siRNA cytotoxicity screen reveals RDH10 association with taxane response in TNBC
We previously identified a 5 Mb copy-number amplification on chromosome 8q (chr8q13.1-.3 and chr8q21.11-.12) in TNBC tumors associated with pCR (12). This region contains 29 genes. We first removed any RNA-coding genes and genes without any known function, leaving 20 protein-coding genes (Figure 1A). We next utilized RNA sequencing data available in TNBC cell lines to eliminate genes with low baseline expression using an RPKM threshold of 0.1, which corresponds to a false-discovery rate (FDR) and false-negative rate of 5% (16-18), narrowing our list to 17 genes (Figure 1A). Finally we eliminated any genes that had poor knockdown efficiency (≤50%) in both TNBC cell lines BT549 and MDA-MB-231 as determined by qRT-PCR, leaving 10 genes for functional study. To identify predictive biomarkers of treatment response to chemotherapy, siRNA cytotoxicity screening was performed in TNBC cell lines, BT549 and MDA-MB-231, with taxane (paclitaxel) or anthracycline (epirubicin)- two classes of drugs used in the BEAUTY study (12) as part of the neoadjuvant chemotherapy regimen (Figure 1B). Following this screening, four genes showed a more resistant phenotype to paclitaxel compared to control (p-value< 0.05) in either BT549 or MDA-MB-231 cells (Figure 1C, Supplementary Table 1). Only RDH10 showed a consistent phenotype in both cell lines.
RDH10 expression regulates cell proliferation, taxane sensitivity and intracellular ATRA in TNBC
We first utilized RNAseq of TNBC cell lines to select three cell lines with varying expression of RDH10 (high (BT549), medium (Hs578t), low (MDA-MB_231)), confirming their expression levels using qRT-PCR and western blot (Figure 2A). To study the role of RDH10 in chemo-response, we generated stable knockdowns in TNBC cell lines BT549 and Hs578t, using two individual shRNA constructs targeting RDH10 (Figure 2B, C). In addition, we performed transient overexpression of RDH10 in BT549 and MDA-MB-231 (Figure 2D, E). Hs578t was utilized for knockdown studies and MDA-MB-231 was utilized for overexpression studies as each had higher and lower endogenous levels of RDH10, respectively. We utilized these lines to determine TNBC response to paclitaxel and epirubicin. We found that loss of RDH10 only increased resistance to paclitaxel in both BT549 and Hs578t with no effect observed with epirubicin (Figure 2B, C). Conversely, overexpression of RDH10 only increased sensitivity to paclitaxel, with no effect with epirubicin (Figure 2D, E). We also determined that loss of RDH10 increased cell proliferation (Figure 2B, C) while overexpression of RDH10 decreased cell proliferation (Figure 2D, E).
RDH10 is the rate limiting step involved in the synthesis of all-trans retinoic acid (ATRA) - the active metabolite in retinoid metabolism (Figure 3A). Since RDH10 is one of over 20 RDH isoforms with known catalytic activity (19), we sought to determine the specific impact RDH10 had on the intracellular concentrations of the RDH precursor, retinol (ROL), and the active metabolite ATRA. We utilized HPLC-UV detection to quantify the endogenous levels of ROL and ATRA in whole cell lysates. We found that following knockdown of RDH10 in BT549 and Hs578t, intracellular ROL increased, while ATRA was reduced by 40-60% (Figure 3B, C), while overexpressing RDH10 in BT549 and MDA-MB-231 resulted in a decrease in ROL and a four-fold increase in intracellular ATRA (Figure 3D, E). These results were further confirmed by determining the ATRA effect on gene transcription through retinoid receptor using a luciferase reporter assay in cells transfected with a luciferase reporter construct with an upstream retinoic acid response element (RARE-luc) (14).
Endogenous ATRA levels can also be regulated through intracellular metabolism by auto-inducible cytochrome P450s (CYPs)(20). While CYP3A4, CYP2C8, CYP26A1 and CYP26B1 contribute to systemic ATRA clearance (21), CYP26A1 and CYP26B1 are also expressed in extrahepatic tissues and influence tissue-specific intracellular ATRA concentrations (20, 22, 23). To determine if ATRA produced by RDH10 overexpression affected its metabolism, we quantified the expression of ATRA-metabolizing CYPs in TNBC cell lines. Utilizing a strict Ct-value cut-off of 30 (24), we found only CYP26A1 and CYP26B1 to be expressed in BT549 and MDA-MB-231 cells, respectively (Figure 3F). Interestingly, overexpression of RDH10 significantly down-regulated transcripts of CYP26A1 in BT549, and CYP26B1 in MDA-MB-231 (Figure 3F).
PIN1 loss increases taxane sensitivity and decreases cell proliferation similar to RDH10 overexpression in TNBC
ATRA was previously identified through a large-scale, small-molecule screen to potently bind to and cause the ablation of peptidyl-prolyl cis/trans isomerase, NIMA interacting-1 (PIN1) (25). PIN1 is often amplified in aggressive forms of cancer, such as TNBC, and is involved in the cis-trans isomerization of phosphorylated serine/threonine-proline residues- activating over 50 oncogenes and inhibiting more than 20 tumor suppressors (26). To further study the mechanism of the RDH10 effect on chemo response, we sought to test the role of PIN1 in this process. First, we knocked down PIN1 in two TNBC cell lines (Supplementary Figure 1A), followed by treatment with increasing doses of paclitaxel or epirubicin. We found that PIN1 knockdown decreased cell proliferation (Supplementary Figure 1C) and increased sensitivity of BT549 and MDA-MB-231 to paclitaxel, but not epirubicin (Supplementary Figure 1B); a phenomenon that was very similar to that of RDH10 overexpression (Figure 2D, E).
RDH10 affects TNBC taxane response and cell proliferation through PIN1
To determine if the ability of RDH10 to modulate intracellular ATRA concentration and response to chemotherapy is through PIN1, we determined endogenous PIN1 levels after knocking down RDH10. We found that when RDH10 was knocked down and intracellular ATRA levels were low (Figure 3B, C), PIN1 levels increased in BT549 and Hs578t (Figure 4A, B), while overexpression of RDH10 increased intracellular ATRA levels (Figure 3D, E) and decreased PIN1 levels (Figure 4C, D); consistent with previous findings that ATRA can degrade PIN1 (25). Furthermore, using TNBC PDX tumor lysates obtained from the BEAUTY patients with either copy number amplification on chromosome 8q or those without, we found that tumors with 8q amplification exhibited higher levels of RDH10 and lower levels of PIN1 compared to those without 8q amplification (Figure 4E).
To further determine if RDH10 effect on cell proliferation and chemo-response is through PIN1 regulation and if PIN1 activity is required for this process, we generated PIN1 stable knockdown cell lines (Supplementary Figure 2) and then introduced either flag-tagged wild-type PIN1 or catalytically inactive S71D PIN1 (27), followed by overexpressing RDH10 (Figure 5). In contrast to WT PIN1, overexpression of the catalytic inactive S71D PIN1 cannot rescue cell proliferation decreased by endogenous PIN1 knockdown. Furthermore, in our experiment, we found the S71D mutation is insufficient to inhibit ATRA from binding and degrading PIN1 which is differs from observations reported previously (25). Additional studies are required to better understand the function of this mutation. Our results, nevertheless, showed that RDH10 decreased PIN1 levels, leading to decreased cell proliferation and increased TNBC sensitivity to paclitaxel (Figure 5; Supplementary Table 2). This suggests that the cell proliferative and cytotoxic effects of RDH10 act through regulation of PIN1 and the effect is also dependent on PIN1 activity.
ATRA sensitizes TNBC to taxane chemotherapy via PIN1 loss.
ATRA has previously been used in the clinic in combination with chemotherapy for the treatment of acute-promyelocytic leukemia (APL), with complete remission rates greater than 90% (28). To determine if ATRA would increase the chemo-sensitivity in TNBC, we first tested the effect of ATRA alone, and found no evidence for cytotoxic effects on BT549 or MDA-MB-231 until concentrations reached as high as 100 uM (Figure 6A). We next determined the effect of ATRA on PIN1 levels with the assumption that PIN1 level is a pharmacodynamic marker for ATRA drug effect. When treated with 1 uM and 10 uM ATRA in BT549 and MDA-MB-231, PIN1 levels decreased (Figure 6B). We next performed HPLC on whole cell lysates of cells treated with 1 uM and 10 uM ATRA. We found with 1 uM ATRA treatment, BT549 exhibited three times greater endogenous levels of ATRA compared to MDA-MB-231. Previous reports have shown that epithelial cells treated with retinoid-receptor agonists, including ATRA, can upregulate gene expression of RDH10 (29). We observed RDH10 to also be upregulated in BT549 with a lesser effect in MDA-MB-231 (Figure 6B), suggesting ATRA-induced RDH10 expression could contribute to the observed differences in intracellular ATRA concentrations and PIN1 levels with 1 uM ATRA treatment between cell lines. However, both cell lines achieved similar endogenous ATRA concentrations with 10 uM ATRA regardless of RDH10 expression. We also noted that 10uM ATRA treatment resulted in similar intracellular ATRA levels that were achieved when RDH10 was overexpressed in both cell lines (Figure 3D, E).
To determine if ATRA treatment could increase sensitivity of TNBC to chemotherapy, we co-treated BT549 and MDA-MB-231 with various concentrations of ATRA and paclitaxel or epirubicin. As expected, addition of 1 or 10 uM ATRA had limited effect on epirubicin sensitivity (Figure 6E); However, there was a dose dependent sensitization to paclitaxel and a decrease in cell proliferation, consistent with that of PIN1 knockdown (Supplementary Figure 1C) or RDH10 overexpression (Figure 2D, E).
Analysis of patient MRI tumor response to taxanes predicts pathological complete response in patients with RDH10 amplification.
MRI imaging and RDH10 expression associations were performed on the 41 TNBC patients in BEAUTY following both the taxane portion and after full neoadjuvant chemotherapy (NAC) (Supplementary Table 3). Following completion of the taxane portion of NAC, 6 (85.7%) of the 7 patients with RDH10 amplification and 16 (47.1%) of 34 patients with wild-type RDH10 had imaging evidence of tumor response. We observed a trend toward a higher tumor response rate after taxane portion of NAC among those with RDH10 amplified TNBC disease than those with RDH10 wildtype TNBC disease (Fisher’s exact test p=0.0994). However, all 7 (100%) TNBC patients with RDH10 amplification following full course of NAC had no invasive disease found in the breast and nodes at surgery (pCR), compared to 15 (44.1%) of 34 TNBC patients with wild-type RDH10. TNBC pCR rate after NAC was significantly higher in those with RDH10 amplification than those with wild-type RDH10 (Fisher’s exact test p=0.0098).