that E2F8 activated the transcription of the RRM2 gene by directly binding with the RRM2 promoter in LUAD cells. Previous studies indicated that inhibition of WEE1 kinase can suppress the phosphorylation of CDK1/2 and promote the degradation of RRM2. We further showed here that the combination of E2F8 knockdown with MK-1775, an inhibitor of WEE1 being evaluated in clinical trials, synergistically suppressed proliferation and promoted apoptosis of LUAD cells in vitro and in vivo. Thus, this study reveals a novel role of E2F8 as a proto-oncogenic transcription activator by activating RRM2 expression in LUAD, and targeting both the transcription and degradation mechanisms of RRM2 could produce a synergistic inhibitory effect for LUAD treatment in addition to conventional inhibition of RR enzyme activity.

The TGFß1-FOXM1-HMGA1-TGFß1 positive feedback loop increases the cisplatin resistance of non-small cell lung cancer by inducing G6PD expression.

Platinum-based chemotherapy is still widely applied for the treatment of advanced non-small cell lung cancer (NSCLC). However, acquired chemoresistance compromises the curative effect of this drug. In this study, we found that glucose-6-phosphate dehydrogenase (G6PD), a critical enzyme of the pentose phosphate pathway, contributed to cisplatin resistance in NSCLC. The experimental results showed that transforming growth factor beta 1 (TGFß1) increased the expression of G6PD by activating the forkhead box protein M1-high mobility group AT-hook 1-G6PD (FOXM1-HMGA1-G6PD) transcriptional regulatory pathway, in which TGFß1 inhibited the ubiquitination and degradation of FOXM1 protein. Additionally, HMGA1 induced TGFß1 expression, and neutralized TGFß1 in the culture medium downregulated HMGA1 levels, suggesting the existence of a TGFß1-FOXM1-HMGA1-TGFß1 positive feedback loop and its role in maintaining G6PD expression. Further investigations showed that exogenous TGFß1 enhanced the cisplatin resistance of NSCLC cells, while disrupting the FOXM1-HMGA1-G6PD pathway, thereby sensitizing the cells to cisplatin. Consistently, the TGFß1-FOXM1-HMGA1-G6PD axis was confirmed in NSCLC tissues, and overactivation of this axis predicted poor survival in NSCLC patients. Collectively, the results of this study demonstrate that the TGFß1-FOXM1-HMGA1-TGFß1 positive feedback loop plays a crucial role in the cisplatin resistance of NSCLC by upregulating the expression of G6PD, providing a potential therapeutic target to restore chemosensitivity in cisplatin-resistant NSCLC.

CREB1 directly activates the transcription of ribonucleotide reductase small subunit M2 and promotes the aggressiveness of human colorectal cancer.

As the small subunit of Ribonucleotide reductase (RR), RRM2 displays a very important role in various critical cellular processes such as cell proliferation, DNA repair, and senescence, etc. Importantly, RRM2 functions like a tumor driver in most types of cancer but little is known about the regulatory mechanism of RRM2 in cancer development. In this study, we found that the cAMP responsive element binding protein 1 (CREB1) acted as a transcription factor of RRM2 gene in human colorectal cancer (CRC). CREB1 directly bound to the promoter of RRM2 gene and induced its transcriptional activation. Knockdown of CREB1 decreased the expression of RRM2 at both mRNA and protein levels. Moreover, knockdown of RRM2 attenuated CREB1-induced aggressive phenotypes of CRC cells in vitro and in vivo. Analysis of the data from TCGA database and clinical CRC specimens with immunohistochemical staining also demonstrated a strong correlation between the co-expression of CREB1 and RRM2. Decreased disease survivals were observed in CRC patients with high expression levels of CREB1 or RRM2. Our results indicate CREB1 as a critical transcription factor of RRM2 which promotes tumor aggressiveness, and imply a significant correlation between CREB1 and RRM2 in CRC specimens. These may provide the possibility that CREB1 and RRM2 could be used as biomarkers or targets for CRC diagnosis and treatment.

Ribonucleotide reductase large subunit M1 plays a different role in the invasion and metastasis of papillary thyroid carcinoma and undifferentiated thyroid carcinoma.

Ribonucleotide reductase (RR) has been reported to be associated with several types of cancer while the expression and role of RR in thyroid carcinoma (TC) has not been investigated. Here, we first examined the expression level of three RR subunit proteins (RRM1, RRM2, and RRM2B) in papillary thyroid carcinoma (PTC) and undifferentiated thyroid carcinoma (UTC) patient samples by immunohistochemistry. The results showed that RRM1 was higher expressed in 95.2 % cancer tissues compared with their adjacent normal tissues in 146 PTC samples. The expression level of RRM1 was positively correlated with T stage, lymph node metastasis (LNM), extrathyroidal invasion (ETI), and TNM stage in PTC patients. However, in 12 UTC samples, RRM1 expression was negatively expressed in six cases. To further determine the biological role of RRM1 in TC, ectopic expression or siRNA-mediated knockdown of RRM1 were carried out in the high-differentiated thyroid carcinoma cell line TPC-1 and the poor-differentiated thyroid carcinoma cell line SW579, respectively. In TPC-1 and SW579 cells, overexpression and siRNA knockdown of RRM1 demonstrated that RRM1 promoted DNA synthesis and proliferation in both cell lines as shown by EdU incorporation and cell viability assays. However, RRM1 enhanced cell migration and invasion in TPC-1 cells but inhibited that in SW579 cells as shown by wound healing and transwell assays. Moreover, we also found that RRM1 promoted PTEN expression and reduced Akt phosphorylation in a RR-activity-independent manner in the low-differentiated TC cells but not in the high-differentiated TC cells. In contrast, RRM2 expression was higher expressed in both PTC and UTC patient samples, consisting with its oncogenic role in other cancers. Therefore, we suggest that RRM1 promotes thyroid carcinoma proliferation as a component of RR but may play a different role in the invasion and metastasis of differently differentiated thyroid carcinomas through a non-RR pathway, which could be meaningful to precision treatment of thyroid carcinoma with RR inhibitors.

E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2.

As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease.