Jason De Melo

PKM2, a Central Point of Regulation in Cancer Metabolism

Aerobic glycolysis is the dominant metabolic pathway utilized by cancer cells, owing to its ability to divert glucose metabolites from ATP production towards the synthesis of cellular building blocks (nucleotides, amino acids, and lipids) to meet the demands of proliferation. The M2 isoform of pyruvate kinase (PKM2) catalyzes the final and also a rate-limiting reaction in the glycolytic pathway. In the PK family, PKM2 is subjected to a complex regulation by both oncogenes and tumour suppressors, which allows for a fine-tone regulation of PKM2 activity. The less active form of PKM2 drives glucose through the route of aerobic glycolysis, while active PKM2 directs glucose towards oxidative metabolism. Additionally, PKM2 possesses protein tyrosine kinase activity and plays a role in modulating gene expression and thereby contributing to tumorigenesis. We will discuss our current understanding of PKM2s regulation and its many contributions to tumorigenesis.

PTEN inhibits BMI1 function independently of its phosphatase activity.

BackgroundPTEN is the second most mutated tumor suppressor gene other than p53. It suppresses tumorigenesis by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate (PIP3) to phosphatidylinositol (4,5)-biphosphate (PIP2), thereby directly inhibiting phosphatidylinositol 3 kinase (PI3K)-mediated tumorigenic activities. Consistent with this model of action, cytosolic PTEN is recruited to the plasma membrane to dephosphorylate PIP3. While nuclear PTEN has been shown to suppress tumorigenesis by governing genome integrity, additional mechanisms may also contribute to nuclear PTEN-mediated tumor suppression. The nuclear protein BMI1 promotes stem cell self-renewal and tumorigenesis and PTEN inhibits these events, suggesting that PTEN may suppress BMI1 function.ResultsWe investigated whether PTEN inhibits BMI1 function during prostate tumorigenesis. PTEN binds to BMI1 exclusively in the nucleus. This interaction does not require PTENs phosphatase activity, as phosphatase-deficient PTEN mutants, PTEN/C124S (CS), PTEN/G129E (GE), and a C-terminal PTEN fragment (C-PTEN) excluding the catalytic domain, all associate with BMI1. Furthermore, the residues 186-286 of C-PTEN are sufficient for binding to BMI1. This interaction reduces BMI1s function. BMI1 enhances hTERT activity and reduces p16INK4A and p14ARF expression. These effects were attenuated by PTEN, PTEN(CS), PTEN(GE), and C-PTEN. Furthermore, knockdown of PTEN in DU145 cells increased hTERT promoter activity, which was reversed when BMI1 was concomitantly knocked-down, indicating that PTEN reduces hTERT promoter activity via inhibiting BMI1 function. Conversely, BMI1 reduces PTENs ability to inhibit AKT activation, which can be attributed to its interaction with PTEN in the nucleus, making PTEN unavailable to dephosphorylate membrane-bound PIP3. Furthermore, BMI1 appears to co-localize with PTEN more frequently in clinical prostate tissue samples from patients diagnosed with PIN (prostatic intraepithelial neoplasia) and carcinoma compared to normal prostate epithelium. While PTEN co-localized with BMI1 in 2.4% of normal prostate epithelial cells, co-localization was observed in 37.6% and 18.5% of cells in PIN and carcinoma, respectively. Collectively, we demonstrate that PTEN inhibits BMI1 function via binding to BMI1 in a phosphatase independent manner.ConclusionWe demonstrate that nuclear PTEN reduces BMI1 function independently of its phosphatase activity. It was recently observed that nuclear PTEN also suppresses tumorigenesis. Our results, therefore, provide a plausible mechanism by which nuclear PTEN prevents tumorigenesis.

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.

Elevation of SIPL1 (SHARPIN) Increases Breast Cancer Risk

SIPL1 (Sharpin) or Sharpin plays a role in tumorigenesis. However, its involvement in breast cancer tumorigenesis remains largely unknown. To investigate this issue, we have systemically analyzed SIPL1 gene amplification and expression data available from Oncomine datasets, which were derived from 17 studies and contained approximately 20,000 genes, 3438 breast cancer cases, and 228 normal individuals. We found a SIPL1 gene amplification in invasive ductal breast cancers compared to normal breast tissues and a significant elevation of SIPL1 mRNA in breast cancers in comparison to non-tumor breast tissues. These results collectively reveal that increases in SIPL1 expression occur during breast cancer tumorigenesis. To further investigate this association, we observed increases in the SIPL1 gene and mRNA in the breast cancer subtypes of estrogen receptor (ER)+, progesterone receptor (PR)+, HER2+, or triple negative. Additionally, a gain of the SIPL1 gene correlated with breast cancer grade and the levels of SIPL1 mRNA associated with both breast cancer stages and grades. Elevation of SIPL1 gene copy and mRNA is linked to a decrease in patient survival, especially for those with PR+, ER+, or HER2- breast cancers. These results are supported by our analysis of SIPL1 protein expression using a tissue microarray containing 224 breast cancer cases, in which higher levels of SIPL1 relate to ER+ and PR+ tumors and AKT activation. Furthermore, we were able to show that progesterone significantly reduced SIPL1 mRNA and protein expression in MCF7 cells. As progesterone enhances breast cancer tumorigenesis in a context dependent manner, inhibition of SIPL1 expression may contribute to progesterones non-tumorigenic function which might be countered by SIPL1 upregulation. Taken together, we demonstrate a positive correlation of SIPL1 with BC tumorigenesis.

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.

SIPL1-facilitated PTEN ubiquitination contributes to its association with PTEN

PTEN is post-translationally modified by ubiquitin via association with multiple E3 ubiquitin ligases, including NEDD4-1, XIAP, and WWP2. Despite the rapid progress made in researching the impact of ubiquitination on PTEN function, our understanding remains fragmented. Building on the previously observed interaction between SIPL1 and PTEN, we report here that SIPL1 promotes PTEN polyubiquitination via lysine 48 (K48)-independent polyubiquitin chains. Substitution of the K48 residue of ubiquitin with arginine (R) enhanced SIPL1-mediated PTEN polyubiquitination. In contrast, the K63R substitution significantly reduced it. The ubiquitin-like (UBL) domain is required for SIPL1-induced PTEN polyubiquitination. This post-translational modification promoted the association of SIPL1 with PTEN. Elevated amounts of the SIPL1/PTEN complex were precipitated in 293T cells co-transfected with PTEN, SIPL1, and ubiquitin compared to cells co-transfected with SIPL1 and PTEN only. Additionally, formation of the SIPL1/PTEN complex was inhibited when either lysine-less (K0) ubiquitin or K63R ubiquitin was co-transfected together with SIPL1+PTEN. The PTEN component in the SIPL1/PTEN complex contained polyubiquitin chains. The ubiquitination reaction may play a structural role, stabilizing the SIPL1/PTEN complex, as a ubiquitin binding-defective SIPL1 mutant (TFLV) is proficient in PTEN association. Collectively, we demonstrate that SIPL1 binds PTEN and enhances PTEN polyubiquitination which in turn promotes the interaction between SIPL1 and PTEN.

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.

Dataset on the effects of CYB5D2 on the distribution of HeLa cervical cancer cell cycle

We have recently reported that CYB5D2 plays a role in suppression of cervical cancer tumorigenesis, “CYB5D2 displays tumor suppression activities towards cervical cancer” [1]. We provide the accompany data here describing the effects of CYB5D2 overexpression and addition of recombinant CYB5D2 on HeLa cell cycle distribution. Furthermore, we will present the conditions used to specifically determine CYB5D2 expression in primary cervical and cervical cancer tissues using immunohistochemistry (IHC) and the patient cohort involved in assessing the CYB5D2 protein levels in primary cervical and cervical cancer tissues.