Search

Supplementary Figure 3 from A Novel Recurrent Chromosomal Inversion Implicates the Homeobox Gene Dlx5 in T-Cell Lymphomas from Lck-Akt2 Transgenic Mice

Supplementary Figure 4 from A Novel Recurrent Chromosomal Inversion Implicates the Homeobox Gene Dlx5 in T-Cell Lymphomas from Lck-Akt2 Transgenic Mice

The oncogene v-akt was isolated from a retrovirus that induced murine thymic lymphomas. Transgenic mice expressing a constitutively activated form of the cellular homologue Akt2 specifically in immature T cells develop spontaneous thymic lymphomas. We hypothesized that tumors from these mice might exhibit oncogenic chromosomal rearrangements that cooperate with activated Akt2 in lymphomagenesis. Cytogenetic analysis revealed a recurrent clonal inversion of chromosome 6, inv(6), in thymic lymphomas from multiple transgenic founder lines, including one line in which 15 of 15 primary tumors exhibited this same rearrangement. Combined fluorescence in situ hybridization, PCR, and DNA sequence analyses showed that the distal inv(6) breakpoint resides at the T-cell receptor β chain locus, Tcrb. The proximal breakpoint maps to a region near a locus comprising the linked homeobox/transcription factor genes Dlx5 and Dlx6. Expression analysis of genes translocated to the vicinity of the Tcrb enhancer revealed that Dlx5 and Dlx6 are overexpressed in tumors exhibiting the inv(6). Experimental overexpression of Dlx5 in mammalian cells resulted in enhanced cell proliferation and increased colony formation, and clonogenic assays revealed cooperativity when both Dlx5 and activated Akt2 were coexpressed. In addition, DLX5, but not DLX6, was found to be abundantly expressed in three of seven human T-cell lymphomas tested. These findings suggest that the Dlx5 can act as an oncogene by cooperating with Akt2 to promote lymphomagenesis. [Cancer Res 2008;68(5):1296–302]

Supplementary Figure 2 from A Novel Recurrent Chromosomal Inversion Implicates the Homeobox Gene Dlx5 in T-Cell Lymphomas from Lck-Akt2 Transgenic Mice

Supplementary Figure 1 from A Novel Recurrent Chromosomal Inversion Implicates the Homeobox Gene Dlx5 in T-Cell Lymphomas from Lck-Akt2 Transgenic Mice

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

Angiogenesis, or the formation of new blood vessels, is stimulated by angiogenic factors such as vascular endothelial growth factor (VEGF). Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis. To explore the mechanism by which PEDF acts, recombinant PEDF was expressed with a 6x-His tag (for purification) and a green fluorescent protein (GFP) tag. The PEDF fusion protein was confirmed to be active in inhibition of endothelial cell proliferation and migration. Direct binding of PEDF to both vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 was demonstrated in an in vitro assay similar to an enzyme-linked immunosorbent assay (ELISA). PEDF was shown by immune-confocal microscopy to be localized within treated endothelial cells. When VEGF-stimulated endothelial cells were incubated with PEDF the VEGF receptors showed intracellular localization. These data suggest that the interaction between PEDF and VEGFR-1 or VEGFR-2 may be a possible mechanism for inhibiting angiogenesis. PEDF may be binding to the VEGF receptors to promote their internalization and/or degradation to limit VEGF responses in treated cells.

Zc3h8 encodes a zinc finger protein that is amplified in 2-6% of human breast tumors. Higher expression of Zc3h8 also results in lower rates of disease-free survival. Amplification of Zc3h8 expression has been shown to increase cell proliferation, migration, invasion and tumor growth in mice. Conversely, decreased expression alters cell behavior by decreasing cell proliferation, migration, invasion, and tumor growth in mice. Zc3h8 comprises three CCCH non-classical zinc finger domains at the C-terminus of the protein that we hypothesize bind to RNA. The interactions between Zc3h8 and nucleic acids may be important for the localization and/or function of Zc3h8 and therefore may be important for the cellular phenotypes observed when the gene is over or under expressed in cells. We used in vitro and in vivo assays to demonstrate that mutations in any of the three zinc finger domains results in a protein that fails to rescue aggressive cellular phenotypes in cells with reduced expression of Zc3h8, while the wild type protein can rescue these phenotypes. This implies that all three zinc finger domains are required for Zc3h8 function in promoting rapid and aggressive cell proliferation. In addition to zinc finger domains, Zc3h8 also contains a casein kinase-2 (CK2) phosphorylation site that may be important for protein localization and/or activation. We have previously shown that CK2 inhibitors alter Zc3h8 localization to nuclear PML Bodies. Mouse cells overexpressing Zc3h8 with a Thr to Ala (T32A) change that prevents CK2 phosphorylation grow very slowly in culture and cannot grow in more challenging 3D or soft agar growth chambers. However, Zc3h8 with a Thr to Glu (T32E) change that mimics constitutive CK2 phosphorylation results in rapidly growing cells that surpass overexpression of Zc3h8 wild type. Zc3h8 phospho-mutants also have altered localization with Zc3h8 T32E becoming diffuse in the nucleus and T32A localizing to nuclear foci. These data suggest that the phosphorylation state of Zc3h8 and its ability to bind nucleic acids via zinc finger domains are critical for the oncogenic behavior that results from amplified expression. We also hypothesize that Zc3h8 has an important role in establishing or maintaining PML Bodies involved in genome maintenance, transcription, and/or post-transcription regulation. Citation Format: John A. Schmidt, Tyler Doan, Emanuel Irizarry, Emily Harris, Keith G. Danielson, Janice E. Knepper. Molecular dissection of Zc3h8 functional domains and the implications for the oncogenic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1713.

Localized in PML nuclear bodies, the fetal liver zinc finger protein (Fliz), or ZC3H8, has been shown to increase cell proliferation, migration, and tumorigenesis in mouse mammary cell lines. While the exact function of ZC3H8 is unclear, PML nuclear bodies are critical centers for genome maintenance. Previous studies have suggested that these nuclear bodies act as deposits of sequestered DNA damage response proteins including SUMO-1, TP53, and BRCA-1. In addition to their localization to PML nuclear bodies, the phosphorylation of ZC3H8 by CK2 leads to dispersion of PML nuclear foci. For these reasons, we speculated that increased expression of Zc3h8, leading to greater protein availability, is involved in the suppression of DNA repair. This in return would increase genome instability and cancer cell aggression. This study uses the single cell comet assay to measure DNA damage repair following treatment with the topoisomerase inhibitor and chemotherapy drug etoposide. Two mouse mammary tumor cells lines, cV1A 01-51 and cV1A 03-31 were transfected to drive an shRNA targeting ZC3H8 (Flizout). In addition, the cV1A 03-31 Flizout line was rescued with a Zc3h8 construct with altered base coding to make transcribed products immune to shRNA targeting (SynFliz). We have also developed a constitutively charged cV1A 03-31 T32E mutant and a non-phosphorylatable T32A mutant. The nontumorigenic Comma1-D cell line was transfected with a vector to increase Zc3h8 expression. Ability to repair after etoposide treatment in these mutants was compared to controls carrying endogenous levels of Zc3h8. Etoposide leads to double strand breaks in the DNA during replication, and all cell lines were sensitive to etoposide and able to undergo repair. For the cV1A 01-51 lines, the control and Flizout mutants had a nonsignificant difference in post-repair comet length (p = 0.06) but a significant difference in tail moment (p = 0.003). Between the control and the mutants, the difference in overall comet length was 4.7%, and the difference in tail moment was 15.8%, with cells expressing higher levels of Zc3h8 being less adept in carrying out repair of double strand DNA breaks. This suggests an inverse relationship between Zc3h8 expression and DNA damage repair. This may indicate that evaluating Zc3h8 expression could be considered for determining the ability of tumor cells to respond to chemotherapy agents. Furthermore, altering its expression may be a novel approach for cancer treatment. Citation Format: Spencer Li, John A. Schmidt, Janice E. Knepper. The effect of Zc3h8 expression levels on sensitivity to DNA damage and repair in mouse mammary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1757.

The zinc finger protein Zc3h8 is overexpressed in many mouse and human solid tumors. We have shown that increased expression of Zc3h8 in cultured mouse mammary cells is associated with increased proliferation rate, increased motility and invasiveness, increased capacity for growth in three dimensions, and increased capacity for tumor formation. Partial knockdown of Zc3h8 expression from mouse mammary tumor cells has the opposite effect. The Zc3h8 protein localizes to both PML bodies and Cajal bodies in the nucleus and features of this localization are altered by treatment of cells with a casein kinase 2 inhibitor, TBB. We have embarked upon an analysis of the contributions of structural features of the Zc3h8 protein through mutagenesis of the gene at key positions. The casein kinase 2 phosphorylation site at position T32 was mutated to either an alanine to prevent phosphorylation, or to glutamic acid to mimic constitutive phosphorylation. Each of the three zinc fingers in the C-terminal region was mutated to replace one cysteine residue. The mutations were introduced into a variant of the zc3h8 gene that has 6 mutations introduced at synonymous positions in order to escape knockdown by RNAi, although the protein sequence generated is identical to the original Zc3h8. Each of the mutant constructs was introduced into mouse mammary tumor cells with knockdown of the endogenous Zc3h8. Mutation of the phosphorylation site T32A led to the formation of fewer, although larger PML bodies, with continued ZC3h8 co-localization. The T32E mutant, with constitutive negative charge at this position led to the formation of a large number of small nuclear foci. Mutant constructs were stably transfected into tumor cells with knockdown of endogenous Zc3h8, and behavior of the mutants was compared to the behavior of cells transfected with the synonymously mutated gene construct. Disruption of each of the zinc fingers individually led to decreased proliferation rate, decreased growth as spheroids or in soft agar, and decreased invasion through Matrigel relative to the synonymous Zc3h8. Tumor cells with Zc3h8 knockdown failed to form tumors in syngeneic BALB/c mice within 10 weeks, while cells transfected with a negative control vector formed tumors in all animals. Rescue of Zc3h8 expression with the synonymous mutant led to tumor formation in 50% of the animals, while no tumors formed from cells transfected with zinc finger mutants. We conclude that the zinc fingers contribute to aggressive tumor cell behavior while the T32 phosphorylation site is essential for proper localization to nuclear bodies. Citation Format: John A. Schmidt, Emily R. Duffner, Emily M. Harris, Tyler Doan, Emanuel Irizarry, Keith G. Danielson, Janice E. Knepper. Structural analysis of features contributing to the oncogenic phenotype conferred by the zinc finger nuclear body protein Zc3h8 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2484.

The ZC3H8 gene encodes a protein with three zinc finger motifs in the C-terminal region, suggesting a role as an RNA binding protein, but whose specific function remains unclear. ZC3H8 protein localizes to both PML bodies and Cajal bodies within the nucleus, as confocal microscopy demonstrates colocalization of ZC3H8 with both PML and coilin proteins. ZC3H8 has a casein kinase 2 phosphorylation site in the N-terminal region, and treatment with a casein kinase 2 inhibitor causes the numerous PML bodies and their associated ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restores PML bodies to their original state. We constructed a mutant of ZC3H8 that removed the predicted CK2 phosphorylation site by replacing the serine with an alanine residue. Cells transfected with this mutant had reduced numbers of PML bodies, similar to cells treated with the CK2 inhibitor. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable serine was predicted to behave as a constitutive mutant. This mutant did not exhibit differences from wild type localization. These experiments suggest that ZC3H8 integrity is key to maintenance of PML bodies. Other studies have identified ZC3H8 as a component of the LEC (little elongation complex), and we found that another identified component, ICE2 (Narg) also localizes to PML bodies. The ZC3H8 gene is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer. We have used RNA silencing to decrease levels of expression in two independent mouse mammary tumor cell lines. Cells with lower ZC3H8 expression have dramatic phenotypic changes, showing decreased rates of proliferation, slower migration in wound healing assays, formation of fewer and smaller colonies in soft agar assays, and decreased ability to invade through a basement membrane in transwell assays. Further, ZC3H8-depleted cells have decreased rates of growth as spheroids and decreased growth in vivo in syngeneic mice. We developed an inducible vector driving expression of ZC3H8. Upon treatment with the inducer, doxycycline, cells assume a more aggressive phenotype. We introduced a synonymous mutant of ZC3H8 unable to be silenced by the RNAi into the knockdown cells, and rescued the aggressive phenotype of the original tumor cells. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells leads to a less pronounced, but opposite, increase in proliferation, motility, and invasion. We have engineered the synonymous ZC3H8 with disrupted zinc finger motifs, and preliminary results suggest that disruption of the first zinc finger is not sufficient to cause loss of the aggressive phenotype. We suggest that ZC3H8 is an oncogene with substantial effects on the cancer cell phenotype. These effects are hypothesized to be associated with ZC3H8 function in PML bodies. Citation Format: John A. Schmidt, Emily Duffner, Gerard Walker, Keith G. Danielson, Janice E. Knepper. ZC3H8 associates with PML bodies and influences aggressive tumor cell behavior [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 2555. doi:10.1158/1538-7445.AM2017-2555

The Zinc Finger CCCH-Type Containing 8 (zc3h8 or Fliz1) gene is overexpressed in many mouse and human breast cancer cell lines that we examined, although the exact function of this gene and the ZC3H8 protein are largely unknown. To characterize ZC3H8 protein, we localized it to distinct sub-nuclear domains that also contain markers for promyelocytic leukemia (PML) nuclear bodies and Cajal bodies. Furthermore, when cells are treated with a casein kinase-2 (CK2) inhibitor, ZC3H8 mislocalizes out of the nuclear bodies and diffuses into the nucleoplasm. CK2 is a known activator of transcription and a potential drug target. ZC3H8 contains a putative CK2 phosphorylation site at position T32, and may be regulated by this kinase. Based on this localization and other preliminary data, we hypothesize that ZC3H8 has a role in transcriptional regulation or RNA modification at these nuclear bodies. This hypothesis is supported by published studies that have found a role for ZC3H8 in the Little Elongation Complex (LEC), which transcribes snRNA involved in RNA splicing in nuclear bodies. Since many tumors and cancer cell lines overexpress zc3h8, we used stably expressed shRNA to knock down expression in two independent mouse breast cancer cell lines for in vitro and in vivo studies. Cells with reduced zc3h8 expression have decreased rates of proliferation and migration compared to overexpressing cells when tested using MTS and wound healing assays. Decreased rates of migration in wound healing assays are reversed by overexpression of the known migration-associated gene MEMO1. Additionally, cells with reduced expression of zc3h8 failed to form colonies in soft agar or pass through an extracellular matrix coating a transwell chamber- a phenotype that can be rescued using recombinant human zc3h8. Conversely, untransformed mouse mammary COMMA-1D cells gain the ability to form colonies in soft agar when transfected to stably overexpress zc3h8. Our in vitro studies suggest that zc3h8 expression has a role in tumorigenesis. These observations have been supported with in vivo studies in mice. We implanted cells knocked down for zc3h8 and control cells into the mammary glands of syngeneic female mice. Mice injected with cells that have reduced expression of zc3h8 form substantially smaller tumors by weight compared to control. When taken together, these data show that zc3h8 has a role in the formation and progression of cancer through its association with nuclear bodies involved in transcriptional regulation and RNA modification. Citation Format: John A. Schmidt, Keith G. Danielson, Jani L. Swiatek, Emily R. Duffner, Janice E. Knepper. Association of ZC3H8 with nuclear bodies and its role in promoting tumor cell behavior in vitro and in vivo. [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 1134.