Overexpression of CA1 mRNA and the CA I Protein in Tumor Cells Does Not Change the Gene Expression of the ECM Proteins

In our study, we performed retroviral transduction to overexpress codon-optimized variant of gene encoding human carbonic anhydrase I (optiCA1) in two tumor cell lines PC3 and MDA-MB-231, derived from human prostatic and breast carcinoma respectively. We achieved significantly enhanced and stable overexpression of exogenous optiCA1 gene. The expression of endogenous, wild CA1 gene was found to be normally low (Ct 28.6 for PC3 cells) or below to the detection limit (Ct 35.5 for MDA-MB-231 cells). No morphological changes and no decreasing viability of tumor cells were observed upon stable overexpression of the optiCA1 gene. In our study we have shown that the overexpression of the optimized human CA1 in engineered PC3 and MDA-MB-231 cells did not induce similar changes as we observed in tumor cells cultivated in the presence of human sera containing extensively high titers of anti-CA I autoantibodies from patients with complete remission of malignant disease. In both optiCA1transduced cell lines, the expression of selected genes responsible for basal lamina assembly, cytoskeleton, extracellular matrix proteins and proto-oncogenes (COL1A1, COL4A4, LAMC2, CTHRC1, and WNT7B) was not changed.


Introduction
Carbonic anhydrase I (CA I) together with other 14 members of family of carbonic anhydrases, catalyzes the reversible hydration of carbon dioxide [1,2]. The CA I enzyme is abundant in erythrocytes (CA I is the second most abundant protein after hemoglobin), but its catalytic efficiency is relatively low [3]. However, the familiar homozygous deficiency of carbonic anhydrase I with virtually absent CA I has no clinical consequences with no hematological and renal defects [4]. The presence of high levels of autoantibodies against CA I (anti-CA I Abs) in the patients' sera was found to be linked with spontaneous tumor regression and improved patient survival, but also with the suppression of hematopoiesis [5,6]. The spectrum of the spontaneously regessed tumors are wide: Hodgkin's disease, non-Hodgkin lymphoma, Ewings's sarcoma and breast cancer [6], multiple myeloma and ovarian cancer (data not shown). Tumor cells of different origin were cultivated in vitro in the media supplemented with patients' sera positive for anti-CA I Abs. As stated previously in the RNA microarray report: "The downregulated genes in cells treated in the presence of patients' sera positive for anti-CA I Abs compared to the negative sera include several genes related to adhesion and cytoskeleton, such as different collagens and keratin family members. In addition, some proto-oncogene WNT7B was downregulated as well" [7]. More precisely, these cells displayed decreased expression of mRNAs of the genes associated with extracellular matrix (ECM) and basal lamina (BL) assembly (COL1A1-collagen I, COL4A4-collagen IV, LAMC2-laminin gamma), cytoskeleton (KRT14-keratin 14 type I), WNT7B and collagen triple helix repeat containing 1 (CTHRC1). Surprisingly, the expression of mRNA of the CA1 gene was increased (up to thirteen times in the PC3 tumor cell line) [7]. This observation led us to examine the possible association of the CA I with the overexpression on the abovementioned "non CA1 genes". Therefore, we have performed retroviral transduction to constantly overexpress codon-optimized variant of gene encoding CA I (optiCA1) in PC3 and MDA-MB-231 tumor cells. We were not able to see any association of the increased CA1 expression (tested with the codon-optimized variant of gene encoding human carbonic anhydrase I) with the decreased mRNA expression of genes associated with the ECM and BL assembly and the WNT7B and CTHRC.

Overexpression of Codon Optimised Human CA1 Gene Reduces Endogenous CA1 Gene Expression but Does Not Change Expression of Genes Encoding Extracellular Matrix Proteins
The optiCA1 version of human CA1 gene was excised from commercial expression vector pSF-CMV-CA1 (Origene) and subcloned into the retroviral vector pJZ308 as described in [8], creating ST44optiCA1 retrovirus (Figure 1a). Because not all tRNAs are expressed equally, codon optimization of CA1 DNA sequence in pSF-CMV-CA1 by changing its codons was made to match the most prevalent tRNAs. This enables more efficient translation of target CA1 gene. Alignment of DNA of codon optimized version (optiCA1) and endogenous native (endoCA1) gene revealed identity of 75%, while amino acid sequences of CA1 remained 100% identical. Transduction using retrovirus ST44optiCA1 guaranteed stable overexpression of codon optimized human optiCA1 gene in prostatic tumor cells PC3 (PC3/optiCA1) and breast carcinoma cells MDA-MB-231 (MDA-MB-231/optiCA1) (Figure 1b). Overexpression of CA1 gene was confirmed by Western blot (Figure 1c), but concurrently, the expression of endoCA1 native gene was found to be decreased at the level of 70-80% ( Figure 1d).
(CTHRC1). Surprisingly, the expression of mRNA of the CA1 gene was increased (up to thirteen times in the PC3 tumor cell line) [7]. This observation led us to examine the possible association of the CA I with the overexpression on the abovementioned "non CA1 genes". Therefore, we have performed retroviral transduction to constantly overexpress codon-optimized variant of gene encoding CA I (optiCA1) in PC3 and MDA-MB-231 tumor cells. We were not able to see any association of the increased CA1 expression (tested with the codon-optimized variant of gene encoding human carbonic anhydrase I) with the decreased mRNA expression of genes associated with the ECM and BL assembly and the WNT7B and CTHRC.

Overexpression of Codon Optimised Human CA1 Gene Reduces Endogenous CA1 Gene Expression but Does Not Change Expression of Genes Encoding Extracellular Matrix Proteins
The optiCA1 version of human CA1 gene was excised from commercial expression vector pSF-CMV-CA1 (Origene) and subcloned into the retroviral vector pJZ308 as described in [8], creating ST44optiCA1 retrovirus ( Figure 1a). Because not all tRNAs are expressed equally, codon optimization of CA1 DNA sequence in pSF-CMV-CA1 by changing its codons was made to match the most prevalent tRNAs. This enables more efficient translation of target CA1 gene. Alignment of DNA of codon optimized version (optiCA1) and endogenous native (endoCA1) gene revealed identity of 75%, while amino acid sequences of CA1 remained 100% identical. Transduction using retrovirus ST44optiCA1 guaranteed stable overexpression of codon optimized human optiCA1 gene in prostatic tumor cells PC3 (PC3/optiCA1) and breast carcinoma cells MDA-MB-231 (MDA-MB-231/optiCA1) ( Figure 1b). Overexpression of CA1 gene was confirmed by Western blot (Figure 1c), but concurrently, the expression of endoCA1 native gene was found to be decreased at the level of 70-80% ( Figure 1d).  The expression of selected genes encoding extracellular matrix and basal lamina components (COL1A1-collagen I, COL4A4-collagen IV, LAMC2-laminin gamma), and proto-oncogenes (CTHRC1-collagen triple helix repeat containing protein 1, WNT7B-Wingless-Type MMTV Integration Site Family, Member 7) were not significantly changed in optiCA1 overexpressing tumor cells (Figure 2a). The cell morphology, which was linked with altered expression profile as we reported previously [7], was not changed when optiCA1 was overexpressed (Figure 2b).

Overexpression of Codon Optimised Human CA1 Gene Doesn't Reduce Short Term Cell Proliferation
After transduction with ST44optiCA1 retrovirus, the growth of transduced tumor cells was not reduced rapidly in the first days (p = 0.02-0.05 on days 7 and 8). During selection with geneticin (G418), the cell confluence of 100 % was reached within 9 days ( Figure 3) and transduced resistant optiCA1 tumor cells proliferated. Dilution of ST44optiCA1 virus containing media revealed successful transduction also with ten-times diluted media (dilution 0.1×; Figure 3), indicating high titer of the virus. We achieved multiplicity of infection (MOI) approx. of 5-10. After long term cultivation (up to 12 weeks), and after four passages, optiCA1 overexpressing PC3 cells were found to be significantly lagging in the growth and proliferation in comparison to the parental cells (data not shown). The viability of the cells was not affected.

Overexpression of Codon Optimised Human CA1 Gene Doesn't Reduce Short Term Cell Proliferation
After transduction with ST44optiCA1 retrovirus, the growth of transduced tumor cells was not reduced rapidly in the first days (p = 0.02-0.05 on days 7 and 8). During selection with geneticin (G418), the cell confluence of 100 % was reached within 9 days (Figure 3) and transduced resistant optiCA1 tumor cells proliferated. Dilution of ST44optiCA1 virus containing media revealed successful transduction also with ten-times diluted media (dilution 0.1×; Figure 3), indicating high titer of the virus. We achieved multiplicity of infection (MOI) approx. of 5-10. After long term cultivation (up to 12 weeks), and after four passages, optiCA1 overexpressing PC3 cells were found to be significantly lagging in the growth and proliferation in comparison to the parental cells (data not shown). The viability of the cells was not affected.

Discussion
In the sera of cancer patients with spontaneous tumor regression after high dose therapy and autologous stem cell transplantation, a high titer of human anti-CA1 autoantibodies (anti-CA I Abs) was discovered [5]. In vitro, in the tumor cell line cultures, the anti-CA I-positive patients' sera induced changes of the cell morphology and downregulated expression of selected genes responsible for the formation of BL, ECM, and cytoskeleton. The expression of proto-oncogenes WNT7B and CTHRC1 was decreased too [7]. Simultaneously, in the presence of sera with anti-CA I Abs, the expression of the CA1 gene mRNA was upregulated among all tested tumor cell lines. In prostatic tumor cells PC3, CA1 mRNA expression increased up to thirteen times during anti-CA I Abs-positive patient's sera treatment. If the cells were grown in the presence of human sera negative for anti-CA I Abs the expression of endogenous CA1 mRNA remained rather low [7].
We were curious about the association between the upregulation of CA1 gene and the downregulation of the genes responsible for the formation of BL, ECM, and some proto-oncogenes (WNT7B and CTHRC). Therefore, we performed retroviral transduction to overexpress codon-optimized variant of gene encoding human carbonic anhydrase I (optiCA1) in two tumor cell

Discussion
In the sera of cancer patients with spontaneous tumor regression after high dose therapy and autologous stem cell transplantation, a high titer of human anti-CA1 autoantibodies (anti-CA I Abs) was discovered [5]. In vitro, in the tumor cell line cultures, the anti-CA I-positive patients' sera induced changes of the cell morphology and downregulated expression of selected genes responsible for the formation of BL, ECM, and cytoskeleton. The expression of proto-oncogenes WNT7B and CTHRC1 was decreased too [7]. Simultaneously, in the presence of sera with anti-CA I Abs, the expression of the CA1 gene mRNA was upregulated among all tested tumor cell lines. In prostatic tumor cells PC3, CA1 mRNA expression increased up to thirteen times during anti-CA I Abs-positive patient's sera treatment. If the cells were grown in the presence of human sera negative for anti-CA I Abs the expression of endogenous CA1 mRNA remained rather low [7].
We were curious about the association between the upregulation of CA1 gene and the downregulation of the genes responsible for the formation of BL, ECM, and some proto-oncogenes (WNT7B and CTHRC). Therefore, we performed retroviral transduction to overexpress codon-optimized variant of gene encoding human carbonic anhydrase I (optiCA1) in two tumor cell lines PC3 and MDA-MB-231. Codon optimization is switching the codons used in a transgene without changing the amino acid sequence that it encodes for. This typically dramatically increases the abundance of the protein the codon optimized gene encodes because it removes "rare" codons and replaces them with abundant codons. So, in order to efficiently express protein in higher quantities, the more abundant of the degenerate tRNAs have to be used. Thus, a gene can be mutated (or synthetized de novo) to change the codons used for coding particular amino acids, without changing the amino acid sequence of the protein itself. Rare codons are replaced by codons that are more abundant in the genes of the host organism. Using transduction with retroviral vector, the overexpression of desired gene can be enhanced more than 100-times and remains stable for weeks [8]. With stable optiCA1 gene overexpression, we intended to achieve increased of CA1 levels analogous to those reported after cultivating anti-CA I Abs positive patients' sera linked with favorable prognosis of malignant disease. However, we observed that optiCA1 overexpressing tumor cells, when compared with the controls, had not statistically changed expression profile of mRNAs (COL1A1, COL4A4, LAMC2, CTHRC1, and WNT7B). Nevertheless, we observed a slight (but not statistically significant) downregulation of the expression of innate endogenous CA1 gene in tumor cells. However, one should be aware although the amino acid sequence of the "opti" CA I is the same as in the wild type CA I enzyme the sequence of CA1 mRNA in the optiCA1 gene is differs from the wild type CA1 mRNA.
The gene encoding carbonic anhydrase I or carbonic anhydrase I protein in humans is not essential, as it has been shown in the study of familial deficiency of CA I synthesis [4]. The CA I role and exact mechanism of CA I action in spontaneous tumor regression remains perplexing. In our pilot study we have shown, that the optiCA1 overexpression alone (in the short-time period after transduction, i.e., up to 10 days) does not mimic the effect of the anti-CA I Abs-positive patients' sera on tumor cells in vitro. The future tasks are further studies of the long-term period effect(s) of the optiCA1 overexpression on cellular processes and to prove the tumorigenicity of engineered optiCA1 overexpressing cells in vivo.
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Construction of Recombinant Retroviral Vector Containing Human CA1 Gene and Retrovirus Production
Intronless human CA1 gene (codon optimized version; optiCA1) was cloned from the pSF-CMV-CA1 expression vector (Oxford Genetics Ltd., Oxford, UK) using standard cloning techniques into the bicistronic retroviral vector pJZ308 derived from Moloney murine leukemia virus (MoMuLV) [8]. The optiCA1 gene encodes identical protein sequence to the endogenous CA1 gene. Retroviral construct named pST44optiCA1 ( Figure 1A) was verified by PCR and the open reading frame of the optiCA1 gene was sequenced. Retroviral vector pST44optiCA1 was used for production of replication deficient retroviral particles ST44optiCA1 in packaging cells GP+E-86 and GP+envAM12 as described in [8]. Virus-containing medium for transduction of target tumor cells was collected from semi-confluent