Autophagy inhibition facilitates the therapeutic efficiency
As therapeutic drug-promoted autophagy is supported by abundant substantial evidence, a new therapeutic strategy considering a combination with inhibiting autophagy has been proposed. Currently, studies to inhibit autophagy commonly employ the approaches of genetic silencing of ATGs and pharmacological inhibitors. Gene silencing using small interfering RNA (siRNA) usually knocks down Beclin1, ATG5, ATG7, ATG8 and ATG12 to inhibit autophagy and sensitizes the drug-resistant cancers [
132‐
135], while pharmacological inhibitors generally use 3-methyladenine (3-MA), bafilomycin A1 (BafA) and chloroquine (CQ) to inhibit the formation of autophagosome [
132‐
134,
136] and sensitize the resistant cancer cells to chemotherapy. 3-MA is an inhibitor of class III PI3K, VPS34, and thus it inhibits autophagy at an early stage. In contrast, BafA, a vacuolar H
+-ATPase inhibitor, and CQ, a fusion inhibitor of autophagosome and lysosome, are in the late stage of autophagy to block cargo degradation.
Specific chemical inhibitors of receptor tyrosine kinase/Class I PI3K/AKT/mTOR pathway have been shown to be effective anti-tumor therapies. Imatinib and dasatinib, as tyrosine kinase inhibitors, are the standard treatments for CML, but a great many of patients don’t respond effectively. Pharmacologically blocking autophagy using CQ, or silencing of ATG5 and ATG7, could significantly enhance imatinib-resistance CML cell death [
132,
136]. Dasatinib combined with autophagy inhibitors resulted in almost complete disappearance of phenotypical and functional CML stem cells [
136]. As the dual Class I PI3K/mTOR inhibitor, NVP-BEZ235, synergized with autophagy inhibitors promotes apoptosis of drug-resistant tumors [
133,
134]. Perifosine is an alkylphospholipid to inhibit AKT activity and exhibit antitumor activity. The combination of perifosine with CQ or NH
4Cl enhanced apoptosis and the inhibition of tumor growth [
137]. In clinical phase I trial, the combination of mTOR inhibitor temsirolimus and autophagy inhibitor hydroxychloroquine (HCQ), augmented cell death in patients with advanced solid tumors and melanoma [
138]. The above discussion shows that the same effect can be achieved at any step in the suppression of receptor tyrosine kinase/Class I PI3K/AKT/mTOR pathway combined with autophagy inhibition. However, choosing which step in the pathway for combination therapy may need to consider tumor type and stage of tumor development.
Preclinical models showed that some proteasome inhibitors stimulated autophagy by the accumulation of misfolded proteins, namely ER stress. In agreement with the viewpoint, the inhibition of proteasome inhibitor (bortezomib and NPI-0052)-induced autophagy using 3-MA or CQ increased the levels of cell death in prostate cancer cell model [
139]. Compared with wild-type animals, proteasome inhibitor epoxomicin exhibited an enhanced antitumor function in autophagydefective Beclin1
+/– mice [
135]. The next-generation proteasome inhibitors carfilzomib and oprozomib also induced autophagy and enhanced cell death when used with autophagy inhibitor CQ in head and neck squamous cell carcinoma tumor [
140]. CQ stimulates a slow protein accumulation, localized to lysosomes, while bortezomib induces a rapid buildup of proteins where aggresome formation is in the cytosol [
141]. The accumulation events often lead to mitochondrial disorder, accompanied by the release of cytochrome
c and the activation of Apaf-1 containing apoptotic complex [
142]. Thus, the combination of proteasome inhibition with autophagy inhibition can achieve stronger antitumor effect.
Cell cycle-mediated drug resistance is described that tumor cells are relatively insensitive to chemotherapeutic drugs because the cell cycle is the mechanism by which cells are divided. Through inducing G2 and M phase arrest and inhibiting cell division, isoliquiritigenin has showed antitumor effect in a variety of tumors [
143]. Using 3-MA to suppress autophagy induced by isoliquiritigenin enhances its antitumor activity in ES-2 cells [
143]. In most cancer cell types, silibinin also causes cell cycle arrest, resulting in cell apoptosis [
144]. Likewise, inhibition of autophagy increases silibinin-induced SW480 and SW620 cell death [
145]. Another important anti-cell cycle chemotherapeutic agent, vincristine, induces apoptosis of gastric cancer cells, and siRNA knock-down Beclin1 or ATG5 sensitizes vincristine-resistance tumor cells [
146]. Maybe the most interesting aspect of those drugs that inhibit cell cycle is how they initiate protective autophagy with one or more specific signal pathway(s) in various cell types. This will be more conducive to remove the drug resistance of tumors by autophagy.
Cetuximab, a therapeutic antibody that blocks the function of epidermal growth factor receptor, induced apoptosis and autophagy, and the knockdown of ATG7 or Beclin1 or treatment with CQ sensitized cancer cells to cetuximab-triggered apoptosis [
147]. Mechanistically, cetuximab acted by downregulating Bcl-2 to promote the association between Beclin1 and VPS34. Human epidermal growth factor receptor 2 (HER2) is highly expressed in a variety of cells, such as breast carcinomas [
148], colon cancer [
149] and stomach adenocarcinoma [
150]. Trastuzumab, as a humanized monoclonal antibody binding to domain IV of HER2, is approved by FDA for the treatment of HER2-positive breast and stomach adenocarcinoma/gastroesophageal junction adenocarcinoma cancer (
https://www.cancer.gov/about-cancer/treatment/drugs/trastuzumab). The state of “autophagy addiction” also occurs in trastuzumab-resistant tumor cells. However, targeted genetic ablation of ATG5, ATG8 or ATG12 notably reduces the intrinsic refractory of trastuzumab [
148]. Table
1 presents several examples of therapeutic anti-cancer antibodies, their targets and primary indications. These reports indicate that combined targeting autophagy can enhance the anti-cancer effect of the therapeutic antibodies.
Table 1
Summary of therapeutic antibodies combined with inhibition of autophagy
Rituximab | CD20 | CQ | Non-Hodgkin lymphoma | |
Bevacizumab | VEGF | CQ | HCC | |
Cetuximab | EGFR | CQ | Vulvar squamous carcinoma | |
| | Overexpressed Bcl-2 | Colorectal adenocarcinoma | |
| | siRNA ATG7 | Lung adenocarcinoma | |
| | siRNA Beclin1 | | |
Gemtuzumab ozogamicin | CD33 | PP242 | Acute myeloid leukemia | |
| | AZD2014 | | |
DN30 | Met | Baf | Cardiomyoblasts | |
DO24 | | | | |
Trastuzumab | HER2 | CQ | Breast cancer | |
Milatuzumab | CD74 | FTY720 | Mantle cell lymphoma | |
Tositumomab | CD20 | Overexpressed Bcl-2 | B cell malignancies | |
| | siRNA Beclin1 | | |
| | siRNA ATG 12 | | |
CH12 | EGFRvIII | siRNA ATG7 | HCC | |
| | siRNA Beclin1 | | |
β2M mAb | β2-microglobulin | Bortezomib | Multiple myeloma | |
PD-1 mAb | PD-1 | Pemetrexed + sildenafil | Non-small cell lung cancer | |
CTLA4 mAb | CTLA4 | | | |
As another important therapeutic strategy, miRNAs have been shown to regulate drug resistance in a wide variety of cancers. miR-23b-3p inhibited autophagy to sensitize vincristine-resistant gastric adenocarcinoma cell SGC7901 via decreasing the expression of ATG12 [
161]. miRNA-29c inhibiting autophagy mediated by USP22 increased the gemcitabine-induced pancreatic cancer cell apoptosis [
162]. Another recent study [
163] showed that miR-409-3p ameliorated the sensitivity of ovarian cancer cells to cisplatin by interrupting the FIP200-mediated autophagy. However, all of them have been found to be downregulated in tumors, which may be one mechanism to result in drug resistance. Also, overcoming drug resistance by using miRNA to target autophagy may prove to be tempting and promising.
Inducing autophagic cell death overcomes drug resistance
A large number of studies have shown that autophagy is necessary to effectively kill tumor cells in certain circumstances. Autophagy in this case is called autophagic cell death, which is regarded as cell death with autophagy rather than cell death by autophagy. Indeed, some drugs, such as rapamycin, bortezomib and butyrate to promote autophagosome formation [
164,
165], and enforced expression of the autophagy-related genes, such as ATG3, ATG4 [
166], ATG5 [
164,
165], ATG9 [
167] and Beclin1 [
127], are demonstrated to induce autophagic cell death, and work in tandem with other chemotherapeutic drugs to conquer cancer in certain cell types.
As described early, mTOR is the central regulator of autophagy and a key target for autophagy regulation. The pharmacological inhibitor rapamycin enhanced isoliquiritigenin-induced autophagic and apoptotic cell death in cancer chemotherapy in adenoid cystic carcinoma cells [
164]. The combination of everolimus, another mTOR inhibitor, and propachlor synergistically enhanced cell death by inducing autophagic cell death in prostate cancer cells [
168]. Similarly, RAPA, a mTOR inhibitor to induce autophagy, increased cell death in temozolomide-treated U251 glioma cells [
169]. However, inhibition of mTOR does not always induce autophagic death, but rather protective autophagy. For instance, given that mTOR inhibitor is a potent inducer of autophagy, HCQ relieved temsirolimus, a mTOR inhibitor, resistance and significantly enhanced antitumor activity with safety and acceptability in cancer patients with advanced solid tumors and melanoma [
138]. Currently, the role of autophagy in cancer patients is being studied in multiple clinical trials with mTOR inhibitors (
https://www.clinicaltrials.gov).
The success of bortezomib, a proteasome inhibitor, has been a standard-of-care therapy for malignancy multiple myeloma [
165]. However, bortezomib is not responded in many patients and those patients commonly develop drug tolerance. A recent study shows that enforced expression of the ATG5 overcomes bortezomib-resistance hematologic malignancies [
165]. A novel proteasome inhibitor marchantin M could directly trigger autophagic cell death via PI3K/AKT/mTOR pathway in prostate cancer cells [
170]. In this regard, induction of sustained autophagy (autophagic cell death) seems to be required to compensate for severely damaged proteasome pathway. Thus, induced autophagic cell death maybe overcomes drug resistance to proteasome inhibitors.
Histone deacetylase (HDAC) is believed to regulate the epigenetic alterations which could restrain tumor suppressor genes and promote tumorigenesis. Consequently, inhibition of HDAC becomes an attractive anti-cancer therapeutic approach. HDAC inhibitor NCO-90/141 increased cell death via cytochrome
c-mediated apoptosis and caspase-independent autophagic cell death in leukemic cell lines [
171]. When apoptosis was inhibited, LAQ824 and panobinostat, two other HDAC inhibitors, triggered autophagic cell death in lymphoma cells and tumor xenograft model [
172]. To overcome EGFR tyrosine kinase inhibitor resistance in T790M mutant lung cancer, the combination of suberoylanilide hydroxamic acid (SAHA) and either BIBW2992 or WZ4002, two tyrosine kinase inhibitors, induces autophagic cell death to enhance anti-tumor effect in PC-9G and H1975 cells and mouse xenografts [
173]. Summing up the above, autophagic cell death is a way of compensation for cell death when the cells fail to undergo apoptosis.
There are also other specific chemical drugs that induce autophagic cell death and then relieve the resistance to therapeutic drugs. For instance, ABT-888, a PPRA inhibitor, increased the therapeutic efficacy of temozolomide-resistance glioma cells by inducing autophagic cell death [
174]. A small-molecule inhibitor of Bcl-2, (-)-gossypol, triggered autophagic cell death and inhibited the growth of androgen-independent prostate cancer xenografts with high levels of Bcl-2 to resist apoptosis [
175]. All of these indicate that induced autophagic cell death, as a strategy of synergistic therapy, can occur in multiple drug resistant tumors to circumvent resistance.