Judy Yan

PKM2 contributes to cancer metabolism

Reprogramming of cell metabolism is essential for tumorigenesis, and is regulated by a complex network, in which PKM2 plays a critical role. PKM2 exists as an inactive monomer, less active dimer and active tetramer. While dimeric PKM2 diverts glucose metabolism towards anabolism through aerobic glycolysis, tetrameric PKM2 promotes the flux of glucose-derived carbons for ATP production via oxidative phosphorylation. Equilibrium of the PKM2 dimers and tetramers is critical for tumorigenesis, and is controlled by multiple factors. The PKM2 dimer also promotes aerobic glycolysis by modulating transcriptional regulation. We will discuss the current understanding of PKM2 in regulating cancer metabolism.

IQGAP2, A candidate tumour suppressor of prostate tumorigenesis.

Loss of IQGAP2 contributes to the tumorigenesis of hepatocellular carcinoma and gastric cancer. However, whether IQGAP2 also suppresses prostate tumorigenesis remains unclear. We report here that IQGAP2 is a candidate tumour suppressor of prostate cancer (PC). Elevated IQGAP2 was detected in prostatic intraepithelial neoplasia (PIN), early stages of PCs (Gleason score ≤3), and androgen-dependent LNCaP PC cells. However, IQGAP2 was expressed at substantially reduced levels not only in prostate glands and non-tumorigenic BPH-1 prostate epithelial cells but also in advanced (Gleason score 4 or 5) and androgen-independent PCs. Furthermore, xenograft tumours that were derived from stem-like DU145 cells displayed advanced features and lower levels of IQGAP2 in comparison to xenograft tumours that were produced from non stem-like DU145 cells. Collectively, these results suggest that IQGAP2 functions in the surveillance of prostate tumorigenesis. Consistent with this concept, ectopic IQGAP2 reduced the proliferation of DU145, PC3, and 293T cells as well as the invasion ability of DU145 cells. While ectopic IQGAP2 up-regulated E-cadherin in DU145 and PC3 cells, knockdown of IQGAP2 reduced E-cadherin expression. In primary PC and DU145 cells-derived xenograft tumours, the majority of tumours with high levels of IQGAP2 were strongly-positive for E-cadherin. Therefore, IQGAP2 may suppress PC tumorigenesis, at least in part, by up-regulation of E-cadherin. Mechanistically, overexpression of IQGAP2 significantly reduced AKT activation in DU145 cells and inhibition of AKT activation upregulated E-cadherin, suggesting that IQGAP2 increases E-cadherin expression by inhibiting AKT activation. Taken together, we demonstrate here that IQGAP2 is a candidate tumour suppressor of PC.

Contactin-1 Reduces E-Cadherin Expression Via Activating AKT in Lung Cancer

Contactin-1 has been shown to promote cancer metastasis. However, the underlying mechanisms remain unclear. We report here that knockdown of contactin-1 in A549 lung cancer cells reduced A549 cell invasion and the cells ability to grow in soft agar without affecting cell proliferation. Reduction of contactin-1 resulted in upregulation of E-cadherin, consistent with E-cadherin being inhibitive of cancer cell invasion. In an effort to investigate the mechanisms whereby contactin-1 reduces E-cadherin expression, we observed that contactin-1 plays a role in AKT activation, as knockdown of contactin-1 attenuated AKT activation. Additionally, inhibition of AKT activation significantly enhanced E-cadherin expression, an observation that mimics the situation observed in contactin-1 knockdown, suggesting that activation of AKT plays a role in contactin-1-mediated downregulation of E-cadherin. In addition, we were able to show that knockdown of contactin-1 did not further reduce A549 cells invasion ability, when AKT activation was inhibited by an AKT inhibitor. To further support our findings, we overexpressed CNTN-1 in two CNTN-1 null breast cancer cell lines expressing E-cadherin. Upon overexpression, CNTN-1 reduced E-cadherin levels in one cell line and increased AKT activation in the other. Furthermore, in our study of 63 primary lung cancers, we observed 65% of primary lung cancers being contactin-1 positive and in these carcinomas, 61% were E-cadherin negative. Collectively, we provide evidence that contactin-1 plays a role in the downregulation of E-cadherin in lung cancer and that AKT activation contributes to this process. In a study of mechanisms responsible for contactin-1 to activate AKT, we demonstrated that knockdown of CNTN-1 in A549 cells did not enhance PTEN expression but upregulated PHLPP2, a phosphatase that dephosphorylates AKT. These observations thus suggest that contactin-1 enhances AKT activation in part by preventing PHLPP2-mediated AKT dephosphrorylation.

Neural Cell Adhesion Protein CNTN1 Promotes the Metastatic Progression of Prostate Cancer

Prostate cancer metastasis is the main cause of disease-related mortality. Elucidating the mechanisms underlying prostate cancer metastasis is critical for effective therapeutic intervention. In this study, we performed gene-expression profiling of prostate cancer stem-like cells (PCSC) derived from DU145 human prostate cancer cells to identify factors involved in metastatic progression. Our studies revealed contactin 1 (CNTN1), a neural cell adhesion protein, to be a prostate cancer-promoting factor. CNTN1 knockdown reduced PCSC-mediated tumor initiation, whereas CNTN1 overexpression enhanced prostate cancer cell invasion in vitro and promoted xenograft tumor formation and lung metastasis in vivo. In addition, CNTN1 overexpression in DU145 cells and corresponding xenograft tumors resulted in elevated AKT activation and reduced E-cadherin (CDH1) expression. CNTN1 expression was not readily detected in normal prostate glands, but was clearly evident on prostate cancer cells in primary tumors and lymph node and bone metastases. Tumors from 637 patients expressing CNTN1 were associated with prostate cancer progression and worse biochemical recurrence-free survival following radical prostatectomy (P < 0.05). Collectively, our findings demonstrate that CNTN1 promotes prostate cancer progression and metastasis, prompting further investigation into the mechanisms that enable neural proteins to become aberrantly expressed in non-neural malignancies.

ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response

DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

Changes in PKM2 Associate with Prostate Cancer Progression

Pyruvate kinase M2 (PKM2) is essential for aerobic glycolysis, the dominant metabolic pathway utilized by cancer cells. To determine the association of PKM2 with prostate cancer (PC), we examined 29 primary PC and three lymph node metastatic tumors; elevation of PKM2 was observed in Gleason 8–10 tumors compared to Gleason 6–7 carcinomas. High PKM2 was detected by immunohistochemistry in more aggressive xenograft tumors derived from PC stem-like cells (PCSCs) compared to those produced from non-PCSCs. While PCSCs and non-PCSCs expressed comparable levels of PKM2, distinct posttranslational modifications were observed. Collectively, upregulation and specific modification to PKM2 associate with PC progression.

Inhibition of ERK activation enhances the repair of double-stranded breaks via non-homologous end joining by increasing DNA-PKcs activation

Non-homologous end joining (NHEJ) is one of the major pathways that repairs double-stranded DNA breaks (DSBs). Activation of DNA-PK is required for NHEJ. However, the mechanism leading to DNA-PKcs activation remains incompletely understood. We provide evidence here that the MEK-ERK pathway plays a role in DNA-PKcs-mediated NHEJ. In comparison to the vehicle control (DMSO), etoposide (ETOP)-induced DSBs in MCF7 cells were more rapidly repaired in the presence of U0126, a specific MEK inhibitor, based on the reduction of γH2AX and tail moments. Additionally, U0126 increased reactivation of luciferase activity, which resulted from the repair of restriction enzyme-cleaved DSBs. Furthermore, while inhibition of ERK activation using the dominant-negative MEK1K97M accelerated the repair of DSBs, enforcing ERK activation with the constitutively active MEK1Q56P reduced DSB repair. In line with MEK activating ERK1 and ERK2 kinases, knockdown of either ERK1 or ERK2 increased DSB repair. Consistent with the activation of DNA-PKcs being required for NHEJ, we demonstrated that inhibition of ERK activation using U0126, MEK1K97M, and knockdown of ERK1 or ERK2 enhanced ETOP-induced activation of DNA-PKcs. Conversely, enforcing ERK activation by MEK1Q56P reduced ETOP-initiated DNA-PKcs activation. Taken together, we demonstrate that ERK reduces NHEJ-mediated repair of DSBs via attenuation of DNA-PKcs activation.

Upregulation of FAM84B during prostate cancer progression

Although the FAM84B gene lies within chromosome 8q24, a locus frequently altered in prostate cancer (PC), its alteration during prostate tumorigenesis has not been well studied. We report here FAM84B upregulation in DU145 cell-derived prostate cancer stem-like cells (PCSLCs) and DU145 cell-produced lung metastases compared to subcutaneous xenograft tumors. FAM84B protein was detected in bone metastases and primary PCs. Nanostring examination of 7 pairs of tumor adjacent normal and PC tissues revealed elevations in FAM84B mRNA levels in all carcinomas. Furthermore, through analysis of FAM84B expression using large datasets within the Gene Expression Omnibus and OncomineTM database, we demonstrate significant increases in FAM84B mRNA in 343 primary PCs versus 181 normal tissues, and elevations in the FAM84B gene copy number (GCN) in 171 primary PCs versus 61 normal tissues. While FAM84B was not detected at higher levels via immunohistochemistry in high grade (Gleason score/GS 8-10) tumors compared to GS6-7 PCs, analyses of FAM84B mRNA and GCN using datasets within the cBioPortal database demonstrated FAM84B upregulation in 12% (67/549) of primary PCs and 18% (73/412) of metastatic castration resistant PCs (mCRPCs), and GCN increases in 4.8% (26/546) of primary PCs and 26% (121/467) of mCRPCs, revealing an association of the aforementioned changes with CRPC development. Of note, an increase in FAM84B expression was observed in xenograft CRPCs produced by LNCaP cells. Furthermore, FAM84B upregulation and GCN increases correlate with decreases in disease free survival and overall survival. Collectively, we demonstrate a novel association of FAM84B with PC tumorigenesis and CRPC progression.

CYB5D2 displays tumor suppression activities towards cervical cancer

Cervical cancer is caused by infections with human papillomaviruses (HPV) and genetic alternations in the cervical epithelium. While the former is well studied, the latter remains unclear. We report here that CYB5D2/Neuferricin possesses tumor suppressing activity towards cervical tumorigenesis. Ectopic expression of CYB5D2 did not affect HeLa cell proliferation and the cells ability to form xenograft tumors, but significantly inhibited HeLa cell invasion in vitro and the cell-produced lung metastasis in NOD/SCID mice. Knockdown of CYB5D2 enhanced HeLa cell invasion. Two mutations in CYB5D2, the substitutions of arginine (R) 7 with either proline (P) or glycine (G), were reported in colon cancer. Both CYB5D2(R7P) and CYB5D2(R7G) were incapable of inhibiting HeLa cell invasion. CYB5D2 binds heme, in which aspartate (D) 86 is required. While CYB5D2(D86G) is heme-binding defective, it inhibited HeLa cell invasion. On the other hand, CYB5D2(R7P) and CYB5D2(R7G) bound heme but did not inhibit HeLa cell invasion. Collectively, CYB5D2 inhibits HeLa cell invasion independently of its heme binding. Furthermore, immunohistochemistry examination of CYB5D2 expression in 20 normal cervical tissues and 40 cervical squamous cell carcinomas (SCC) revealed a CYB5D2 reduction in 87.5% (35/40) of SCC. Analysis of CYB5D2 gene expression and genomic alteration data available from Oncomeine™ detected significant reductions of CYB5D2 mRNA in 40 SCCs and CYB5D2 gene copy number in 107 SCCs. Collectively, we provide evidence that CYB5D2 is a candidate tumor suppressor of cervical tumorigenesis.

SIPL1 enhances the proliferation, attachment, and migration of CHO cells by inhibiting PTEN function

The PTEN tumour suppressor plays critical roles in inhibiting cell proliferation, adhesion and migration through downregulation of the PI3K-AKT pathway. SIPL1 is a novel PTEN‑negative regulator (PTEN-NR) that contributes to PTEN inactivation during tumorigenesis. However, whether SIPL1 plays a role in inhibiting PTEN function in the process of cell adhesion and migration remains unclear. The aim of this study was to investigate this possibility using CHO-K1 cells, and western blotting, qPCR analyses and microscopy. Results showed that the overexpression of SIPL1 in CHO-K1 cells decreased the amount of PTEN protein. The downregulation was not caused by an obvious reduction in PTEN mRNA levels or ubiquitin-dependent protein degradation. Nonetheless, the reduction was functional, as SIPL1 overexpression increased the activation of AKT under serum‑starved conditions, promoting CHO-K1 cell proliferation in an AKT‑dependent manner. Furthermore, SIPL1 increased the migration and attachment of CHO-K1 cells. Taken together, the evidence suggested that SIPL1 promotes AKT activation by decreasing the amount of PTEN protein in CHO-K1 cells, thereby promoting cell proliferation and migration.