Colin J. Daniel

The Axin1 scaffold protein promotes formation of a degradation complex for c‐Myc

Expression of the c‐Myc proto‐oncoprotein is tightly regulated in normal cells. Phosphorylation at two conserved residues, threonine58 (T58) and serine62 (S62), regulates c‐Myc protein stability. In cancer cells, c‐Myc can become aberrantly stabilized associated with altered T58 and S62 phosphorylation. A complex signalling cascade involving GSK3β kinase, the Pin1 prolyl isomerase, and the PP2A‐B56α phosphatase controls phosphorylation at these sites. We report here a novel role for the tumour suppressor scaffold protein Axin1 in facilitating the formation of a degradation complex for c‐Myc containing GSK3β, Pin1, and PP2A‐B56α. Although knockdown of Axin1 decreases the association of c‐Myc with these proteins, reduces T58 and enhances S62 phosphorylation, and increases c‐Myc stability, acute expression of Axin1 reduces c‐Myc levels and suppresses c‐Myc transcriptional activity. Moreover, the regulation of c‐Myc by Axin1 is impaired in several tested cancer cell lines with known stabilization of c‐Myc or loss of Axin1. This study provides critical insight into the regulation of c‐Myc expression, how this can be disrupted in three cancer types, and adds to our knowledge of the tumour suppressor activity of Axin1.

Targeting c-MYC by antagonizing PP2A inhibitors in breast cancer

Significance Increased kinase activity and suppressed phosphatase activity are hallmarks of oncogenic signaling. The transcription factor c-MYC, a master driver of human cancer, is stabilized and activated by persistent serine 62 phosphorylation. The tumor suppressor protein phosphatase 2A (PP2A) targets this site and negatively regulates c-MYC. Here, we show that two cellular inhibitors of PP2A, the SET oncoprotein and cancerous inhibitor of PP2A (CIP2A), are overexpressed in breast cancer, and depletion or inhibition of SET or CIP2A reduces c-MYC expression and activity and decreases the tumorigenic potential of breast cancer cells. These findings strongly suggest that inhibiting SET or CIP2A to reactivate PP2A may be an effective therapeutic strategy for targeting c-MYC in breast cancer. The transcription factor c-MYC is stabilized and activated by phosphorylation at serine 62 (S62) in breast cancer. Protein phosphatase 2A (PP2A) is a critical negative regulator of c-MYC through its ability to dephosphorylate S62. By inactivating c-MYC and other key signaling pathways, PP2A plays an important tumor suppressor function. Two endogenous inhibitors of PP2A, I2PP2A, Inhibitor-2 of PP2A (SET oncoprotein) and cancerous inhibitor of PP2A (CIP2A), inactivate PP2A and are overexpressed in several tumor types. Here we show that SET is overexpressed in about 50–60% and CIP2A in about 90% of breast cancers. Knockdown of SET or CIP2A reduces the tumorigenic potential of breast cancer cell lines both in vitro and in vivo. Treatment of breast cancer cells in vitro or in vivo with OP449, a novel SET antagonist, also decreases the tumorigenic potential of breast cancer cells and induces apoptosis. We show that this is, at least in part, due to decreased S62 phosphorylation of c-MYC and reduced c-MYC activity and target gene expression. Because of the ubiquitous expression and tumor suppressor activity of PP2A in cells, as well as the critical role of c-MYC in human cancer, we propose that activation of PP2A (here accomplished through antagonizing endogenous inhibitors) could be a novel antitumor strategy to posttranslationally target c-MYC in breast cancer.

Mechanistic insight into Myc stabilization in breast cancer involving aberrant Axin1 expression

High expression of the oncoprotein Myc has been linked to poor outcome in human tumors. Although MYC gene amplification and translocations have been observed, this can explain Myc overexpression in only a subset of human tumors. Myc expression is in part controlled by its protein stability, which can be regulated by phosphorylation at threonine 58 (T58) and serine 62 (S62). We now report that Myc protein stability is increased in a number of breast cancer cell lines and this correlates with increased phosphorylation at S62 and decreased phosphorylation at T58. Moreover, we find this same shift in phosphorylation in primary breast cancers. The signaling cascade that controls phosphorylation at T58 and S62 is coordinated by the scaffold protein Axin1. We therefore examined Axin1 in breast cancer and report decreased AXIN1 expression and a shift in the ratio of expression of two naturally occurring AXIN1 splice variants. We demonstrate that this contributes to increased Myc protein stability, altered phosphorylation at S62 and T58, and increased oncogenic activity of Myc in breast cancer. Thus, our results reveal an important mode of Myc activation in human breast cancer and a mechanism contributing to Myc deregulation involving unique insight into inactivation of the Axin1 tumor suppressor in breast cancer.

Pin1 Regulates the Dynamics of c-Myc DNA Binding To Facilitate Target Gene Regulation and Oncogenesis

ABSTRACT The Myc oncoprotein is considered a master regulator of gene transcription by virtue of its ability to modulate the expression of a large percentage of all genes. However, mechanisms that direct Mycs recruitment to DNA and target gene selection to elicit specific cellular functions have not been well elucidated. Here, we report that the Pin1 prolyl isomerase enhances recruitment of serine 62-phosphorylated Myc and its coactivators to select promoters during gene activation, followed by promoting Mycs release associated with its degradation. This facilitates Mycs activation of genes involved in cell growth and metabolism, resulting in enhanced proproliferative activity, even while controlling Myc levels. In cancer cells with impaired Myc degradation, Pin1 still enhances Myc DNA binding, although it no longer facilitates Myc degradation. Thus, we find that Pin1 and Myc are cooverexpressed in cancer, and this drives a gene expression pattern that we show is enriched in poor-outcome breast cancer subtypes. This study provides new insight into mechanisms regulating Myc DNA binding and oncogenic activity, it reveals a novel role for Pin1 in the regulation of transcription factors, and it elucidates a mechanism that can contribute to oncogenic cooperation between Pin1 and Myc.

Targeting Inhibitors of the Tumor Suppressor PP2A for the Treatment of Pancreatic Cancer

Pancreatic cancer is a deadly disease that is usually diagnosed in the advanced stages when few effective therapies are available. Given the aggressive clinical course of this disease and lack of good treatment options, the development of new therapeutic agents for the treatment of pancreatic cancer is of the upmost importance. Several pathways that have shown to contribute to pancreatic cancer progression are negatively regulated by the tumor suppressor protein phosphatase 2A (PP2A). Here, the endogenous inhibitors of PP2A, SET (also known as I2PP2A) and cancerous inhibitor of PP2A (CIP2A), were shown to be overexpressed in human pancreatic cancer, contributing to decreased PP2A activity and overexpression and stabilization of the oncoprotein c-Myc, a key PP2A target. Knockdown of SET or CIP2A increases PP2A activity, increases c-Myc degradation, and decreases the tumorigenic potential of pancreatic cancer cell lines both in vitro and in vivo. Moreover, treatment with a novel SET inhibitor, OP449, pharmacologically recapitulates the phenotypes and significantly reduces proliferation and tumorigenic potential of several pancreatic cancer cell lines, with an accompanying attenuation of cell growth and survival signaling. Furthermore, primary cells from patients with pancreatic cancer were sensitive to OP449 treatment, indicating that PP2A-regulated pathways are highly relevant to this deadly disease. Implications: The PP2A inhibitors SET and CIP2A are overexpressed in human pancreatic cancer and are important for pancreatic cancer cell growth and transformation; thus, antagonizing SET and/or CIP2A may be an innovative approach for the treatment of human pancreatic cancer. Mol Cancer Res; 12(6); 924–39. ©2014 AACR.

Control of Translocation through the Sec61 Translocon by Nascent Polypeptide Structure within the Ribosome

During polytopic protein biogenesis, multiple transmembrane segments (TMs) must pass through the ribosome exit tunnel and into the Sec61 translocon prior to insertion into the endoplasmic reticulum membrane. To investigate how movement of a newly synthesized TM along this integration pathway might be influenced by synthesis of a second TM, we used photocross-linking probes to detect the proximity of ribosome-bound nascent polypeptides to Sec61α. Probes were inserted at sequential sites within TM2 of the aquaporin-1 water channel by in vitro translation of truncated mRNAs. TM2 first contacted Sec61α when the probe was positioned ∼38 residues from the ribosome peptidyltransferase center, and TM2-Sec61α photoadducts decreased markedly when the probe was >80 residues from the peptidyltransferase center. Unexpectedly, as nascent chain length was gradually extended, photocross-linking at multiple sites within TM2 abruptly and transiently decreased, indicating that TM2 initially entered, withdrew, and then re-entered Sec61α. This brief reduction in TM2 photocross-linking coincided with TM3 synthesis. Replacement of TM3 with a secretory reporter domain or introduction of proline residues into TM3 changed the TM2 cross-linking profile and this biphasic behavior. These findings demonstrate that the primary and likely secondary structure of the nascent polypeptide within the ribosome exit tunnel can influence the timing with which topogenic determinants contact, enter, and pass through the translocon.

The Tumor Suppressor Protein HBP1 Is a Novel c-Myc-binding Protein That Negatively Regulates c-Myc Transcriptional Activity

c-Myc is an important transcription factor that regulates cellular proliferation, cell growth, and differentiation. A number of transcriptional co-factors for c-Myc have been described that have binding sites within highly conserved regions of the c-Myc transactivational domain (TAD). Given the importance of the c-Myc TAD, we set out to identify new proteins that interact with this region using a yeast two-hybrid assay. HBP1 was identified in our screen as a protein that interacts with full-length c-Myc but not a c-Myc mutant lacking the TAD. HBP1 is a transcriptional repressor and has been shown to negatively regulate the cell cycle. A correlation between HBP1 under-expression and breast cancer relapse has been described, suggesting that HBP1 may be an important tumor suppressor protein. We have found that HBP1 binds c-Myc in cells, and expression of HBP1 inhibits c-Myc transactivational activity at least partly by preventing c-Myc binding to target gene promoters. c-Myc binds to the C terminus of HBP1, a region lost in some breast tumors, and some HBP1 mutants found in breast cancer weakly interact with and/or no longer negatively regulate c-Myc. This work adds to our understanding of c-Myc regulation and mechanisms of tumor suppression by HBP1.

A critical role for Mnt in Myc-driven T-cell proliferation and oncogenesis

Mnt (Maxs next tango) is a Max-interacting transcriptional repressor that can antagonize both the proproliferative and proapoptotic functions of Myc in vitro. To ascertain the physiologically relevant functions of Mnt and to help define the relationship between Mnt and Myc in vivo, we generated a series of mouse strains in which Mnt was deleted in T cells in the absence of endogenous c-Myc or in the presence of ectopic c-Myc. We found that apoptosis caused by loss of Mnt did not require Myc but that ectopic Myc expression dramatically decreased the survival of both Mnt-deficient T cells in vivo and Mnt-deficient MEFs in vitro. Consequently, Myc-driven proliferative expansion of T cells in vitro and thymoma formation in vivo were prevented by the absence of Mnt. Consistent with T-cell models, mouse embryo fibroblasts (MEFs) lacking Mnt were refractory to oncogenic transformation by Myc. Tumor suppression caused by loss of Mnt was linked to increased apoptosis mediated by reactive oxygen species (ROS). Thus, although theoretically and experimentally a Myc antagonist, the dominant physiological role of Mnt appears to be suppression of apoptosis. Our results redefine the physiological relationship between Mnt and Myc and requirements for Myc-driven oncogenesis.

Overcoming hypoxia-induced apoptotic resistance through combinatorial inhibition of GSK-3β and CDK1

Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we conducted a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds, we identified were a structurally related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3β and cyclin-dependent kinase (CDK) 1. Combinatorial inhibition of these proteins in hypoxic cancer cells and tumors increased levels of c-Myc and decreased expression of c-IAP2 and the central hypoxia response regulator hypoxia-inducible factor (HIF) 1α. In mice, these compounds augmented the hypoxic tumor cell death induced by cytotoxic chemotherapy, blocking angiogenesis and tumor growth. Taken together, our findings suggest that combinatorial inhibition of GSK-3β and CDK1 augment the apoptotic sensitivity of hypoxic tumors, and they offer preclinical validation of a novel and readily translatable strategy to improve cancer therapy.

The tumor suppressor phosphatase PP2A-B56α regulates stemness and promotes the initiation of malignancies in a novel murine model

Protein phosphatase 2A (PP2A) is a ubiquitously expressed Serine-Threonine phosphatase mediating 30–50% of protein phosphatase activity. PP2A functions as a heterotrimeric complex, with the B subunits directing target specificity to regulate the activity of many key pathways that control cellular phenotypes. PP2A-B56α has been shown to play a tumor suppressor role and to negatively control c-MYC stability and activity. Loss of B56α promotes cellular transformation, likely at least in part through its regulation of c-MYC. Here we report generation of a B56α hypomorph mouse with very low B56α expression that we used to study the physiologic activity of the PP2A-B56α phosphatase. The predominant phenotype we observed in mice with B56α deficiency in the whole body was spontaneous skin lesion formation with hyperproliferation of the epidermis, hair follicles and sebaceous glands. Increased levels of c-MYC phosphorylation on Serine62 and c-MYC activity were observed in the skin lesions of the B56αhm/hm mice. B56α deficiency was found to increase the number of skin stem cells, and consistent with this, papilloma initiation was accelerated in a carcinogenesis model. Further analysis of additional tissues revealed increased inflammation in spleen, liver, lung, and intestinal lymph nodes as well as in the skin lesions, resembling elevated extramedullary hematopoiesis phenotypes in the B56αhm/hm mice. We also observed an increase in the clonogenicity of bone marrow stem cells in B56αhm/hm mice. Overall, this model suggests that B56α is important for stem cells to maintain homeostasis and that B56α loss leading to increased activity of important oncogenes, including c-MYC, can result in aberrant cell growth and increased stem cells that can contribute to the initiation of malignancy.