Glenn Hegamyer

A Novel Function of the MA-3 Domains in Transformation and Translation Suppressor Pdcd4 Is Essential for Its Binding to Eukaryotic Translation Initiation Factor 4A

ABSTRACT Αn α-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4E249K, Pdcd4D253A, Pdcd4D414K, Pdcd4D418A, Pdcd4E249K,D414K, and Pdcd4D253A,D418A) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4D253A and Pdcd4D253A,D418A do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4D253A, Pdcd4D418A, or Pdcd4D235A,D418A. Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.

Translation Inhibitor Pdcd4 Is Targeted for Degradation during Tumor Promotion

Inactivation of tumor suppressors is among the rate-limiting steps in carcinogenesis that occur during the tumor promotion stage. The translation inhibitor programmed cell death 4 (Pdcd4) suppresses tumorigenesis and invasion. Although Pdcd4 is not mutationally inactivated in human cancer, the mechanisms controlling Pdcd4 inactivation during tumorigenesis remain elusive. We report that tumor promoter 12-O-tetradecanoylphorbol-13-acetate exposure decreases protein levels of Pdcd4 in mouse skin papillomas and keratinocytes as well as in human HEK293 cells. This decrease is attributable to increased proteasomal degradation of Pdcd4 and is mediated by protein kinase C-dependent activation of phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin-p70(S6K) and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling. Both Akt and p70(S6K) phosphorylate Pdcd4, allowing for binding of the E3-ubiquitin ligase beta-TrCP and consequently ubiquitylation. MEK-ERK signaling on the other hand facilitates the subsequent proteasomal degradation. We further show that Pdcd4 protein levels in vivo are limiting for tumor formation, establishing Pdcd4 as a haploinsufficient tumor suppressor in Pdcd4-deficient mice. Thus, because endogenous Pdcd4 levels are limiting for tumorigenesis, inhibiting signaling to Pdcd4 degradation may prove a valid strategy for cancer prevention and intervention.

Pdcd4 repression of lysyl oxidase inhibits hypoxia-induced breast cancer cell invasion

Metastasis to bone, liver and lungs is the primary cause of death in breast cancer patients. Our studies have revealed that the novel tumor suppressor Pdcd4 inhibits breast cancer cell migration and invasion in vitro. Loss of Pdcd4 in human nonmetastatic breast cancer cells increased the expression of lysyl oxidase (LOX) mRNA. LOX is a hypoxia-inducible amine oxidase, the activity of which enhances breast cancer cell invasion in vitro and in vivo. Specific inhibition of LOX activity by β-aminopropionitrile or small interfering RNA decreased the invasiveness of T47D and MCF7 breast cancer cells attenuated for Pdcd4 function. Most significantly, loss of Pdcd4 augments hypoxia induction of LOX as well. Conversely, overexpression of Pdcd4 significantly reversed the hypoxia induction of LOX expression in T47D cells attenuated for Pdcd4. However, Pdcd4 did not affect hypoxia-inducible factor-1 (HIF-1) protein expression or HIF-1-responsive element-luciferase activity in response to hypoxia, suggesting that Pdcd4 regulation of LOX occurs through an HIF-independent mechanism. Nevertheless, the loss of Pdcd4 early in cancer progression may have an important role in the increased sensitivity of cancer cells to hypoxia through increased LOX activity and concomitant enhanced invasiveness.

A transforming activity not detected by DNA transfer to NIH 3T3 cells is detected by JB6 mouse epidermal cells

Transfection of four different mouse epidermal tumor cell DNAs into NIH 3T3 cells yielded neither morphologically altered foci nor anchorage independence. However, promotion-sensitive, but not promotion-insensitive, JB6 mouse epidermal cell lines were permissive for the expression of anchorage independence after transfection of DNA from three of these tumor cell lines. This transforming activity and the promotion-sensitive activity that confers sensitivity to promotion of transformation show differences in restriction enzyme sensitivity. In view of this difference and the differences in both recipient cells and 12-O-tetradecanoyl-phorbol-13-acetate dependence of expression, it appears that the transforming activity and the promotion-sensitive activity are specified by different genes. The JB6 promotion-sensitive cell lines may be useful for detecting and cloning transforming genes that escape detection in the NIH 3T3 cell focus assay.

The Role of Phorbol Ester Receptor Binding in Responses to Promoters by Mouse and Human Cells

One of the major unanswered questions in carcinogenesis today concerns the rate-limiting steps that determine premalignant progression during the long latent period from the onset of carcinogen exposure until tumor appearance. Since tumor promoters apparently act to increase both the probability of occurrence and the rate of traversing events leading to malignancy, attempts to counter these promoter-induced events might offer a promising means of cancer prevention. In this connection, an understanding of the basis for resistance to tumor promoters could lead to an exploitable strategy. Our laboratory has developed the JB6 mouse epidermal cell model system for studying late-stage irreversible promotion of transformation by phorbol esters and other tumor promoters. We have recently described the isolation of promotion-resistant variants of JB6 cells which permit us to study the molecular and cellular basis for resistance (1,2). Since phorbol esters bind to specific cellular receptors and since resistance to a variety of hormones that also bind to specific receptors is associated with receptor deficiency (3), we have investigated whether the resistance of phorbol ester-resistant JB6 mouse cells can be attributed to a lack of phorbol diester receptors. This inquiry has been extended to phorbol ester-resistant variants of human hematopoietic cells.

Abstract 3079: Identification of the translational targets of tumor suppressor Pdcd4

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Programmed-cell-death-4 (Pdcd4) is a novel protein that functions as a transformation suppressor. Pdcd4 deficiency renders mice susceptible to increased tumor multiplicity in initiation-promotion skin carcinogenesis. On the contrary transgenic expression of Pdcd4 in mice inhibits DMBA-TPA induced skin tumorigenesis and tumor progression. Pdcd4 interacts with translation initiation factors eIF4A and eIF4G to inhibit translation in an mRNA-specific fashion and consequently blocks pro-oncogenic events such as activation of activator protein-1 (AP-1)-dependent transcription, anchorage-independent transformation and invasion. Pdcd4 expression decreases during carcinogenesis in several human cancer sites including colon, esophagus, lung, pancreas and brain and its activation appears to be important in the mechanism by which some cancer therapeutic drugs work. While Pdcd4 is not mutationally inactivated, its expression is post translationally regulated. Pdcd4 expression is decreased during carcinogenesis by mechanisms involving microRNA-21 (miR21) inhibition of translation and by S6 kinase-dependent proteasome degradation. Loss of Pdcd4 has been shown to affect transcription of TIMP-2, MAP4K1, LOX and u-PAR. Although the tumor suppressor Pdcd4 acts directly on the translation initiation complex, translational targets of Pdcd4 have yet to be identified. In order to identify and characterize functionally significant translational targets of tumor suppressor Pdcd4 when it inhibits translation during tumor promotion, tumorigenesis and invasion of malignant tumors, we knocked down Pdcd4 in T47D breast cancer cells. Loss of Pdcd4 increases cell migration and invasion as well as activity of luciferase reporters of AP-1-dependent transcription and translation initiation. To identify translational targets of Pdcd4, translationally active mRNA species were fractionated by sucrose gradient. Polyribosome bound mRNA fractions were pooled and analyzed for mRNA levels by microarray. Knockdown of Pdcd4 produced more than 2 fold increases in 14 mRNAs in the translationally active polyribosomal fractions compared to the control. The polysome shifts in Pdcd4 translational targets are being confirmed by quantitative RT-PCR and Western blot analysis of protein. Pdcd4 targets recently confirmed by quantitative RT-PCR are PPARGC1B and delta p73, both known to be oncogenic, as well as zinc finger protein ZNF281. The functional significance of these targets is being determined. Discovery of the mRNAs selectively targeted by Pdcd4 will facilitate the elucidation of the mechanism by which Pdcd4 suppresses tumorigenesis and may provide additional targets for intervention. 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 3079. doi:10.1158/1538-7445.AM2011-3079