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Autophagy promotes resistance to CRC-therapy by specifically promoting GSK-3β/Wnt/β-catenin signaling to promote CSC survival, and 36-077, a PIK3C3/VPS34 inhibitor, helps promote efficacy of CRC therapy.
- autophagy
- 5-FloroUracil
- PI3KC3
- chemoresistance
- cancer stem cells
1. Introduction
Despite significant advances in its clinical management, colorectal cancer (CRC) remains the second leading cause of cancer-related death [1]. As per recent estimates, approximately a million new colon cancer cases are reported worldwide every year, and about half of these patients die from the disease [2]. The five-year survival for CRC patients depends largely on the disease stage as it ranges from 90% for patients with localized disease (stage I) to ~10% when the tumor becomes distant and invasive, and spreads to distant organs (stage IV) [3][4][3,4]. The basis of the advanced CRC treatment consists primarily of chemotherapy; however, drug resistance remains a major hurdle [5]. Thus, there is an urgent need for novel therapeutics that can help overcome chemotherapy resistance and restrict CRC progression.
Cancer stem cells (CSCs) are a specific cellular subpopulation within a cancer and possess the capacity to self-renew and generate cancer cell lineages [6]. The CSCs play a central role in cancer progression and therapy resistance including in CRC [7], and therefore are attractive therapeutic targets. Both intracellular and extracellular signals including metabolic stress regulate CSC self-renewal and plasticity [8]. Remarkably, CSCs lose self-renewal properties when removed from their microenvironment, which implies an essential role for microenvironment in directing their fate [9].
Autophagy, a catabolic process initiated in response to cellular and/or metabolic stress is a survival-promoting program [10][11][10,11]. Autophagy plays a context-specific role in tumorigenesis; however, in the majority of the oncogenic environments, autophagy promotes cancer progression and drug (therapy) resistance [12]. Recent studies have shown that inhibiting autophagy can restore the chemosensitivity in resistant cancer cells [13][14][15][13,14,15]. However, chloroquine (CQ) and its derivatives, currently used to interfere with autophagy by inhibiting lysosomal degradation of the autophagic cargo, have not proven sufficiently effective and specific in clinical trials. In this regard, recent studies have identified phosphatidylinositol 3-kinase (PIK3C3/VPS34 kinase), a key regulator of autophagy, as a specific and potent target for inhibiting autophagy, including in cancer cells [15][16][17][15,16,17]. However, the premise of inhibiting CSCs through interference with the PIK3C3/VPS34 function to prevent CRC malignancy remains ill understood.
2. Chemotherapy Induces Autophagy in Colon Cancer
The 5-FU therapy remains the state-of-the-art chemotherapy option for treating colon cancer; however, its cancer cell killing activity is limited in the drug-resistant colon cancer [18][22]. To elucidate if autophagy helps CRC cells in developing resistance to chemotherapy, we employed a fluorescent reporter system (LC3/GFP/RFP) that measures autophagy in a dynamic fashion (details in Section 2). In brief, green fluorescent protein (GFP) expression coincides with autophagy inhibition, and red fluorescent protein (RFP) expression represents autophagy induction [19][23]. Colon cancer cells harboring this reporter system were cultured in the presence of 5-FU for twenty-four hours. The relative GFP/RFP expressions between control (vehicle-treated) and 5-FU treated cells were determined using Inverted fluorescence microscopy and puncta were quantitated. The 5-FU treatment led to a robust increase in LC3-RFP puncta (versus control cells) in both Caco-2 and HCT116 cells (Figure 1A,B). To validate that 5-FU treatment induced autophagy, we further determined the status of the established autophagy markers, P62/SQSTM1 and conversion of LC3-I (microtubule-associated proteins light chain-I) to LC3-II (microtubule-associated proteins light chain-II). Immunoblotting using cell lysates from control and 5-FU-treated cells showed a dose-dependent decrease in P62/SQSTM1 expression (*** p < 0.001) while the LC3-II/LC3-I ratio was significantly upregulated (*** p < 0.001) in the 5-FU-treated cells (Figure 1C–F). Similar changes in P62/SQSTM1 and LC3-II/I ratio in 5-FU-treated colon tumoroids (from the APCMin/+ mice) (** p < 0.01 at 10 µM of 5-FU), supported the data from the colon cancer cells (Figure 1G,H). Taken together, our data suggested that the 5-FU-treatment induces autophagy in colon cancer.
3. Che uced Autophagy Is Specific to Cancer Stem Cells
Cancer stem cells (CSCs) are central to the resistance to cancer therapy and are thought to be resistant to the chemotherapy-associated apoptotic stress (reviewed by Nguyen et al. and Beck et al. [20][21][24,25]). Hence, we determined if 5-FU treatment-induced autophagy was associated with the CSCs. Colon cancer cells expressing the autophagy reporter system, as in above studies, were subjected to 5-FU treatment for 24-h and then subjected to FACS-sorting to obtain GFP- and RFP-enriched cell populations (schematic representation in Figure 2A). The GFP-enriched population was significantly higher compared to the RFP positive cell population (Figure 2B,C; *** p < 0.001). We then performed qPCR analysis using total RNA isolated from the GFP- and RFP-enriched cells for cancer stem cell markers, LGR5, CD133, CD166, and OLFM4. WAs shown in Figure 2D, we found significant upregulation in LGR5 (*** p < 0.001), CD133 (** p < 0.01), CD166 (* p < 0.05), and OLFM4 (* p < 0.05) expressions in the RFP-positive cells compared to the GFP-positive cells. Taken together, these data suggested that chemotherapy-induced autophagy was limited to a subset of colon cancer cells with CSC characteristics.
4. 36-077, a PIK3C3/VPS34 Inhibitor, Is a Highly Specific and Potent Autophagy Inhibitor
Currently, hydroxychloroquine (HCQ) is the only FDA-approved drug that is known to inhibit autophagy. However, clinical trials using HCQ for improving cancer therapy have not been promising [22][26]. Recent studies have shown the potential of inhibiting VPS34-activity as a highly potent means for inhibiting autophagy [23][27]. Hence, based on the recently published reports focusing on VPS34 inhibition [24][25][26][28,29,30] we synthesized a highly potent and specific VPS34 inhibitor that was potent, selective and potentially effective in vivo [27][31], and named it 36-077. The chemical structure of 36-077 is presented in Figure 3A and the synthesis route has been described in the Figure S1 and Section 2. We first validated the efficacy of 36-077 in inhibiting autophagy in colon cancer cells. Using Caco-2 cells, we determined the dose-response effects of 36-077 treatment upon autophagy. As shown in Figure 3B, accumulation of vacuoles, a cellular phenomenon associated with the inhibition of autophagy, was sharply upregulated in 36-077 treated cells compared to the vehicle-treated cells. Immunoblot analysis using total cell lysates further showed a significant and dose-dependent upregulation of P62/SQSTM1 expression in 36-077-treated cells (versus control cells; *** p < 0.001). The LC3-II/LC3-I ratio was sharply downregulated in same samples (Figure 3C,D; *** p < 0.001 at 10 or 50 nM). We found a similar dose-dependent effect of 36-077-treatment upon autophagy in the 3D-culture of colon tumors from APCMin/+ mice (Figure 3E–G; *** p < 0.001 at 10 nM and 50 nM). Overall, the above data confirmed the efficacy of 36-077 in inhibiting autophagy in colon cancer cells and colon tumors.
5. Co-Treatment with 36-077 Improves Therapeutic Efficacy of 5-FU Treatment
Based on above findings, we postulated that the combination treatment with 36-077 would improve the efficacy of 5-FU therapy. To test, we treated CRC cells with 5-FU or 36-077, or in combination. The dose response effect was determined upon cell survival. CAs demonstrated in Figure 4A, co-treatment of HCT116 cells with 5-FU+36-077 markedly lowered the EC50 (relative global growth inhibition) for both 5-FU and 36-077 from 10.44 and 29.75 nM, respectively, to 4.19 nM. To determine whether the effects of above treatments are dependent on the changes in cell survival and/or apoptosis, we determined expression of cyclin-D1, a cell proliferation marker, and cleaved caspase-3, a marker of cell death. These studies were done using Caco-2, HCT116 cells, and colon tumoroids. The 5-FU treatment induced a dose-dependent decrease in cyclin-D1 expression, which was in line with its known effect in inhibiting the cell cycle progression (Figure 4B–G). However, 36-077, even at 50 nM, was only modestly effective in inhibiting cyclin-D1 expression or inducing the expression of cleaved caspase-3. The combination treatment (5-FU+36-077) not only resulted in a significant increase in the expression of cleaved caspase-3, but also a contrasting downregulation of cyclin-D1 expression (Figure 4B–G). Both cyclin-D1 inhibition and caspase-3 induction can inhibit cancer cell survival. We therefore calculated the “cell proliferation index”, the ratio of cell proliferation versus apoptosis for an unbiased view of the potential impact of the changes in these proteins upon cell survival. The ratio of cyclin-D1/caspase-3 expressions showed that the co-treatment of 36-077 significantly improved the therapeutic efficacy of 5-FU (*** p < 0.001 at 5-FU (10 µM) and 36-077 (50 nM)).
6. Co-Treatment of 5-FU and 36-077 Inhibits GSK-3β/Wnt/β-Catenin Signaling
We further examined how 36-077 co-treatment potentiates the efficacy of 5-FU therapy. Based on an initial screening of oncogenic signaling pathways, we focused on Wnt/β-catenin signaling considering its central role in CRC and in promoting the CSC niche [28][32]. To analyze the status of Wnt/β-catenin signaling, we used the highly sensitive TOP-FLASH reporter assay, as described previously [29][20]. Both 5-FU and 36-077-treatments significantly inhibited the TOP-FLASH reporter activity; however, combination treatment inhibited the reporter activity to its nadir (Figure 5A; **** p < 0. 0001). GSK-3β regulates the Wnt/β-catenin signaling and destabilizes β-catenin expression by phosphorylating the protein at positions Ser33/Ser37/Thr41 [30][31][32][33,34,35]. We therefore determined if 5-FU+36-077 treatment affected the β-catenin phosphorylation (Ser33/Ser37/Thr41) to regulate Wnt-signaling. Immunoblotting was done using the total cell lysates and phospho-specific antibody, which demonstrated that the co-treatment of 5-FU and 36-077 significantly upregulated p-β-catenin (Ser33/Ser37/Thr41) expression (*** p < 0.001) (Figure 5B–D). Accompanying changes in the β-catenin protein expression supported its degradation. Based on the above findings, we examined if GSK-3β expression and phosphorylation (Ser9) were modulated by the 5-FU+36-077 treatment (Figure 5B–D). Immunoblotting using the p-GSK-3β (Ser9) and GSK-3β antibodies showed that the combinatorial treatment also inhibited expression of p-GSK-3β (Ser9) (*** p < 0.001) (Figure 5B–D). These findings were consistent amongst the colon cancer cells and colon tumoroids (Figure 5B–D). Immunolocalization studies for β-catenin cellular localization further demonstrated aggregate formation and membrane-localized β-catenin expression in CRC cells treated with 5-FU+36-077 compared to the 5-FU or 36-077 treatment (Figure 5E). Based on the above findings, we proposed that the effects of the co-treatment with 36-077 in promoting the efficacy of 5-FU-treatment should be overcome by forced activation of the Wnt/β-catenin signaling. Of note, Wnt-3A is a ligand of the Wnt-signaling [33][34][36,37]. Caco-2 cells were treated with 5-FU or 36-077 or in combination and in the presence or absence of Wnt-3A (10 nM). Immunoblotting using the cell lysates from the respective cell lines demonstrated an expected downregulation in the expression of p-β-catenin (Ser33/Ser37/Thr41) in cells treated with 5-FU+36-077 + Wnt-3A compared to the cells that received 5-FU+36-077 (Figure 5F,G; *** p < 0.001). We also found robust increases in cyclin-D1 expression in these cells compared to the cells receiving only 5-FU+36-077 (Figure 5F,G, ** p< 0.01). The cleaved caspase-3 expression was also significantly downregulated in cells receiving the 5-FU+36-077 + Wnt-3A compared to the cells receiving 5-FU+36-077. Wnt signaling regulates the CSC phenotypes, which are resistant to the anti-cancer therapy. Therefore, we further analyzed the expression of CSCs markers in cells treated with 5-FU+36-077 + Wnt-3A. The qPCR analysis demonstrated significant (*** p < 0.001) upregulation of the CSC markers in cells receiving 5-FU+36-077 + Wnt-3A versus 5-FU+36-077 (Figure S2). Taken together, our data suggested that inhibiting autophagy using 36-077, a PIK3C3/VPS34 inhibitor, along with 5-FU treatment, has a profound effect on the GSK-3β/Wnt/β-catenin signaling to inhibit cancer stem cells.
7. Combination Treatment of 5-FU+36-077 Inhibits Survival of Cancer Stem Cells
Since the above data suggested that combination treatment of 5-FU+36-077 targets the CSC population, we further utilized the spheroid assay commonly used for CSC culture, as described in [35][38], to further test our hypothesis. Effects of 5-FU (10 µM) treatment alone or with 36-077 (50 nM) were examined. As shown in Figure 6A, 5-FU+36-077 treatment inhibited the growth of spheroids significantly more than 5-FU or 36-077 alone. A similar outcome arose upon treating the spheroid cultures generated using single cell preparation from the colon tumoroids (Figure 6B). Subsequent qPCR analysis for CSC markers (LGR5, CD166, and CD133) using total RNA isolated from the resultant spheroid cultures, further confirmed that combinatorial treatment was significantly more effective in inhibiting the CSC population than 5-FU or 36-077 treatments (Figure 6C).
To further validate, we used side population (SP) cells that we have isolated and established in culture from DLD-1 cells (Figure 6D and Section 2). Notably, SP cells are considered CSCs based on their characteristics including expression of CSC markers, and high level of tumorigenicity [36][37][39,40]. Immunoblot analysis of P62/SQSMT1 and LC3-I/LC3II expressions showed a significant upregulation of autophagy flux in SP cells compared to the non-side population DLD-1 cells (NSP) (Figure 6E). We treated the SP cells with 5-FU, 36-077, or the combination. Phase contrast images of SP cells treated for 48 h showed that the combination treatment significantly inhibited their colony formation ability compared to the control cells. (Figure 6F,G). Immunoblotting of the total cell lysate demonstrated a significant decrease in OLFM4, a stem cell marker, and survivin, a proliferation marker in cells receiving 5-FU+36-077 compared to their individual treatment (Figure 6H,I). The proliferative index (CyclinD1/Cleaved caspase-3 expression) was also sharply downregulated in the cells receiving 5-FU+36-077 treatment compared to their individual treatment (*** p < 0.001) (Figure 6H,I). In same samples, expression of p-β-catenin (Ser33/Ser37/Thr41) expression was significantly upregulated while p-GSK3-β (Ser9) expression was sharply downregulated. Taken together, these data demonstrated that co-treatment of 36-077 improves efficiency of 5-FU-treatment by inhibiting the CSC population by modulating the GSK-3β-catenin signaling.
8. 36-077 Treatment Increased the Therapeutic Efficacy of 5-FU by Inhibiting the WNT Signeling in CSC
In summary, heterogenous populations of cancer cells represent the differential autophagy flux. CSC represent high autophagy flux and treatment of 5-FU leads to induction of cells death in cancer cells except CSC. These cells have self-renewal properties and upregulated WNT signaling. These properties make the CSC resistance to 5-FU and are responsible for cancer reoccurrence. Treatment of 36-077 in combination with 5-FU significantly inhibits WNT signaling pathway and induces the cells death in CSC.