ALDH1A1 is a direct target of AURKA
The chemical genetic approach involves an engineered kinase, which in the presence of a radioactive orthogonal ATP analog (e.g., N
6-(benzyl) ATP, N
6-(phenethyl) ATP), specifically transfers the radioactive tag (32P) to its substrates. The modified pocket in the engineered kinase is created by replacing a conserved bulky residue in the active site with glycine. The complementary substituent on ATP is created by attaching bulky groups at the N-6 position of ATP. These ATP analogs are not accepted by wild-type kinases, permitting unbiased identification of direct substrates of the engineered kinase in a global environment [9–12, 14, 16–22]. Using the aforementioned design criteria, we generated an AURKA mutant (called AURKA-as7) that efficiently accepted N(6)phenethyl-ATP (PE-ATP) as the orthogonal ATP analog. Using AURKA-as7 and [32P] PE-ATP, we previously identified several novel AURKA substrates including PHLDA1 and LIMK2 [12, 20]. In this study, we focused on ALDH1A1 as the direct target of AURKA. To confirm the results obtained from the chemical genetic screen, we conducted an in vitro kinase assay using recombinant ALDH1A1 and AURKA, which revealed that AURKA directly phosphorylates ALDH1A1 (Fig. 1a).
AURKA regulates the subcellular localization of ALDH1A1 in pancreatic cancer cells
We next examined the subcellular localization of AURKA and ALDH1A1 in BxPC3 and Panc1 cells. ALDH1A1 displayed cytoplasmic localization, which is consistent with previous findings showing it to be a cytoplasmic enzyme (Fig. 1b and c). AURKA too displayed cytoplasmic localization in both BxPC3 and Panc1 cells (Fig. 1d and e). More importantly, when AURKA levels were knocked down using AURKA-shRNA or inhibited using MLN8237 (aka alisertib, an AURKA-specific inhibitor), ALDH1A1 adopted somewhat perinuclear localization (compare actin staining versus ALDH1A1 staining), suggesting that AURKA regulates the subcellular localization of ALDHI1A1 to some extent in BxPC3 cells (Fig. 1f and h). As shown in Fig. 1g and i, ~35% and 22% of BxPC3 cells displayed perinuclear localization of ALDH1A1 in AURKA shRNA-treated and MLN8237-treated cells, respectively. To examine whether AURKA-mediated regulation of ALDH1A1 was common in other pancreatic cancer cells, we investigated ALDH1A1 subcellular localization in Panc1 cells in the absence or presence of either AURKA shRNA or MLN8237. Similar to the results obtained in BxPC3 cells, AURKA depletion or inhibition resulted in moderate perinuclear localization of ALDH1A1 (Fig. 1j–m). Data used to generate the summary statistics shown in Fig. 1g, i, k, and m are reported in Additional file 2.
AURKA and ALDH1A1 associate in pancreatic cancer cells
To investigate whether AURKA associates with ALDH1A1 in BxPC3 cells, we isolated ALDH1A1 immune complex, which pulled down AURKA (Fig. 2a). This finding was corroborated by isolating AURKA immune complex, which too revealed significant association with ALDH1A1 (Fig. 2b). Similarly, we observed robust association of AURKA and ALDH1A1 in Panc1 cells (Fig. 2c and d). These results confirm that both AURKA and ALDH1A1 associate with each other in pancreatic cancer cells.
AURKA positively regulates ALDH1A1 levels
Our previous studies revealed that AURKA-mediated phosphorylation of PHLDA1 at S98 degrades it, whereas AURKA-mediated phosphorylation of LIMK2 at S283, T494, and T505 increases its protein stability [12, 20]. Thus, we examined whether AURKA affects the protein levels of ALDH1A1. Ectopic overexpression of AURKA increased the levels of ALDH1A1 (Fig. 2e), and its depletion using corresponding shRNAs decreased it (Fig. 2f), suggesting that AURKA positively regulates ALDH1A1 levels. Figure 2g shows quantification of alterations in ALDH1A1 levels upon AURKA knock-down from three independent experiments. We also inhibited AURKA using MLN8237, which too resulted in substantial decrease in ALDH1A1 levels, suggesting that AURKA regulates ALDH1A1 using its kinase activity (Fig. 2h). We observed similar AURKA-mediated positive regulation of ALDH1A1 in Panc1 cells, suggesting that it is a common mechanism in pancreatic cancer cells (Fig. 2i–n). Data used to generate the summary statistics shown in Fig. 2g, j, l, and n are reported in Additional file 3.
AURKA inhibits ALDH1A1 degradation
As AURKA directly phosphorylates ALDH1A1, we hypothesized that AURKA might increase ALDH1A1 levels by inhibiting its degradation via phosphorylation. Thus, we examined the profile of ALDH1A1 degradation in BxPC3 and AURKA-overexpressing BxPC3 (AURKA-BxPC3) cells using cycloheximide. As shown in Fig. 3a–c, AURKA overexpression reduced ALDH1A1 degradation, suggesting that it regulates the level of ALDH1A1 by inhibiting its degradation. This study also revealed that the half-life of ALDH1A1 was less than 2 h in BXPC3 cells. ALDH1A1 degradation could be mediated by ubiquitin or non-ubiquitin mechanisms. We transfected 6x-His-ubiquitin into BxPC3 and AURKA-depleted-BxPC3 cells, and analyzed the ubiquitylation of ALDH1A1. Knock-down of AURKA led to increased ubiquitylation of ALDH1A1 (Fig. 3d), thus confirming that AURKA stabilizes ALDH1A1 levels by inhibiting its degradation by ubiquitylation.
AURKA increases the enzymatic activity of ALDH1A1
Increased ALDH1A1 levels and activity are hallmarks of cancer stem cells. Thus, we investigated whether AURKA modulates the enzymatic activity of ALDH1A1. We designed a two-step approach to phosphorylate ALDH1A1 and subsequently measure its dehydrogenase activity. Due to the nature of the assay, we selected five controls to account for any phosphorylation-independent change in dehydrogenase activity. Upon phosphorylation by AURKA, we observed a robust increase in ALDH1A1 activity relative to the phosphorylated controls (Fig. 3e). During our initial experiments, we observed an increase in activity when ALDH1A1 was incubated with ATP alone; however, this change was less than that observed when in the presence of AURKA and ATP (Fig. 3e). We hypothesized that this increase was likely due to phosphorylation by bacterial kinases co-purified with ALDH1A1 and/or a direct interaction with ATP itself. We thus carried out a high-stringency purification of ALDH1A1 to eliminate potential impurities, which resulted in a robust increase in ALDH1A1 activity only when AURKA was able to phosphorylate ALDH1A1 (Fig. 3f).
We observed that the increase in ALDH1A1 activity in these experiments is highly influenced by the activity of AURKA. Accordingly, if the ratio of phosphorylated to unphosphorylated ALDH1A1 was too low, the change in activity could not be observed. Thus, we needed to address the stoichiometry of phosphorylation and correlate it with the observed increase in activity.
To answer this question, we measured the activity and the percent mol phosphate incorporated per mole of ALDH1A1 (Fig. 3g). As expected, there was virtually no observable change in ALDH1A1 activity after 1.5 h (Fig. 3h). This was not surprising, as the stoichiometry of phosphorylation was a mere 3.1% (Fig. 3g). However, after a 6-h kinase reaction, the stoichiometry of phosphorylation reached 38%, which was accompanied by an observable increase in ALDH1A1 activity (Fig. 3i). This increase was prevalent in the first several hours of the ALDH1A1 activity, as seen in the projections of each plot after 2 h (Fig. 3h, i, right panel).
As an additional approach to confirm that the increase in ALDH1A1 activity was indeed due to direct phosphorylation by AURKA, we treated phosphorylated ALDH1A1 with calf-intestinal alkaline phosphatase (CIP) and monitored the impact on dehydrogenase activity (Fig. 3j). As predicted, the addition of CIP caused a decrease in the overall activity, supporting that the change in activity observed following the kinase assay was the result of direct phosphorylation by AURKA. Combined, these findings serve to validate that ALDH1A1 enzymatic activity is regulated via phosphorylation.
ALDH1A1 also positively regulates AURKA levels, triggering a feedback activation loop
Interestingly, several AURKA substrates are known to regulate the levels or activity of AURKA in a feedback mechanism. Thus, we examined whether ALDH1A1 exhibits a similar impact on AURKA levels. We overexpressed ALDH1A1, which revealed a concomitant increase in AURKA levels, suggesting that a positive feedback activation loop exists between the two proteins (Fig. 4a). This finding was confirmed by depleting ALDH1A1 using two different ALDH1A1 shRNAs, which led to a robust decrease in AURKA levels (Fig. 4b). Figure 4c shows quantification of alterations in AURKA levels upon ALDH1A1 knock-down from three independent experiments. Similar results were obtained using Panc1 cells, suggesting that the AURKA-ALDH1A1 feedback activation loop is a common mechanism in pancreatic cancer cells (Fig. 4d–g). Data used to generate the summary statistics shown in Fig. 4c, e, and g are reported in Additional file 4. We next examined AURKA and ALDH1A1 levels in cycloheximide-treated BxPC3 and ALDH1A1-overexpressing BxPC3 cells. ALDH1A1 overexpression significantly reduced the degradation of AURKA, suggesting that ALDH1A1 stabilizes AURKA protein levels (Fig. 4h–j). To further corroborate this finding, we investigated the ubiquitylation of AURKA in BxPC3 and ALDH1A1-depleted BxPC3 cells, which showed increased ubiquitylation of AURKA upon ALDH1A1 depletion (Fig. 4k). Together, these results confirm that ALDH1A1 increases AURKA levels by preventing its degradation, thereby triggering a positive feedback activation loop.
AURKA phosphorylates ALDH1A1 at T267, T442, and T493
AURKA preferentially phosphorylates the R/K/N-R-X-S/T-Φ consensus sequence, where Φ denotes a hydrophobic residue except for Pro [23]. This preference suggested T267, T442, and T493 as potential AURKA sites on ALDH1A1. We generated the corresponding phosphorylation-resistant mutants, T267A, T442A, and T493A and analyzed their phosphorylation using AURKA in vitro. AURKA phosphorylates all the three sites on ALDH1A1 (Fig. 5a). To investigate whether AURKA phosphorylates any additional sites, we generated the corresponding phosphorylation-resistant triple mutant (T267A, T442A, T493A, denoted as 3A) and conducted an in vitro kinase assay. AURKA did not phosphorylate the 3A mutant, confirming that T267, T442, and T493 are the only AURKA sites on ALDH1A1 (Fig. 5b).
AURKA regulates the subcellular localization of ALDH1A1 via phosphorylation
To investigate the significance of AURKA-mediated phosphorylation of ALDH1A1, we initially investigated the subcellular localization of HA-tagged wild-type and mutant 3A-ALDH1A1 (T267A, T442A, T493A) in BxPC3 cells. While wild-type ALDH1A1 displayed cytoplasmic localization similar to the endogenous enzyme, the 3A mutant revealed perinuclear localization, showing that AURKA-mediated phosphorylation contributes to the cytoplasmic residence of ALDH1A1 (Fig. 5c). We further analyzed total ALDH1A1 (including endogenous levels) and ectopically expressed wild-type and mutant ALDH1A1 in the corresponding cell lines using ALDH1A1 and HA antibodies, respectively. While wild-type ALDH1A1-expressing cells showed diffused cytoplasmic staining of both endogenous and ectopically expressed ALDH1A1, 3A mutant-expressing cells showed distinct perinuclear staining of the 3A mutant but cytoplasmic staining for the endogenous enzyme (Fig. 5d). Together, these results underscore a role of AURKA in maintaining the cytoplasmic localization of ALDH1A1.
To investigate the contribution of each of the ALDH1A1 phosphorylation sites in regulating its subcellular localization, we generated HA-tagged wild-type and phospho-resistant single mutants of ALDH1A1-expressing BxPC3 cells, and examined their subcellular localization. Surprisingly, all single phospho-resistant mutants of ALDH1A1 showed predominantly cytoplasmic localization, suggesting that inhibition of phosphorylation at all sites is required for its perinuclear localization (Fig. 5e).
AURKA increases ALDH1A1 levels via phosphorylation at all three sites
We next examined the consequences of AURKA-mediated phosphorylation of ALDH1A1 on its expression levels in BxPC3 cells. These cells were transfected with wild-type ALDH1A1 and the 3A-ALDH1A1 allele for 30 h, and their protein levels analyzed. As shown in Fig. 5f, while wild-type ALDH1A1 was highly expressed, 3A-ALDH1A1 was expressed at a much lower level, suggesting that AURKA-mediated phosphorylation of ALDH1A1 is required to stabilize its protein levels. Furthermore, as 3A-ALDH1A1 was expressed at a lower level, it led to a concomitant decrease in the AURKA level, presumably due to the feedback activation loop.
Our data revealed that AURKA increases ALDH1A1 levels by inhibiting its ubiquitylation; thus, we examined whether 3A-ALDH1A1 was impervious to AURKA-mediated protein stability. We transiently depleted AURKA from wild-type ALDH1A1-BxPC3 and 3A-ALDH1A1-BxPC3 cells and examined the relative ubiquitylation levels of ALDH1A1. While wild-type ALDH1A1 was significantly degraded via ubiquitylation upon AURKA depletion, 3A-ALDH1A1 showed slight ubiquitylation, confirming that AURKA-mediated phosphorylation is responsible for increased ALDH1A1 stability (Fig. 5g).
AURKA-mediated stabilization of ALDH1A1 via phosphorylation prompted us to analyze potential degron sites in the ALDH1A1 sequence, which revealed T267 within a D box motif (RVT*L). Although APC-Cdh1-mediated degradation of D-box-containing proteins can occur independently of any modification, we hypothesized that AURKA-mediated stabilization of ALDH1A1 is likely due to the phosphorylation at T267. Thus, we generated stably expressing T267A-ALDH1A1-BxPC3 cells. As controls, we also generated the other two single-mutant expressing cells (T442A-ALDH1A1-BxPC3 and T493A-ALDH1A1-BxPC3 cells). ALDH1A1 levels were analyzed in wild-type, the three single mutant expressing BxPC3 cells, and 3A-BxPC3 cells. As hypothesized, the ALDH1A1 level was reduced in T267A-ALDH1A1-BxPC3 cells compared to wild-type ALDH1A1-expressing cells; however, interestingly, it was also reduced in T442A-ALDH1A1-BxPC3 and T493A-ALDH1A1-BxPC3 cells, with the triple mutant showing minimal protein levels, suggesting that phosphorylation of each of the three sites contributes to increased ALDH1A1 levels (Fig. 5h and i).
As our data showed that AURKA increases ALDH1A1 levels by inhibiting its ubiquitylation, we analyzed the contribution of each of these three sites in affecting protein stability. The mutant ALDH1A1-expressing cell lines displayed a similar steady state decrease in ALDH1A1 levels compared to wild-type ALDH1A1-expressing cells, as shown in Fig. 5h and i. We then transiently knocked down AURKA in these cells using AURKA shRNA, which resulted in robust ubiquitylation of wild-type ALDH1A1, followed by each of the three single mutants with relatively less ubiquitylation (Fig. 5j). The 3A-ALDH1A1 mutant displayed minimal ubiquitylation upon AURKA depletion, confirming its independence from AURKA-mediated phosphorylation. These results demonstrate that AURKA-mediated phosphorylation of ALDH1A1 at each of the three sites (T267, T442, and T493) contributes to increased protein stability.
AURKA increases ALDH1A1 activity predominantly via phosphorylation at the T267 site
ALDH1A1 has an NAD+ binding pocket (from amino acids 8–135 and 159–270), a catalytic site (271–470) and an oligomerization domain (amino acids 140–158 and 486–495). T267 is within the NAD+ binding pocket (Fig. 6a). The neighboring E269 residue in the active site is essential for catalysis, suggesting that phosphorylation at T267 may have a direct impact on the catalytic activity of ALDH1A1. T442 is within the catalytic domain and T493 is part of the oligomerization domain, which is involved in oligomer formation (Fig. 6a). Thus, we hypothesized that all the three phosphorylation sites may participate in regulating ALDH1A1 enzymatic activity. Although all single mutants exhibited a robust decrease in activity relative to the wild-type enzyme, the T267 mutant showed no activity, suggesting that phosphorylation at T267 primarily governs ALDH1A1 activity (Fig. 6b).
AURKA-mediated phosphorylation of ALDH1A1 regulates its oligomeric states
ALDH1A1 can exist in monomeric, dimeric or tetrameric forms; however, their relative activities remain unknown. As phosphorylation is known to regulate the oligomeric distribution of several proteins, we hypothesized that AURKA-mediated phosphorylation of ALDH1A1 alters its oligomeric state, and that this change was coupled with the observed change in activity. Similar to most multimeric proteins, ALDH1A1 exists predominantly as a tetramer at high concentrations; however, as the dilution increases, the equilibrium becomes more dynamic and begins to populate the monomeric form. Surprisingly, very little dimer was observed at all concentrations, suggesting that the tetramer and the monomer are its most abundant forms.
We monitored the abundance of each species upon phosphorylation as a function of time to analyze the kinetics of the phosphorylation-dependent oligomeric redistribution of ALDH1A1. To this end, we chose a concentration which contained both tetrameric and monomeric species. As the phosphorylation progressed, the ALDH1A1 oligomeric distribution drastically shifted to the monomeric form (Fig. 6c). The concomitant decrease in tetramer abundance serves as an internal control to validate that it is indeed dissociating upon phosphorylation. While Fig. 6d shows average relative changes in tetramer and monomer abundance, Fig. 6e displays the average ratio of normalized monomer to tetramer as a function of time derived from three independent experiments.
We then sought to explore which oligomeric species had the highest activity with and without phosphorylation. To this end, we conducted a similar kinetic analysis and measured the dehydrogenase activity using a native in-gel activity assay (Fig. 6f, g). Upon phosphorylation the monomeric species that formed harbored the highest activity (a greater than sevenfold increase), whereas the tetramer showed little to no change. Figure 6h shows quantification of oligomer-specific ALDH1A1 activities from three independent experiments. This is the first example to identify the oligomer-specific catalytic activities of ALDH1A1 complexes following phosphorylation.
We next sought the oligomeric species that was most phosphorylated to examine a potential correlation between phosphorylation and activity. As predicted, the monomeric population that formed was highly phosphorylated (Fig. 6i–k). Collectively, these data suggest that phosphorylation of ALDH1A1 triggers the dissociation of the tetramer into its monomeric form and that this highly phosphorylated monomer harbors the highest enzymatic activity among all of the oligomeric species. Data used to generate the summary statistics shown in Fig. 6d, e, h, and k are reported in Additional file 5.
ALDH1A1 and AURKA feedback activation loop promotes highly aggressive pancreatic cancer phenotypes
Since AURKA is crucial for mitosis, we examined whether ALDH1A1 impacts the cell cycle. A fluorescence-activated cell sorting (FACS) analysis was conducted using unsynchronized stable scrambled shRNA-expressing BxPC3 and stable ALDH1A1-depleted BxPC3 cells. Both cell types showed a similar distribution of cells in different cell cycle phases and no aneuploidy, which suggests that ALDH1A1 does not affect the cell cycle (Fig. 7a). We next investigated the effect of AURKA-mediated phosphorylation of ALDH1A1 on cellular proliferation. As expected, ectopic expression of either AURKA or wild-type ALDH1A1 increased cellular proliferation in both BxPC3 (Fig. 7b) and Panc1 cells (Fig. 7c). In contrast, expression of 3A-ALDH1A1 showed a significantly impaired proliferation rate, which was lower than either BxPC3 (Fig. 7b) or Panc1 cells (Fig. 7c). More importantly, depletion of AURKA in BxPC3, ALDH1A1-BxPC3, and 3A-ALDH1A1-BxPC3 cells significantly reduced proliferation in ALDH1A1-BxPC3 cells but not in 3A-ALDH1A1-BxPC3 cells, suggesting that the ALDH1A1-triggered increase in cell proliferation is predominantly due to an AURKA-mediated feedback activation loop (Fig. 7d). We further tested this hypothesis by overexpressing AURKA in BxPC3, ALDH1A1-BxPC3, and 3A-ALDH1A1-BxPC3 cells. In agreement with our previous result, AURKA overexpression increased cell proliferation in wild-type ALDH1A1-BxPC3 cells, but not in 3A-ALDH1A1-BxPC3, thereby confirming that AURKA-mediated phosphorylation of ALDH1A1 is crucial for an enhanced growth rate (Fig. 7e). Similar results were obtained in Panc1 cells, where AURKA depletion significantly reduced proliferation in ALDH1A1-BxPC3 cells, but not in 3A-ALDH1A1-BxPC3 cells (Fig. 7f), whereas the AURKA overexpression mediated increase in cell proliferation in wild-type ALDH1A1-Panc1 cells, but not in 3A-ALDH1A1-Panc1 cells (Fig. 7g).
The impact of ALDH1A1 was further evaluated in BxPC3, ALDH1A1-BxPC3 and 3A-ALDH1A1-BxPC3 cells under anchorage-independent conditions. ALDH1A1 expression led to a robust increase in colony formation in BxPC3 cells compared to parental BxPC3 cells. By contrast, expression of 3A-ALDH1A1 acted as dominant negative and exhibited minimal number of colonies in the soft agar assay (Fig. 7h). Similar results were also obtained in Panc1 cells (Fig. 7i). These findings show that AURKA-mediated phosphorylation of ALDH1A1 is crucial for promoting cell proliferation both under attached and anchorage-independent conditions in pancreatic cancer cells.
ALDH1A1 and AURKA feedback activation loop enhances cell motility
We next determined the contribution of ALDH1A1 in promoting chemotaxis using serum-starved BxPC3, ALDH1A1-BxPC3, and 3A-ALDH1A1-BxPC3 cells. A robust increase in cell motility was observed upon wild-type ALDH1A1 expression (Fig. 7j). However, expression of 3A-ALDH1A1 significantly impaired cell motility compared to vector-expressing BxPC3 cells, suggesting that 3A-ALDH1A1 may act as a dominant negative and inhibit cell motility. To further explore the impact of AURKA on ALDH1A1-mediated motility, we depleted AURKA in BxPC3, ALDH1A1-BxPC3, and 3A-ALDH1A1-BxPC3 cells, which considerably reduced cell motility in ALDH1A1-BxPC3 cells, but not in 3A-ALDH1A1-BxPC3 cells, suggesting that the ALDH1A1-triggered increase in cell motility is due to AURKA-mediated positive regulation (Fig. 7k). We further tested this hypothesis by overexpressing AURKA in BxPC3, ALDH1A1-BxPC3, and 3A-ALDH1A1-BxPC3 cells. AURKA overexpression increased cell migration in wild-type ALDH1A1-BxPC3 cells, but not in 3A-ALDH1A1-BxPC3 cells (Fig. 7l). This result was further validated in Panc1 cells, where wild-type ALDH1A1 expression showed a drastic increase in cell motility while 3A-ALDH1A1 showed significant inhibition when compared to wild-type Panc1 cells (Fig. 7m, n). Similarly, AURKA depletion significantly reduced cell motility in ALDH1A1-BxPC3 cells, but not in 3A-ALDH1A1-BxPC3 cells (Fig. 7o, p), while AURKA overexpression increased cell migration in wild-type ALDH1A1-Panc1 cells, but not in 3A-ALDH1A1-Panc1 cells (Fig. 7q, r). Data used to generate the summary statistics shown in Fig. 7c, f, g, i, n, p, and r are reported in Additional file 6. Together, these results corroborate that AURKA-mediated phosphorylation of ALDH1A1 plays a key role in increasing cell motility. As chemotaxis plays a key role in cancer metastasis, these results underscore a crucial oncogenic role of the AURKA-ALDH1A1 feedback activation loop in pancreatic malignancy.
ALDH1A1 upregulation is a key mechanism by which AURKA increases EMT and CSC phenotypes
A pivotal role of ALDH1A1 in inducing EMT and CSC is well delineated. A recent study has also reported that AURKA leads to EMT using MLN8237; however, the mechanism remains unclear [24]. Our data suggest that ALDH1A1 upregulation may be a key mechanism by which AURKA leads to EMT and CSC. Thus, to test this hypothesis, we examined potential modulation of E-cadherin, Snail, Slug, and CD44p using corresponding luciferase plasmids which measure their promoter activities. As shown in Fig. 8a, expression of wild-type ALDH1A1 led to a robust increase in the activities of Snail, Slug, and CD44p, but had minimal effect on E-cadherin activity. More importantly, ectopic expression of the phosphorylation-resistant triple mutant not only prevented the increase in promoter activities of CD44, Slug, and Snail, but in effect resulted in even less activity compared to parental cells, thereby underscoring a crucial role of AURKA in ALDH1A1-induced aggressive oncogenic phenotypes.
To further confirm these results, we analyzed the protein levels of N-cadherin, CD44, Slug, Snail, and E-cadherin in BxPC3, wild-type ALDH1A1-BxPC3, and 3A-ALDH1A1-BxPC3 cells. E-cadherin is an epithelial cell marker, which is downregulated upon EMT, while Snail, Slug, and CD44p are increased in cells undergoing EMT and CSC. Similar to the promoter activation assays, N-cadherin, CD44, Slug, and Snail levels increased considerably upon ALDH1A1 expression, but were prevented in the presence of 3A-ALDH1A1 in BxPC3 cells, confirming that the phosphorylation-resistant mutant serves as a dominant negative (Fig. 8b). In addition, E-cadherin levels decreased in ALDH1A1-overexpressing cells, but showed a significant increase in 3A-ALDH1A1 cells. We also analyzed the levels of vimentin and matrix metalloproteinase-2 (MMP-2), levels of which are known to increase in EMT-undergoing cells and promote tumor invasion. ALDH1A1 overexpression increased both vimentin and MMP-2 levels in BxPC3 cells, whereas phospho-resistant mutant expression abolished their expression. Collectively, these results validate that AURKA-mediated phosphorylation plays a key role in ALDH1A1-mediated aggressive phenotypes.
In parallel, we also analyzed the levels of E-cadherin, N-cadherin, CD44, vimentin, MMP-2, Slug, and Snail in BxPC3 cells treated either with DMSO control or MLN8237. AURKA inhibition reduced the levels of N-cadherin, CD44, vimentin, MMP-2, Slug, and Snail, but increased E-cadherin levels in pancreatic cancer cell lines (Fig. 8c). These findings further underscored a key role of AURKA in promoting EMT in pancreatic cancer, presumably by modulating ALDH1A1 levels and activity.
We next conducted a sphere-forming assay to gauge the self-renewal capacity of BxPC3, ALDH1A1-BxPC3, and 3A-ALDH1A1-BxPC3 cells using ultralow attachment conditions. Under these conditions, cancer stem cells grow in suspension and form independent spheres or colonies. As shown in Fig. 8d, parental BxPC3 mainly formed aggregates of cells with no pancreatosphere formation. In contrast, overexpression of ALDH1A1 induced large pancreatosphere formation, confirming that ALDH1A1 overexpression causes the CSC phenotype. Importantly, 3A-ALDH1A1-BxPC3 cells also showed no pancreatosphere formation, thereby confirming that AURKA-mediated phosphorylation of ALDH1A1 is crucial for inducing the CSC phenotype.
ALDH1A1 phosphorylation contributes to drug resistance
As both EMT and CSC contribute to drug resistance, we examined doxorubicin sensitivity in BxPC3 cells, which revealed about 50% loss in cell viability in 72 h at 1 μM concentration (Fig. 8e). Expression of wild-type ALDH1A1 in BxPC3 rendered these cells significantly resistant to doxorubicin (~30% loss in cell viability in 72 h), whereas 3A-ALDH1A1 expression conferred high sensitivity to doxorubicin-induced toxicity (~70% loss in cell viability) (Fig. 8e). These findings suggest that AURKA-mediated phosphorylation of ALDH1A1 considerably contributes to the drug resistance observed in patients with pancreatic ductal adenocarcinoma (PDAC). These results led us to investigate whether ablation of AURKA sensitizes BxPC3 cells to ALDH1A1 inhibition. We used N,N-diethylaminobenzaldehyde (DEAB) to inhibit ALDH1A1, although it is not very selective and inhibits other ALDH isozymes as well. As shown in Fig. 8f, AURKA-depleted cells exhibit higher sensitivity to ALDH1A1 inhibition compared to scrambled shRNA-expressing cells, suggesting that targeting the AURKA-ALDH1A1 axis is likely to be more effective than individually targeting either AURKA or ALDH1A1.
To test this hypothesis, we treated BxPC3 cells with 100 μM DEAB for 24 h, which showed minimal toxicity, suggesting inhibition of ALDH1A1 alone does not confer toxicity to cells (Fig. 8g, h, bar graph and line graph, respectively). In contrast, AURKA inhibition showed ~32% and ~35% loss in cell viability at 1 and 1.5 μM MLN8237 concentrations, respectively. More importantly, co-inhibition of ALDH1A1 and AURKA was highly effective, particularly, at 1.5 μM MLN8237 concentration, which showed more than 65% loss in cell viability. These findings underscore the significance of the AURKA-ALDH1A1 feedback activation loop in pancreatic carcinoma and suggest that concurrent inhibition of AURKA and ALDH1A1 is likely to be highly effective in targeting highly chemoresistant PDAC (Fig. 8i).