ABCB1 Amplicon Contains Cyclic AMP Response Element-Driven TRIP6 Gene in Taxane-Resistant MCF-7 Breast Cancer Sublines

A limited number of studies are devoted to regulating TRIP6 expression in cancer. Hence, we aimed to unveil the regulation of TRIP6 expression in MCF-7 breast cancer cells (with high TRIP6 expression) and taxane-resistant MCF-7 sublines (manifesting even higher TRIP6 expression). We found that TRIP6 transcription is regulated primarily by the cyclic AMP response element (CRE) in hypomethylated proximal promoters in both taxane-sensitive and taxane-resistant MCF-7 cells. Furthermore, in taxane-resistant MCF-7 sublines, TRIP6 co-amplification with the neighboring ABCB1 gene, as witnessed by fluorescence in situ hybridization (FISH), led to TRIP6 overexpression. Ultimately, we found high TRIP6 mRNA levels in progesterone receptor-positive breast cancer and samples resected from premenopausal women.

Due to various modules, TRIP6 interacts with a plethora of partners extensively summarized elsewhere [4]. TRIP6 resides in the cell cytoplasm, where it accumulates at focal adhesions [5] and adherent junctions [6][7][8], the sites associated with the actin cytoskeleton. Additionally, TRIP6 has been described to shuttle between the cell nucleus and cytoplasm [2]. The N-terminally truncated isoform of TRIP6 (nTRIP6) entirely localizes in

Collection and Processing of Breast Cancer Tissue Samples
Breast cancer tissue samples (N = 95) were collected and snap-frozen during primary surgery in The Faculty Hospital Motol and Institute for the Care for Mother and Child (Prague, Czech Republic) between 2003 and 2009. Sample processing was described in detail previously [35,36]. Samples from 82 patients were collected during the primary surgery before any chemotherapy or hormonal therapy (adjuvant group; ACT group). Samples from the second group of patients (N = 13) were collected during the primary surgery after neoadjuvant cytotoxic therapy with regimens containing taxanes or taxanes in combination with 5-fluorouracil and/or anthracycline, and cyclophosphamide (NACT group), a standard regimen in the period of sample collection. Noteworthy, the current guidelines do not support the addition of 5-fluorouracil to the anthracycline (Doxorubicin/Epirubicin)cyclophosphamide regimen.
A response to NACT was evaluated pre-and post-therapy by ultrasonography. Histological classification of carcinomas was performed according to standard diagnostic procedures [37]. The expression of estrogen and progesterone receptors was assessed immunohistochemically (IHC) with the 1% cut-off value for classification of tumors as hormone receptor positive. ERBB2 status was defined as positive in samples with IHC score 2+ or 3+ confirmed by fluorescence in situ hybridization or silver in situ hybridization. The cut-off between high and low expression of proliferative marker Ki-67 was 13.25% [38]. Samples were subtyped according to hormone receptor and ERBB2 expression as triplenegative (TNBC) subtype, ERBB2 subtype and luminal subtype [39]. Disease-free survival (DFS) was defined as the time elapsed between surgery and disease recurrence.

Isolation of Nucleic Acids and Proteins
Cultured cells were harvested by trypsin-EDTA solution and washed. Breast cancer tissue samples were grounded to powder by mortar and pestle under liquid nitrogen. Nucleic acids and protein were isolated using Allprep DNA/RNA/protein Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Nucleic acids were quantified using Quanti-iT TM PicoGreen TM dsDNA Assay Kit (Invitrogen TM , Carlsbad, CA, USA) and Quant-iT RiboGreen RNA Assay Kit (Invitrogen) in Infinite M200 microplate reader (Tecan Group Ltd., Männendorf, Switzerland). RNA integrity was checked by Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Assay Kit (Agilent Technologies, Inc., Santa Clara, CA, USA).

Quantitative Reverse Transcription PCR (qRT-PCR)
The real-time PCR study design adhered to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines [40]. The synthesis of complementary DNA (cDNA) is described in Table S1. The used TaqMan ® Gene Expression probes and PCR conditions are specified in Tables S2 and S3. IPO8 and MRPL19 were used as reference genes in patient cohorts based on their stability, as previously published [35]. To achieve the best reaction efficiency (>90%), we optimized the cycling conditions of each assay using a calibration curve as described previously [41,42]. For cell lines, the threshold cycle (Ct) of the gene of interest (GOI) was normalized to the reference gene (REF) by the following Genes 2023, 14, 296 4 of 23 formula ∆Ct = Ct REF − Ct GOI . To compare gene expression between MCF-7 cells and taxane-resistant MCF-7 sublines, we calculated the ∆∆Ct value by the following formula ∆∆Ct = ∆Ct RES − ∆Ct MCF−7 . Otherwise, to compare gene expression, fold change was calculated using the 2 −∆∆Ct method [43].

Assessment of Gene Copy Number
Genomic DNA ( For ABCB1 in the 0035R subline, data were not corrected by the TERT reference gene DNA level as the TERT copy number likely altered in these cells ( Figure A1). To estimate gene copy number gain or loss in taxane-resistant sublines, we subtracted the normalized (∆Ct) values as follows this formula ∆∆Ct = ∆Ct RES − ∆Ct MCF−7 , where RES means taxane-resistant MCF-7 subline.

Dual-Luciferase Reporter Assay
Cells (2.0 × 10 5 ) were seeded into wells of Nunc TM F96 MicroWell TM plate (Cat. No. 236105, Thermo Fisher Scientific) in paclitaxel-free medium. After 24 h, enabling cells to attach to a surface, the cells were co-transfected with 100 ng of DNA per well at a 100:1 ratio (reporter to co-reporter) using jetPRIME ® transfection reagent (Polyplus-Transfection, Illkirch, France) following the manufacturer's instructions. After 4 h, the transfection mix was replaced by a fresh paclitaxel-free cell culture medium. After 48 h post-transfection, samples were assayed by Nano-Glo ® Dual-Luciferase Assay System I (Promega). Plates were read after 10 min of incubation in M200 Pro Plate Reader (Tecan Group Ltd.).

DNA Methylation Profiling
Bisulfite conversion of 500 ng DNA was performed with a EZ DNA Methylation TM Kit (Zymo Research, Irvine, CA, USA), according to the manufacturer's protocol. The genomewide DNA methylation was assessed by the Infinium Human MethylationEPIC BeadChip platform (Illumina, San Diego, CA, USA) following the manufacturer's instructions. The microarray was scanned by the Illumina iScan system. The obtained data were further processed using the R language [46]. Quality control and data normalization were carried out in the minfi package as described previously [47,48]. Raw data were converted into β values for further analysis [49,50]. Probes mapped to single nucleotide polymorphism were removed from the analysis [51]. Differentially methylated probes were defined with |∆β| > 0.2 (20% difference). The β value is defined as the ratio between methylated versus unmethylated alleles.

Statistical Analysis
Graphs and statistical analysis were generated in Graph Pad Prism 9.2.0 (GraphPad Software, San Diego, CA, USA) regarding the recommendations described by others [53]. The SPSS v16.0 program (SPSS Inc, Chicago, IL, USA) was used for whole gene CpG methylation data and associations with breast cancer clinic pathological data. The normality of data was tested by the Shapiro-Wilk test prior to statistical analysis. Associations of transcripts with clinical data were assessed by the non-parametric Mann-Whitney, Kruskal-Wallis, and Spearman rank test. All p-values were obtained by two-sided tests. A p-value of <0.05 was considered statistically significant.
Variances were compared by F-test prior to unpaired t-test analysis. The distribution of residuals was checked by residual plot, homoscedasticity plot, and QQ plot. Individual statistical analysis is specified in each figure or table legend.

TRIP6 as Well as ABCB1 Are Overexpressed in Taxane-Resistant MCF-7 Sublines
Recently, we established Stony Brook 0035-resistant MCF-7 subline (0035R subline) from the same parental MCF-7 cells as paclitaxel-resistant MCF-7 subline (PacR) [34]. SB-T-0035 is a paclitaxel derivative in that a dimethyl carbamoyl group replaces the methyl group at the C10 position of the baccatin core ( Figure 1). scripts with clinical data were assessed by the non-parametric Mann-Whitney, Kruskal-Wallis, and Spearman rank test. All p-values were obtained by two-sided tests. A p-value of <0.05 was considered statistically significant.
Variances were compared by F-test prior to unpaired t-test analysis. The distribution of residuals was checked by residual plot, homoscedasticity plot, and QQ plot. Individual statistical analysis is specified in each figure or table legend.

TRIP6 as well as ABCB1 are Overexpressed in Taxane-resistant MCF-7 Sublines
Recently, we established Stony Brook 0035-resistant MCF-7 subline (0035R subline) from the same parental MCF-7 cells as paclitaxel-resistant MCF-7 subline (PacR) [34]. SB-T-0035 is a paclitaxel derivative in that a dimethyl carbamoyl group replaces the methyl group at the C10 position of the baccatin core ( Figure 1). We were interested in whether the TRIP6 gene (7q22.1, 100.8Mb) is overexpressed in 0035R subline similarly to the already described PacR subline [21], and we wanted to identify what mechanisms underpin TRIP6 overexpression in taxane-resistant MCF-7 sublines. Regarding de novo expression of the adjacent ABCB1 gene (7q21.12, 87.5Mb), we hypothesized that co-amplification could drive enhanced levels of TRIP6 and ABCB1 in PacR cells [33,34].
We compared TRIP6 and ABCB1 copy number (Figure 2A), mRNA level ( Figure 2B), and protein level ( Figure 2C) between MCF-7 cells and taxane-resistant MCF-7 sublines. Due to both the target (TRIP6, ABCB1) and reference (RPPH1, TERT) gene copy numbers being unknown in all assayed cell samples, we roughly estimated copy number gain or loss from ΔΔCt values obtained by duplex real-time TaqMan ® PCR ( Figure 2A, Figure  A1). The ABCB1 and TRIP6 gene copy number increased in 0035R cells (ΔΔCtABCB1 = 2.32, ΔΔCtTRIP6 = 2.38) although less than in PacR cells (ΔΔCtABCB1 = 3.42, ΔΔCtTRIP6 = 3.50). The level of TRIP6 mRNA increased in 0035R cells (ΔΔCtTRIP6 = 1.35) although less than in PacR cells (ΔΔCtTRIP6 = 2.16) compared to MCF-7 cells. The level of TRIP6 protein increased approximately by 3.5-fold in both taxane-resistant MCF-7 sublines compared to MCF-7 cells ( Figure 2B). Furthermore, we also found markedly elevated levels of ABCB1 mRNA and protein in 0035R cells, although it was two-fold lower compared to PacR cells ( Figure 2B and 2C). We were interested in whether the TRIP6 gene (7q22.1, 100.8Mb) is overexpressed in 0035R subline similarly to the already described PacR subline [21], and we wanted to identify what mechanisms underpin TRIP6 overexpression in taxane-resistant MCF-7 sublines. Regarding de novo expression of the adjacent ABCB1 gene (7q21.12, 87.5Mb), we hypothesized that co-amplification could drive enhanced levels of TRIP6 and ABCB1 in PacR cells [33,34].
Collectively, TRIP6 copy number, mRNA level, and protein level increased in line with ABCB1 copy number, mRNA, and protein level, suggesting that co-amplification is accountable for their increased expression.

A Few TRIP6 Loci Pre-Exists in MCF-7 Cell Line
Variation in TRIP6 copy number might underlie high TRIP6 mRNA and protein expression even in parental MCF-7 cells. In addition, the massive distribution and subcultivation of MCF-7 cells during the last 50 years resulted in enormous MCF-7 cell line heterogeneity [54]. Thus, we first determined the karyotype of MCF-7 cells used in this study.

A Few TRIP6 Loci pre-exists in MCF-7 Cell Line
Variation in TRIP6 copy number might underlie high TRIP6 mRNA and protein expression even in parental MCF-7 cells. In addition, the massive distribution and subcultivation of MCF-7 cells during the last 50 years resulted in enormous MCF-7 cell line heterogeneity [54]. Thus, we first determined the karyotype of MCF-7 cells used in this study.

Chromosome 7 is Rearranged in Taxane-resistant MCF-7 Sublines
To validate amplification of the region encompassing TRIP6 and ABCB1, we carried out similar FISH analyses in taxane-resistant MCF-7 sublines.

Chromosome 7 Is Rearranged in Taxane-Resistant MCF-7 Sublines
To validate amplification of the region encompassing TRIP6 and ABCB1, we carried out similar FISH analyses in taxane-resistant MCF-7 sublines.
To identify cis-acting regulatory elements in the active human TRIP6 proximal promoter, we scanned the −200 to −1 sequence with Jaspar 2022 transcription factor binding profiles (≥93% relative profile score threshold) (Table S9) [55]. We manually identified core elements within most of the predicted binding sites ( Figure 6A). Remarkably, we discovered a full cyclic AMP response element (CRE) motif at position −60 to −53, corresponding to the −72/−12 construct with marked activity. Mutagenesis of CRE demonstrated a 6to 21-fold reduction in Fluc2P/Nluc activity in 5 truncated constructs (−157/−12∆CRE, −117/−12∆CRE, and −72/−12∆CRE) ( Figure 6B), indicating that CRE is crucial to TRIP6 transcription in MCF-7 cells. The sequence also includes a 5' untranslated region and TRIP6 start codon. CpG dinucleotides and start codon are highlighted in bold. TSS means TRIP6 transcription start. E box refers to enhancer box, CT box refers to CT-rich sequence, AP-1 site refers to Activator protein 1, M-CAT refers to muscle-CAT element (in reverse orientation), CRE refers to cyclic AMP response element, GC-box refers to GC-rich sequence. Schematic diagrams of the TRIP6 proximal promoter with wild-type CRE or mutated CRE motif (on the right). Colored rectangles correspond to predicted cis-acting gene regulatory elements positioned at the 5' flanking sequence of TRIP6. (B) Scatter dot plots showing Fluc2P/Nluc activities relative to the -157/-12 construct (on the right). minP refers to synthetic minimal TATA box promoter. The mean and 95% confidence interval (CI) are displayed for each construct (N = 4, 3 technical replicates). Empty vector pGL4.24[luc2P/minP] served as a mock. Statistical significance was tested using the one-way blocked ANOVA with Geisser-Greenhouse correction followed by Tukey's post hoc test on log-transformed data. * p<0.05, ** p<0.01, *** p<0.001.
Furthermore, the -117/-72 construct exhibited a two-fold increase in Fluc2P/Nluc activity compared to the -72 /-12 construct in all tested cells ( Figure 6B). The region -117 to -72 encompasses an enhancer box (E box) and CT box [56]. Furthermore, activating protein 1 (AP-1) motif located within the region -157 to -117 weakly stimulated (1.6-fold, p = 0.036) The sequence also includes a 5 untranslated region and TRIP6 start codon. CpG dinucleotides and start codon are highlighted in bold. TSS means TRIP6 transcription start. E box refers to enhancer box, CT box refers to CT-rich sequence, AP-1 site refers to Activator protein 1, M-CAT refers to muscle-CAT element (in reverse orientation), CRE refers to cyclic AMP response element, GC-box refers to GC-rich sequence. Schematic diagrams of the TRIP6 proximal promoter with wild-type CRE or mutated CRE motif (on the right). Colored rectangles correspond to predicted cis-acting gene regulatory elements positioned at the 5 flanking sequence of TRIP6. (B) Scatter dot plots showing Fluc2P/Nluc activities relative to the −157/−12 construct (on the right). minP refers to synthetic minimal TATA box promoter. The mean and 95% confidence interval (CI) are displayed for each construct (N = 4, 3 technical replicates). Empty vector pGL4.24[luc2P/minP] served as a mock. Statistical significance was tested using the one-way blocked ANOVA with Geisser-Greenhouse correction followed by Tukey's post hoc test on log-transformed data. * p < 0.05, ** p < 0.01, *** p < 0.001. Furthermore, the −117/−72 construct exhibited a two-fold increase in Fluc2P/Nluc activity compared to the −72 /−12 construct in all tested cells ( Figure 6B). The region −117 to −72 encompasses an enhancer box (E box) and CT box [56]. Furthermore, activating protein 1 (AP-1) motif located within the region −157 to −117 weakly stimulated (1.6-fold, p = 0.036) Fluc2P/Nluc activity in MCF-7 cells but not in PacR and 0035R cells (1.2-fold, p = 0.60, 1.1-fold, p = 0.33, respectively). Yet, the other element(s), probably the GC box [57] or M-CAT [58], increased basal expression as seen in the −72/−12∆CRE construct. We recurrently detected no stimulatory activity in the region −45 to −12 in all tested cells.
Collectively, CRE unambiguously promotes TRIP6 transcription in MCF-7 cells and taxane-resistant MCF-7 sublines. The predicted E box, GC box, CT box, and M-CAT might contribute to the TRIP6 promoter activity; however, there would still be other unidentified motifs. In addition, the AP1 site likely enhances TRIP6 transcription only in MCF-7 cells, as it does not appear to modulate the response in PacR and 0035R cells.

TRIP6 Proximal Promoter Is Hypomethylated in Taxane-Resistant MCF-7 Sublines
Methylation of CpG site in CRE motif hampers transcription in cis [59]. Considering TRIP6 dependence on the CRE motif ( Figure 6), we assessed the methylation of 8 CpG sites within the TRIP6 proximal promoter by bisulfite PCR. As it is shown in Figure 7A, the analyzed region exhibits hypomethylation in MCF-7 cells (3.8%), PacR subline (6.0 %), and 0035R subline. Importantly, we detected an unmethylated CpG in the CRE motif in all tested cells ( Figure 7A). Methylation of CpG site in CRE motif hampers transcription in cis [59]. Considering TRIP6 dependence on the CRE motif ( Figure 6), we assessed the methylation of 8 CpG sites within the TRIP6 proximal promoter by bisulfite PCR. As it is shown in Figure 7A, the analyzed region exhibits hypomethylation in MCF-7 cells (3.8%), PacR subline (6.0 %), and 0035R subline. Importantly, we detected an unmethylated CpG in the CRE motif in all tested cells ( Figure 7A). Furthermore, we assessed TRIP6 methylation in clinical breast cancer samples (TCGA study) ( Figure 7B). TRIP6 mRNA expression negatively correlated (Spearman's coefficient = -0.52, p < 0.001) with CpG methylation level, indicating that DNA methylation might regulate TRIP6 expression also in breast tumors.

Associations of TRIP6 mRNA Level with Clinicopathological Features of Breast Cancer
In a recent study, Zhao et al. postulated TRIP6 as a putative prognostic biomarker in breast cancer [20]. Therefore, we aimed to validate this finding by evaluating TRIP6 expression in 95 breast tumor tissue samples and 6 non-tumor tissues collected in the Czech Republic. Table 4 summarizes clinical data, response to the therapy, and survival of patients who provided breast cancer tissues. The median age (± SD) of patients with a breast cancer diagnosis was 56.0 ± 10.7 years. Most individuals were diagnosed with invasive ductal carcinoma (84.2%), grade 1 or 2 (75.8%), and stage II (62.1%). Nearly all breast cancer tissues expressed estrogen receptor (ER, 90.5% of samples) and progesterone receptor (PR, 70% of samples), meaning that luminal molecular subtype (91.6%) prevailed in evaluated samples. The median of disease-free survival (DFS) (± SD) of patients was 61.1 ± 28.4 months, and overall survival was 70.9 ± 28 months. Unfortunately, disease progression occurred in 9 of the 95 patients, and 8 patients died.
We assessed TRIP6 mRNA expression in all collected breast tissue samples (N = 95) and protein expression only in a small number of samples (N = 20) due to limitations in sample size. Whereas all breast tumor tissues expressed TRIP6 mRNA, we detected TRIP6 protein expression by immunoblotting in 17 of the 20 examined samples ( Figure 8A). TRIP6 mRNA and protein level correlated intermediately (Spearman's coefficient 0.594, p = 0.032) in breast cancer tissues. (Figure 8B).

Discussion
An early study demonstrated a ubiquitous 1.8-Kb TRIP6 mRNA expression in human organs except for skeletal muscle, brain, and leukocytes [60]. Recently, Shukla et al. detected TRIP6 in ependymal and choroid plexus cells of embryonic and early post-natal (to P10) mice brains [12]. Additionally, observations of enhanced TRIP6 expression in various neoplasms might indicate disrupted gene regulatory mechanisms during cancerogenesis [20,61]. Furthermore, we found TRIP6 overexpression in paclitaxel-resistant MCF-7/PacR subline [62], yet the molecular mechanism(s) that drive TRIP6 expression in MCF-7 cells as well in paclitaxel-resistant cells have not been described in detail.
Herein, we revealed that TRIP6 copy number gain and the activity of the cyclic-AMP response element in the hypomethylated TRIP6 proximal promoter contribute to the high TRIP6 protein level in parental MCF-7 cells. Although the AP-1 motif seems more important in parental MCF-7 cells, copy number gain but not altered regulation of the TRIP6 promoter instead contribute to TRIP6 overexpression in both taxane-resistant MCF-7 sublines ( Figure 9).
While TRIP6 mRNA levels differed between PacR cells and 0035R cells, TRIP6 protein abundance was identical and markedly higher compared to parental MCF-7 cells (Figure 2). This discrepancy might indicate the differential TRIP6 post-transcription regulation, for instance, by putative differential TRIP6 mRNA base modifications, miRNA, or recently observed TRIP6 ubiquitin-mediated degradation [24]. Concerning miRNA, it was reported that miR-7, miR-138-5p, miR-485-3p, and miR-589-5p regulate TRIP6 gene expression; unfortunately, their function related to TRIP6 in breast cancer has not been investigated [21][22][23]63]. However, our preliminary data suggest that miR-138-5p We found no difference in TRIP6 mRNA expression levels between adjuvant (N = 82) and neoadjuvant (N = 13) cohorts (p = 0.86). Furthermore, we found no statistically significant correlation between TRIP6 mRNA level and DFS or OS, independent of the type of therapy. High TRIP6 mRNA expression was observed in premenopausal (p = 0.033) and progesterone receptor positive (p = 0.020) breast cancer in the adjuvant cohort of breast cancer patients but not in the neoadjuvant cohort (p = 0.50 and p = 0.77, respectively) ( Table 5).

Discussion
An early study demonstrated a ubiquitous 1.8-Kb TRIP6 mRNA expression in human organs except for skeletal muscle, brain, and leukocytes [60]. Recently, Shukla et al. detected TRIP6 in ependymal and choroid plexus cells of embryonic and early post-natal (to P10) mice brains [12]. Additionally, observations of enhanced TRIP6 expression in various neoplasms might indicate disrupted gene regulatory mechanisms during cancerogenesis [20,61]. Furthermore, we found TRIP6 overexpression in paclitaxel-resistant MCF-7/PacR subline [62], yet the molecular mechanism(s) that drive TRIP6 expression in MCF-7 cells as well in paclitaxel-resistant cells have not been described in detail.
Herein, we revealed that TRIP6 copy number gain and the activity of the cyclic-AMP response element in the hypomethylated TRIP6 proximal promoter contribute to the high TRIP6 protein level in parental MCF-7 cells. Although the AP-1 motif seems more important in parental MCF-7 cells, copy number gain but not altered regulation of the TRIP6 promoter instead contribute to TRIP6 overexpression in both taxane-resistant MCF-7 sublines (Figure 9).

Conclusions
This study presents compelling evidence that the cyclic AMP response element (CRE) located within the stable hypomethylated proximal promoter controls TRIP6 expression in MCF-7 cells. Furthermore, increased TRIP6 copy number contributes to high TRIP6 expression in MCF-7 cells in vitro. Co-amplification of TRIP6 with ABCB1 underlies TRIP6 upregulation in two taxane-resistant MCF-7 sublines. Cytogenetic analyses showed that amplicon arose from intact chromosome 7. In addition, we observed a loss of derivative chromosome der(18)t (18;22) in both sublines, with an unknown relation to taxane resistance. Moreover, the present study has not found direct prognostic or predictive relevance of TRIP6 for better tailoring breast cancer management at the clinics. Instead, the analysis of breast tumor of a neoadjuvant cohort revealed TRIP6 mRNA expression level associations with positive progesterone receptor expression status and premenopausal status.
Collectively, we propose that TRIP6 proximal promoter might act as another important regulatory site in regulation of TRIP6 expression. The relevance of our functionally valid observation for clinical course of breast and other cancer(s), including While TRIP6 mRNA levels differed between PacR cells and 0035R cells, TRIP6 protein abundance was identical and markedly higher compared to parental MCF-7 cells (Figure 2). This discrepancy might indicate the differential TRIP6 post-transcription regulation, for instance, by putative differential TRIP6 mRNA base modifications, miRNA, or recently observed TRIP6 ubiquitin-mediated degradation [24]. Concerning miRNA, it was reported that miR-7, miR-138-5p, miR-485-3p, and miR-589-5p regulate TRIP6 gene expression; unfortunately, their function related to TRIP6 in breast cancer has not been investigated [21][22][23]63]. However, our preliminary data suggest that miR-138-5p is not expressed in MCF-7 cells and taxane-resistant MCF-7 sublines (personal communication Dr. R. Václavíková).
Strikingly, the TRIP6 (100.8 Mb, 7q22.1) gene copy number and mRNA level increased in parallel with the ABCB1 (87.5 Mb, 7q21.12) gene copy number and mRNA level. In agreement, FISH analyses unambiguously validate TRIP6/ABCB1 co-amplification in taxane-resistant MCF-7 sublines (Figure 4). Despite TRIP6 amplification, our findings indicate that TRIP6 is not involved in resistance to taxanes, as silencing of the TRIP6 does not seem to affect the response of the 0035R cells to SB-T-0035 compound ( Figure S6). To date, upregulation of the TRIP6 gene occurred in daunorubicin-(EPG85-257RDB) and mitoxantrone-resistant (EPG85-257RNOV) human gastric carcinoma cells, the former cells having also upregulated ABCB1 [64].
The most famous breakage-fusion-break (BFB) mechanism of amplicon formation leverages specific sequences referred to as fragile sites [68]. What mechanism specifies fragile site selection is not well known. The breaks likely occurred at the FRA7F aphidicolin site (98.7-107.4 Mb) and the 7q11.2 region. The order of events is challenging due to the utilization of multiple selection steps and no direct observation of fusion bridges. Nevertheless, numerical aberration of chromosome 7 in taxane-resistant MCF-7 sublines might be a remnant of dicentric chromosome 7.
Beyond ABCB1/TRIP6 co-amplification, we noticed the loss of der(18)t (18;22) in both taxane-resistant MCF-7 sublines. The impact of this aberration in the context of taxane resistance is unknown.
Since the enhanced TRIP6 expression could theoretically be caused by different transcriptional regulations at the TRIP6 promoter level, we analyzed the responsiveness of the TRIP6 promoter by dual-luciferase assay. The TRIP6 proximal promoter region (−157 to −45) controlled luciferase expression in MCF-7 cells ( Figure 5). The most active segment spanning −72 to −45 nucleotides harbors the M-CAT motif, cyclic AMP response element (CRE), and GC box ( Figure 6). Disrupting the CRE motif by mutagenesis reduced luciferase activity, highlighting its pivotal role in TRIP6 transcription regulation ( Figure 6). A genomewide analysis has previously identified identical CRE motif within the TRIP6 proximal promoter [69], but its role has remained elusive. The cis-regulating activity of the CRE motif relies on its position (< 250 bases) relative to gene transcription start [70] and methylation of the inner CpG site [59]. As tested by bisulfite sequencing (Figure 7A), the CpG site within the CRE motif was not methylated in MCF-7 cells and in both taxane-resistant cells; however, whether this particular methylation affects the expression of TRIP6 remains to be determined in further studies.
Finally, we evaluated the clinical data of breast cancer patients with TRIP6 mRNA expression. Recently, we revealed no clinicopathological association of the TRIP6 mRNA expression level in ovarian cancer [71]. To highlight our findings concerning the regulation of TRIP6 expression in sensitive and taxane-resistant MCF-7 breast cancer cell lines, we evaluated TRIP6 mRNA expression against clinical data of breast cancer patients who had undergone taxane-containing regimens. So far, Zhao et al. have analyzed TRIP6 protein expression in breast cancer from the Chinese cohort [20]. Unfortunately, our data did not validate most of the published results, likely due to the small number of patients in our study and the heterogenous nature of breast cancer.

Conclusions
This study presents compelling evidence that the cyclic AMP response element (CRE) located within the stable hypomethylated proximal promoter controls TRIP6 expression in MCF-7 cells. Furthermore, increased TRIP6 copy number contributes to high TRIP6 expression in MCF-7 cells in vitro. Co-amplification of TRIP6 with ABCB1 underlies TRIP6 upregulation in two taxane-resistant MCF-7 sublines. Cytogenetic analyses showed that amplicon arose from intact chromosome 7. In addition, we observed a loss of derivative chromosome der(18)t (18;22) in both sublines, with an unknown relation to taxane resistance. Moreover, the present study has not found direct prognostic or predictive relevance of TRIP6 for better tailoring breast cancer management at the clinics. Instead, the analysis of breast tumor of a neoadjuvant cohort revealed TRIP6 mRNA expression level associations with positive progesterone receptor expression status and premenopausal status.
Collectively, we propose that TRIP6 proximal promoter might act as another important regulatory site in regulation of TRIP6 expression. The relevance of our functionally valid observation for clinical course of breast and other cancer(s), including eventual utility of TRIP6 as a target for new therapy design, shall be evaluated by follow-up studies.