Paul Mellor

Multiple roles for the p85α isoform in the regulation and function of PI3K signalling and receptor trafficking

The p85α protein is best known as the regulatory subunit of class 1A PI3Ks (phosphoinositide 3-kinases) through its interaction, stabilization and repression of p110-PI3K catalytic subunits. PI3Ks play multiple roles in the regulation of cell survival, signalling, proliferation, migration and vesicle trafficking. The present review will focus on p85α, with special emphasis on its important roles in the regulation of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and Rab5 functions. The phosphatidylinositol-3-phosphatase PTEN directly counteracts PI3K signalling through dephosphorylation of PI3K lipid products. Thus the balance of p85α-p110 and p85α-PTEN complexes determines the signalling output of the PI3K/PTEN pathway, and under conditions of reduced p85α levels, the p85α-PTEN complex is selectively reduced, promoting PI3K signalling. Rab5 GTPases are important during the endocytosis, intracellular trafficking and degradation of activated receptor complexes. The p85α protein helps switch off Rab5, and if defective in this p85α function, results in sustained activated receptor tyrosine kinase signalling and cell transformation through disrupted receptor trafficking. The central role for p85α in the regulation of PTEN and Rab5 has widened the scope of p85α functions to include integration of PI3K activation (p110-mediated), deactivation (PTEN-mediated) and receptor trafficking/signalling (Rab5-mediated) functions, all with key roles in maintaining cellular homoeostasis.

The signalling factor PI3K is a specific regulator of the clathrin-independent dynamin-dependent endocytosis of IL-2 receptors.

Summary Receptor-mediated endocytosis is an essential process used by eukaryotic cells to internalise many molecules. Several clathrin-independent endocytic routes exist, but the molecular mechanism of each pathway remains to be uncovered. The present study focuses on a clathrin-independent dynamin-dependent pathway used by interleukin 2 receptors (IL-2R), essential players of the immune response. Ras-related C3 botulinum toxin substrate (Rac1) and its targets, the p21-activated kinases (Pak), are specific regulators of this pathway, acting on cortactin and actin polymerization. The present study reveals a dual and specific role of phosphatidylinositol 3-kinase (PI3K) in IL-2R endocytosis. Inhibition of the catalytic activity of PI3K strongly affects IL-2R endocytosis, in contrast to transferrin (Tf) uptake, a marker of the clathrin-mediated pathway. Moreover, Vav2, a GTPase exchange factor (GEF) induced upon PI3K activation, is specifically involved in IL-2R entry. The second action of PI3K is through its regulatory subunit, p85&agr;, which binds to and recruits Rac1 during IL-2R internalisation. Indeed, the overexpression of a p85&agr; mutant missing the Rac1 binding motif leads to the specific inhibition of IL-2R endocytosis. The inhibitory effect of this p85&agr; mutant could be rescued by the overexpression of either Rac1 or the active form of Pak, indicating that p85&agr; acts upstream of the Rac1-Pak cascade. Finally, biochemical and fluorescent microscopy techniques reveal an interaction between p85&agr;, Rac1 and IL-2R that is enhanced by IL-2. In summary, our results indicate a key role of class I PI3K in IL-2R endocytosis that creates a link with IL-2 signalling.

CREB3L1 is a metastasis suppressor that represses expression of genes regulating metastasis, invasion, and angiogenesis.

ABSTRACT The unfolded protein response (UPR) is activated in response to hypoxia-induced stress such as in the tumor microenvironment. This study examined the role of CREB3L1 (cyclic AMP [cAMP]-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis. Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low-metastasis or nonmetastatic cell lines. When metastatic cells were transfected with CREB3L1, they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under nonadherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, not only did CREB3L1-expressing cells fail to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and chromatin immunoprecipitation with microarray technology (ChIP on Chip) analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis. These data suggest that CREB3L1 plays an important role in suppressing tumorigenesis and that loss of expression is required for the development of a metastatic phenotype.

Identification of Mutations in Distinct Regions of p85 Alpha in Urothelial Cancer

Bladder cancers commonly show genetic aberrations in the phosphatidylinositol 3-kinase signaling pathway. Here we have screened for mutations in PIK3R1, which encodes p85α, one of the regulatory subunits of PI3K. Two hundred and sixty-four bladder tumours and 41 bladder tumour cell lines were screened and 18 mutations were detected. Thirteen mutations were in C-terminal domains and are predicted to interfere with the interaction between p85α and p110α. Five mutations were in the BH domain of PIK3R1. This region has been implicated in p110α-independent roles of p85α, such as binding to and altering the activities of PTEN, Rab4 and Rab5. Expression of these mutant BH-p85α forms in mouse embryonic fibroblasts with p85α knockout indicated that all forms, except the truncation mutants, could bind and stabilize p110α but did not increase AKT phosphorylation, suggesting that BH mutations function independently of p110α. In a panel of 44 bladder tumour cell lines, 80% had reduced PIK3R1 mRNA expression relative to normal urothelial cells. This, along with mutation of PIK3R1, may alter BH domain functioning. Our findings suggest that mutant forms of p85α may play an oncogenic role in bladder cancer, not only via loss of ability to regulate p110α but also via altered function of the BH domain.

The role of PTEN - HCV core interaction in hepatitis C virus replication

Hepatitis C virus (HCV) infection leads to severe liver diseases including hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumour suppressor, is frequently mutated or deleted in HCC tumors. PTEN has previously been demonstrated to inhibit HCV secretion. In this study, we determined the effects of PTEN on the other steps in HCV life cycle, including entry, translation, and replication. We showed that PTEN inhibits HCV entry through its lipid phosphatase activity. PTEN has no effect on HCV RNA translation. PTEN decreases HCV replication and the protein phosphatase activity of PTEN is essential for this function. PTEN interacts with the HCV core protein and requires R50 in domain I of HCV core and PTEN residues 1–185 for this interaction. This interaction is required for PTEN-mediated inhibition of HCV replication. This gives rise to a reduction in PTEN levels and intracellular lipid abundance, which may in turn regulate HCV replication. HCV core domain I protein increases the lipid phosphatase activity of PTEN in an in vitro assay, suggesting that HCV infection can also regulate PTEN. Taken together, our results demonstrated an important regulatory role of PTEN in the HCV life cycle.

Epigenetic silencing of CREB3L1 by DNA methylation is associated with high-grade metastatic breast cancers with poor prognosis and is prevalent in triple negative breast cancers

BackgroundCREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the unfolded protein response, has recently been identified as a metastasis suppressor in both breast and bladder cancer.MethodsQuantitative real time PCR (qPCR) and immunoblotting were used to determine the impact of histone deacetylation and DNA methylation inhibitors on CREB3L1 expression in breast cancer cell lines. Breast cancer cell lines and tumor samples were analyzed similarly, and CREB3L1 gene methylation was determined using sodium bisulfite conversion and DNA sequencing. Immunohistochemistry was used to determine nuclear versus cytoplasmic CREB3L1 protein. Large breast cancer database analyses were carried out to examine relationships between CREB3L1 gene methylation and mRNA expression in addition to CREB3L1 mRNA expression and prognosis.ResultsThis study demonstrates that the low CREB3L1 expression previously seen in highly metastatic breast cancer cell lines is caused in part by epigenetic silencing. Treatment of several highly metastatic breast cancer cell lines that had low CREB3L1 expression with DNA methyltransferase and histone deacetylase inhibitors induced expression of CREB3L1, both mRNA and protein. In human breast tumors, CREB3L1 mRNA expression was upregulated in low and medium-grade tumors, most frequently of the luminal and HER2 amplified subtypes. In contrast, CREB3L1 expression was repressed in high-grade tumors, and its loss was most frequently associated with triple negative breast cancers (TNBCs). Importantly, bioinformatics analyses of tumor databases support these findings, with methylation of the CREB3L1 gene associated with TNBCs, and strongly negatively correlated with CREB3L1 mRNA expression. Decreased CREB3L1 mRNA expression was associated with increased tumor grade and reduced progression-free survival. An immunohistochemistry analysis revealed that low-grade breast tumors frequently had nuclear CREB3L1 protein, in contrast to the high-grade breast tumors in which CREB3L1 was cytoplasmic, suggesting that differential localization may also regulate CREB3L1 effectiveness in metastasis suppression.ConclusionsOur data further strengthens the role for CREB3L1 as a metastasis suppressor in breast cancer and demonstrates that epigenetic silencing is a major regulator of the loss of CREB3L1 expression. We also highlight that CREB3L1 expression is frequently altered in many cancer types suggesting that it could have a broader role in cancer progression and metastasis.

Abstract 4852: Targeting cancer progression genes upregulated in CREB3L1-deficient breast cancer cells

Introduction: Our laboratory is interested in determining ways to block breast cancer metastasis. We discovered that CREB3L1 (cAMP responsive element binding protein 3-like protein), a stress-activated transcription factor, acts as a metastasis suppressor in breast cancer. CREB3L1 is expressed ubiquitously in noncancerous human breast cells and restricts expression of genes that promote cell growth, angiogenesis, and migration. Loss of CREB3L1 expression is a frequent event in high-grade metastatic human breast tumors and can result in enhanced metastatic properties. Experimental Procedures: To establish the mechanisms by which CREB3L1 exerts its tumor suppressive effects, a gene expression microarray analysis was performed to determine which genes are upregulated in CREB3L1-deficient breast cancer cells. Six hundred and eighty genes were statistically significantly upregulated at least 2-fold in all 4 of the CREB3L1-deficient cell lines assessed. To focus our analysis, 4 genes were identified as potential targets for this study, given that they have previously been shown to have a role in cancer progression. Knockdowns were generated for each of the 4 genes in a CREB3L1-deficient breast cancer cell line (HCC1806) and matched CREB3L1-expressing cells (HCC1806 + HA-CREB3L1), as well as a nontumorigenic control breast cell line (MCF10A). To knockdown expression of each of the 4 targets, shRNA directed at the gene of interest was transduced via a lentiviral system into the cells. Cell-based assays were performed to characterize the properties of these cell lines, which included the Boyden chamber assay to measure migration, soft agar assay to assess anchorage-independent growth, and the MTT assay to evaluate cell proliferation. Results: Successful knockdown at both the mRNA and protein level for each target gene was validated using quantitative real time PCR and immunoblotting. Results from the MTT assay verified that knockdown of each of the 4 gene targets did not affect cell survival in MCF10A cells, confirming that these test genes are not essential for survival in noncancerous breast cells (p > 0.05). Similarly, knockdown of each of the test genes did not affect cell proliferation in HCC1806 (± CREB3L1) cells, except for a significant decrease in proliferation in HCC1806 CREB3L1-deficient cells with Gene 1 knockdown (p =0.02). Preliminary results indicate that knockdown of Genes 1, 2, and 4 in HCC1806 CREB3L1-deficient cells reduces cancer cell properties, including cell migration and anchorage-independent growth (p Conclusions: Promising candidates will be tested in follow-up mouse xenograft studies. Encouraging results in these pre-clinical models would provide a strong rationale for the development and testing of inhibitors to one or more of these potential targets as a new therapeutic for the 30% of breast cancers that are CREB3L1-deficient. Citation Format: Deborah Anderson, Shari Smith, Farah Goubran, Paul Mellor. Targeting cancer progression genes upregulated in CREB3L1-deficient breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4852. doi:10.1158/1538-7445.AM2017-4852

Abstract 2860: Loss of expression of the metastasis suppressor CREB3L1 is associated with high-grade metastatic breast cancer and poorer prognosis

The unfolded protein response (UPR) is required for proper protein folding in the endoplasmic reticulum under conditions of stress such as those encountered in the tumor microenvironment. Recently, a number of studies have suggested a role for the unfolded protein response in the development of cancer, more specifically in regulating the balance between cell death, dormancy and the growth of cancer cells in the tumor microenvironment. This study examined the role of CREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis. Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low- or non-metastatic cell lines. When metastatic cells were transfected with CREB3L1 they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under non-adherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, CREB3L1 expressing cells not only failed to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and ChIP on Chip analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis. To determine if these findings translated to human breast cancer, real-time PCR analysis of CREB3L1 expression in human breast cancer tissues of differing grade (n = 213) was performed. Expression of CREB3L1 was elevated in low and medium grade tissue but substantially reduced in high grade tissue, compared to normal. Analysis of the on-line TCGA database of breast cancer samples identified a strong inverse relationship between CREB3L1 gene methylation and the loss of CREB3L1 mRNA expression. Low expression of CREB3L1 was associated with a poorer prognosis with a shorter relapse-free survival for luminal A and triple negative breast cancer patients. These data suggest that CREB3L1 plays an important role in suppressing tumorgenesis and loss of expression is required for the development of a metastatic phenotype. In addition CREB3L1 expression may be a useful marker for determining patient prognosis. Citation Format: Paul Mellor, Alison Ward, Deborah Anderson. Loss of expression of the metastasis suppressor CREB3L1 is associated with high-grade metastatic breast cancer and poorer prognosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2860.

Abstract 1450: Loss of the UPR member CREB3L1 is vital for the survival of breast cancer cells in the tumor microenvironment and development of the metastatic phenotype

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The unfolded protein response (UPR) is required for proper protein folding in the endoplasmic reticulum under conditions of stress such as those encountered in the tumor microenvironment. Recently, a number of studies have suggested a role for the unfolded protein response in the development of cancer, more specifically in regulating the balance between cell death, dormancy and the growth of cancer cells in the tumor microenvironment. This study examined the role of the transcription factor CREB3L1, a member of the UPR machinery, in breast cancer development and metastasis. Northern blotting analysis identified a loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low- or non-metastatic cell lines, which was subsequently shown, through bisulphate sequencing, to be the result of enhanced promoter methylation. When metastatic cells were transfected with CREB3L1 they demonstrated a significant decrease in survival in hypoxic and non-adherent growth conditions, and reduced in vitro invasion and migration in chemotaxis assays. Interestingly, in an in vivo rat mammary tumor model where rats were injected with cells in the hind footpad, the CREB3L1 expressing rat cells not only failed to form metastases but tumor regression was seen in 70% of the animals. In contrast, metastases were seen in the popliteal lymph node of 90% of rats injected with wild-type and empty vector-transfected cells. Microarray and ChIP on Chip analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis when CREB3L1 was expressed. We also observed a significant decrease in the number of migrating endothelial cells in scratch and chemotaxis assays in media conditioned by CREB3L1 expressing cells, compared to conditioned media from untransfected cells. To determine if these findings translated to human breast cancer, real-time PCR analysis of CREB3L1 expression in human breast cancer tissues of differing grade (n=30) was performed. Expression of CREB3L1 was elevated in low and medium grade tissue but substantially reduced in high grade tissue, compared to normal. The high grade breast cancer tissues demonstrated a 6.6 fold decrease in the expression of CREB3L1 compared to the low grade tissues (p<0.001), with about a 2 fold decrease compared to normal tissue. In conclusion, CREB3L1 expression is elevated in low and medium grade breast cancer tissue, perhaps as the cancer cells respond to various stresses. However, loss of CREB3L1 expression is required for the long-term survival of breast cancer cells in the tumor microenvironment and the progression to a highly metastatic phenotype. CREB3L1 expression may be a useful marker in predicting tumor grade and patient prognosis, in addition to providing a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1450. doi:10.1158/1538-7445.AM2011-1450