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In the absence of apparent mutations, alteration of gene expression patterns represents the key mechanism by which normal cells evolve to cancer cells.
Gene expression is tightly regulated by posttranscriptional processes. Within this context, RNA-binding proteins (RBPs) represent fundamental factors, since they control mechanisms, such as mRNA-stabilization, -translation and -degradation. Human antigen R (HuR) was among the first RBPs that have been directly associated to carcinogenesis. HuR modulates the stability and translation of mRNAs which encode proteins facilitating various ‘hallmarks of cancer’, namely proliferation, evasion of growth suppression, angiogenesis, cell death resistance, invasion and metastasis. Furthermore, it is well established that tumor-promoting inflammation contributes to tumorigenesis. In this process, monocytes are attracted to the site of the tumor and educated towards a tumor-promoting macrophage phenotype. While HuR has been extensively studied in various tumor cell types, little is known about HuR in hepatocellular carcinoma (HCC). Thus, the aim of my work was to characterize the contribution of HuR to the development of cancer characteristics in HCC. I was particularly interested to investigate if HuR facilitates tumor-promoting inflammation, since a role for HuR has not been described in this context. To this end, I depleted HuR in HepG2 cells (HuR k/d) and used a co-culture model of HepG2 tumor spheroids and infiltrating monocytes to study the impact of HuR on the tumor microenvironment. I could show that depletion of HuR resulted in the reduction of cell numbers. Additionally, the expression of proliferation marker KI-67 and proto-oncogene c-Myc was reduced, supporting a proliferative role of HuR. Furthermore, exposure to cytotoxic staurosporine elevated apoptosis in HuR k/d cells compared to control cells. Concomitantly, the expression of the anti-apoptotic mediator B-cell lymphoma protein-2 (Bcl-2) was markedly reduced in the HuR k/d cells, pointing to an involvement of HuR in cell survival processes.
Accordingly, a pro-survival function of HuR was also observed in tumor spheroids, since HuR k/d spheroids exhibited a larger necrotic core region at earlier time points and showed elevated numbers of dead cells compared to control (Ctr.) spheroids. Interestingly, HuR k/d spheroids isplayed reduced numbers of infiltrated macrophages, suggesting that HuR contributes to a tumor-promoting, inflammatory microenvironment by recruiting monocytes/macrophages to the tumor site. Aiming at identifying HuR-regulated factors responsible for the recruitment of monocytes, I found reduced levels of the chemokine interleukin 8 (IL-8) in supernatants of HuR k/d spheroids, supporting a critical involvement of HuR in the chemoattraction of monocytes. Analyzing supernatants of co-cultures of macrophages and HuR k/d or Ctr. spheroids revealed additional differences in chemokine secretion patterns. Interestingly, protein levels of many chemokines were elevated in co-cultures of HuR k/d spheroids compared to control co-cultures. Albeit enhanced chemokine secretion was observed, less monocytes are recruited into HuR k/d spheroids, further underlining the necessity of HuR in cancer related monocyte/macrophage attraction and infiltration. Differences between chemokine profiles of mono- and co-cultured spheroids could be attributable to changes in spheroid-derived chemokines as a result of the crosstalk with the immune cells. Provided the chemokines originate from monocytes/macrophages, the different secretion patterns suggest that HuR contributes to the modulation of the functional phenotype of infiltrated macrophages, since the tumorenvironment is critically involved in the shaping of macrophage phenotypes. Regions of low-oxygen (hypoxia) represent another critical feature of tumors. Therefore, I next analyzed the impact of HuR on the hypoxic response. Loss of HuR attenuated hypoxia-inducible factor (HIF) 2α expression after exposure to hypoxia, while HIF-1α protein levels remained unaltered. Considering previous results of our group, showing that HIF-2α depletion (HIF-2α k/d) resulted in the enhanced expression of HIF-1α protein, I aimed to determine the involvement of HuR in the compensatory upregulation of HIF-1α protein in HIF-2α k/d cells. I could demonstrate that not only total HuR protein levels, but specifically cytoplasmic HuR was elevated in HIF-2α depleted cells pointing to enhanced HuR activity. Silencing HuR in HIF-2α deficient cells attenuated enhanced HIF-1α protein expression, thus confirming a direct role of HuR in the compensatory upregulation of HIF-1α. This as also reflected on HIF-1α target gene expression. I further investigated the mechanism underlying the compensatory HIF-1α expression in HIF-2α deficient cells. Analyzing HIF-1α mRNA expression, I excluded enhanced HIF1-α transcription and stability to account for elevated HIF-1α expression in HIF-2α k/d cells. HIF-1α promoter activity assays confirmed the mRNA data. Furthermore, HIF-1α protein half-life was not elevated in HIF-2α k/d cells compared to control cells, indicating that HIF-1α protein stability is not altered in HIF-2α k/d cells. Analysis of the association of HIF-1α with the translational machinery using polysomal fractionation finally revealed an increased istribution of HIF-1α mRNA in the heavier polysomal fractions in HIF-2α k/d cells compared to control cells. Since augmented ribosome occupancy is an indicator for more efficient translation, I propose enhanced HIF-1α translation as underlying principle of the compensatory increase in HIF-1α protein levels in HIF-2α k/d cells. In summary, my results demonstrate that HuR is critical for the development of cancer characteristics in HCC. Future work analyzing the impact of HuR on tumor-promoting inflammation, specifically macrophage attraction and activation could provide new trategies to inhibit macrophage-driven tumor progression. Furthermore, I provide evidence that HuR contributes to the hypoxic response by regulating the expression of HIF-1α and HIF-2α. Targeting single HIF-isoforms for tumor therapy should be carefully considered, because of their compensatory regulation when one α-subunit is depleted. Thus, therapeutic strategies targeting factors such as HuR that control both α-subunits and at the same time prevent compensation might be more promising.
Orthopoxviruses are large DNA viruses that replicate within the cytoplasm of infected cells encoding over a hundred different proteins. The orthopoxviral 68k ankyrin‐like protein (68k‐ank) is highly conserved among orthopoxviruses, and this study aimed at elucidating the function of 68k‐ank. The 68k‐ank protein is composed of four ankyrin repeats (ANK) and an F‐box‐like domain; both motifs are known proteinprotein interaction domains. The F‐box is found in cellular F‐box proteins (FBP), crucial components of cellular E3 ubiquitin (Ub) ligases. With yeast‐two‐hybrid screens and subsequent co‐immunoprecipitation analyses, it was possible to identify S‐phase kinase‐associated protein 1a (Skp1a) as a cellular counterpart of 68k‐ank via binding to the F‐box‐like domain. Additionally, Cullin‐1 was co‐precipitated, suggesting the formation of a viral‐cellular SCF E3 Ub ligase complex. Modified Vaccinia virus Ankara (MVA) ‐ being attenuated and unable to replicate in most mammalian cell lines due to a block in morphogenesis – nevertheless, expresses its complete genetic information attributing to its properties as promising vector vaccine. Conservation of 68k‐ank as the only ANK protein encoded by MVA implied a substantial role of this viral factor. Hence, its function in the viral life cycle was assessed by studying a 68k‐ank knock‐out MVA. A mutant phenotype manifested in nonpermissive mammalian cells characterized by a block succeeding viral early gene expression and by a reduced ability of the virus to shutoff host protein synthesis. Studies with MVA encoding a 68k‐ank F‐box‐like domain truncated protein revealed that viral‐cellular SCF complex formation and maintenance of viral gene expression are two distinct, unrelated functions fulfilled by 68k‐ank. Moreover, K1, a well‐described VACV host range factor of the ANK protein family, is able to complement 68k‐ank function. This suggests that gene expression of MVA putatively depends on the ANKs encoded in 68k‐ank. In addition to the important findings in vitro, first virulence studies with the mouse pox agent, ectromelia virus (ECTV) deleted of the 68k‐ank ortholog (C11) suggested that this factor contributes to ECTV virulence in vivo.
Unlimited self-renewal is an absolute prerequisite for any malignancy, and is the ultimate arbiter of the continuous growth and metastasis of tumors. It has been suggested that the self-renewal properties of a tumor are exclusively contained within a small population, i.e., the so-called cancer stem cells. Enhanced self-renewal potential plays a pivotal role in the development of leukemia. My data have shown that APL associated translocation products PML/RARalpha and PLZF/RARalpha increased the replating efficiency of mouse lin-/Sca1+ hematopoietic stem cells (HSCs). This effect is partly mediated by induction of gamma–catenin which is an important mediator of the Wnt signaling pathway and has been shown to be up regulated by the AML associated translocation products(AATPs). Suppression of gamma–catenin by siRNA can abrogate the increased replating efficiency induced by AATPs. Transduction of gamma–catenin in lin-/Sca1+ HSCs led to increased replating efficiency and the expression of stem cell markers Sca1 and c-kit. Additionally it induced accelerated cell cycle progression of mouse bone marrow HSCs. Transduction/transplantation mouse models have shown that ectopic expression of gamma–catenin in HSCs led to acute myeloid leukemia without maturation. These data suggest important roles of Wnt signaling pathway in the leukemogenesis induced by PML/RARalpha, PLZF/RARalpha and AML1/ETO. In contrast to AATPs, CML and Ph+-ALL associated translocation products p185(BCR-ABL) and p210(BCR-ABL) did not affect the self-renewal potential of hematopoietic stem/progenitor cells. However my studies indicated that their reciprocal translocation products p40(ABL/BCR) and p96(ABL/BCR) actually increased the replating efficiency of hematopoietic stem/progenitor cells. The effect is stronger when induced by p96(ABL/BCR) than by p40(ABL/BCR). It is very intriguing that p96(ABL/BCR) can activate Wnt signaling and up regulate the expression of HoxB4. Transduction/transplantation mouse model has shown that p40(ABL/BCR) and p96(ABL/BCR) both have their own leukemogenic potential. Given the fact that leukemic stem cells maintain the growth of tumor and are the origin of relapse, the cure of leukemia is dependent on the eradication of the leukemic stem cell and abrogation of aberrantly regulated self-renewal capability. Both t-RA and As2O3 have been shown to induce complete remission in APL patients with PML/RARalpha translocation product. However, t-RA as a single agent achieves completeremission (CR) but not complete molecular remissions (CMR). Therefore, virtually all patients will experience a relapse within a few months. In contrast to t-RA, As2O3 as a single agent is able to induce CR as well as CMR followed by long-term relapse-free survival in about 50% of APL patients even if relapsed after treatment with t-RA-containing chemotherapy regimens. Nothing is known about the mechanisms leading to the complete different clinical outcomes by the two compounds although both have been shown to induce differentiation of blast cells, proliferation arrest, induction of apoptosis and degradation of PML/RARalpha. We investigated the effect of t-RA and arsenic on PML/RARalpha-expressing cell population with stem cell capacity derived from the APL cell line NB4 as well as Sca1+/lin- murine bone marrow cells. We found that t-RA did not reduce the replating efficiency in PML/RARalpha- and PLZF/RARalpha-infected Sca1+/lincells whereas it selected small compact colonies representing very early progenitor cells. T-RA was unable to reduce the capacity to form colony forming units-spleen (CFU-S) of Sca1+/lin-cells expressing PML/RARalpha, additionally t-RA did not impair the capability of engraftment of NB4 cells in NOD/SCID mouse. On the contrary to t-RA, As2O3 abolished the aberrant self-renewal potential of Sca1+/lin- cells expressing PML/RARalpha. As2O3 not only abolished the replating efficiency of PML/RARalpha positive cells but also completely abrogated the ability of PML/RARalpha-positive HSC to produce CFU-S in vivo. On the contrary to As2O3, t-RA increased the absolute cell number and the percentage of cells in the side population with respect to the whole cell population in NB4 cells. Taken together these data suggest that arsenic but not all-trans retinoic acid overcomes the aberrant stem cell capacity of PML/RARalpha positive leukemic stem cells. My data prove for the first time that there is a direct relationship between the capacity of compounds to effectively target the LSC and their capacity to eradicate the leukemia, and, thereby, to induce complete molecular remission and long-term relapse-free survival. Thus, in order to increase the curative potential of leukemia therapies, future studies need to include the effect of given compounds on the stem cell compartment to determine their ability to eradicate the LSC.