2.4.3. Anticancer
HCQ explains its antitumor activity thanks to its ability to inhibit autophagy. HCQ is, indeed, an FDA-approved drug inhibiting autophagy [135]. Several types of tumors develop chemoresistance by enhancing autophagic flux. Autophagy consists in the sequestration of materials in autophagic vesicles to be eliminated through lysosomal fusion and allows cells to overcome metabolic and therapeutic stresses. By recycling intracellular components, cells may maintain an energy balance and increase their growth. If it occurs in cancer cells, resistance mechanisms may establish. One of the mechanisms responsible for drug resistance is related to increased drug efflux by ATP-binding cassette (ABC) transporters [136,137]. It has been observed that HCQ is significantly reduced the increase in P-gp (ABCB1) expression and, in combination with several anticancer drugs, induced higher cytotoxicity in refractory cancers by inhibiting autophagic activity [138]. However, the role of autophagy in cancer is controversial and depends on genotype and tumor stage development [139]. Many clinical trials examined the synergistic effects of the addition of HCQ to conventional chemotherapic drugs, finding that the role of autophagy is complex and is influenced by several factors. Depending on genetic concomitant alterations, autophagy may possess both pro-tumorigenic and tumor-suppressive roles. It has been confirmed in murine models of pancreatic ductal adenocarcinoma, a type of cancer with a high mortality rate, due to its refractoriness to therapies. Mice presenting activated oncogenic KRAS and normal expression of p53 oncosuppressor experienced a critical regression of tumor developing under HCQ (60 mg/kg/day i.p.). By contrast, in those with a deficiency of p53, the inhibition of autophagy by HCQ increased the tumor progression, demonstrating that autophagy’s role in tumorigenesis is strictly related to the expression of p53 [140]. The expression of p53 is often altered in cancer, so as to be found mutated or absent in the 75% of pancreatic ductal adenocarcinomas [141]. This issue highlights the necessity to carefully evaluate the use of HCQ in certain tumor types. Different outcomes have been previously described, it has been found that inhibition of autophagy by HCQ might arise as a valuable adjuvant in pancreatic ductal adenocarcinoma chemotherapy, regardless of p53 status [142,143]. Given the same doses and route of administration, these inconsistencies between the two reported studies could probably derive from the use of p53 homozygous and heterozygous models of mice, respectively. Regarding KRAS oncoprotein, its downstream pathway is one of the major players of pancreatic carcinogenesis. The inhibition of this pathway by cytotoxic drugs, as well as trametinib, is often associated with an increase in autophagy. For this reason, Drucker and Rosen [144] performed an off-label trial with an association of 400–1200 mg of HCQ and a constant dose of trametinib, observing a partial response with a general reduction of tumor lesion size, circulating tumor markers and cancer-associated pain.
In other cancer types, such as ovarian, prostatic, and human breast cancer, the anticancer or pro-tumorigenic effects of HCQ are determined by tumor stage. In the early stages of the disease, the inhibition of autophagy results in an inhibition of tumorigenesis, while in the advanced phase, it enhances cancer survival [145]. Then, it is important in assessing the contextual role of HCQ in cancer resistance mechanisms. Epirubicin in triple-negative breast cancer therapy often lost efficacy, due to chemoresistance acquiring. It has been shown that this cytotoxic agent induced autophagic flux, increasing cancer cell survival. The combination with HCQ (120 mg/kg by i.p.), thanks to the anti-autophagic properties, significantly suppressed tumor growth by up to 50% with respect to the monotherapy [142]. In addition, estrogen receptor-positive breast cancers developed resistance to treatment with tamoxifen, due to the enhancement of autophagy. The coadministration of HCQ (1–2 mg/day/mice in drinking water) restored the susceptibility of cancer cells to tamoxifen [146]. In mice with thyroid gland xenograft carcinoma, HCQ (150 mg/kg/day p.o. for two weeks) did not provide significant results on tumor growth, while the combination of HCQ with the chemotherapic agent vemurafenib potentiated the anticancer properties of both drugs [147]. Similarly, the two weeks coadministration of HCQ (65 mg/kg) and CCI-779 resulted in a synergism that significantly enhanced their in vivo activity against melanoma tumor growth, in terms of tumor size, with respect to their single treatment [148]. HCQ was revealed also to be active against chemoresistant lung cancer. In this type of cancer, the hypoxic conditions led to lesser susceptibility of cancer cells towards lymphocyte T-mediated cytolysis, thanks to the activation of autophagy. HCQ intake, at doses of 30 mg/kg/day i.p. for 10 days, sensitized tumor cells to lysis and allowed, together with conventional treatment, the eradication of the tumor [149].
Together with autophagy, glycolysis plays a pivotal role in satisfying the increased energetic demand. The dual targeting of the processes may provide a new therapeutic approach in cancer cells. Emonet, et al. [150] performed a randomized preclinical study on Earlic ascites hepatoma-bearing mice, showing that the coadministration of HCQ (60 mg/kg i.p.) and the antiglycolytic inhibitor 3-bromopyruvate possessed a synergistic effect on tumor growth inhibition. Moreover, this treatment is associated with an improvement of oxidative status in hepatic tissue, with a decrement in the number of cancer cells, without affecting the total cell count [151].
Resistance mechanisms also involved alterations in β-Cell Lymphoma (Bcl) Bcl-2 and Bcl-xL and anti-apoptotic gene expression. To evaluate the validity of the dual approach, targeting both apoptosis and autophagy, HCQ (50 mg/kg i.p.) and an apoptosis inhibitor, ABT-737, were administered to prostatic cancer xenograft mice for 15 days. Tumor growth was significantly suppressed by a combination of drugs, with respect to HCQ or ABT-737 alone [152]. In the same way, Fenollar, et al. [153] demonstrated the efficacy of Obatoclax, a pan-Bcl-2 inhibitor, used in association with HCQ (3–60 mg/kg) or conventional in neuroblastoma-bearing mice. Positive outcomes regarded the diminution of tumor size and the complete absence of metastases in cotreated mice with respect to Obatoclax alone or with respect to control [154]. Apoptosis is also at the base of the anticancer activity of interferon-alpha, but the cancer treatment with this drug alone often leads to chemoresistance. It has been demonstrated that autophagy is in the main responsible for chemoresistance, thus the combination of interferon-alpha with inhibitors of autophagic flux may be a useful therapeutic approach. In 30 xenograft mice with head and neck squamous carcinoma, the combination of interferon-alpha with HCQ (60 mg/kg/day i.g.) and wortmannin synergistically promoted apoptosis and inhibited tumor growth [155]. In a similar fashion, Le Goff, et al. [156] investigated the potential synergic role of HCQ (30 mg/kg) in enhancing the anticancer activity of melatonin on tongue squamous cell carcinoma mouse models. The anticancer activity of melatonin depends on its pro-apoptotic effects. Nevertheless, this activity is accompanied by a pro-autophagic activity that caused chemoresistance. The coadministration of the autophagy inhibitor HCQ strongly enhanced melatonin anticancer efficacy, resulting in a smaller tumor size and weight. The effect of inhibition of autophagy on tumor growth may be enhanced if the inhibition of autophagic flux occurs when the process of autophagy is quite completed. This hypothesis has been evaluated by Brönnimann, et al. [157], administering by intravenously TAT–Beclin 1 peptide and HCQ (65 mg/kg) in murine models of breast cancer. Initially, the first agent induced the autophagic flux with the production of autophagosomes, while in the final phase of the process, the second stopped the autophagy by deacidification of lysosomes, causing the accumulation of autophagic vesicles and tumor death. HCQ was administered as HCQ-loaded liposomes, to modulate the onset of autophagy inhibition [158]. This formulation allows us to overcome the limits of HCQ usage, related to the high doses required, which is often unachievable in humans. Relatively high doses of HCQ were loaded in nanoparticles, together with the cytotoxic drug chlorambucil, demonstrating it to be safe and efficient in killing leukemia/lymphoma cancer cells in a human-mouse model of Burkitt’s lymphoma. Eight injections of nanoparticles containing 400 mg of HCQ and chlorambucil led to the overall survival of mice. These concentrations of free drugs are inapplicable, due to their high toxicity [159]. As demonstrated by Naso, Wong, Wong, Chen and Hoang [72], HCQ liposomes (60 mg/kg), together with a pH-sensitive targeting peptide that delivered HCQ into the tumor cells and lysosomes, enhanced the chemotherapic effect of conventional anticancer drug doxorubicin in animal models of melanoma. Likewise, Vayssade, et al. [160] conceived a nanogel (CA4-FeAlg/HCQ) for co-addressing vascular blocker CA4 and anti-autophagic agent HCQ (30 mg/kg) in tumor blood vessels, to synergistically treat A549 lung cancer in mice. Firstly, the release of CA4 exerted anti-angiogenic effects in the vascular site, then FeAlg/HCQ were released into small nanogels and entered in the tumor, where HCQ inhibited autophagy and iron generated ROS with a synergic antitumor effect [161]. Similarly, De Jong, et al. [162] evaluated the response of an animal model of pancreatic cancer to HCQ (5 mg/kg) and paclitaxel administration, loaded in liposomes, modified with an acid environmental sensitive peptide that is responsible for site-specific delivery. Tumor weight, together with the number of liver metastases, was significantly reduced. The administration of HCQ is associated with the inhibition of autophagy and the reduction of IL-6 that is responsible for cross-talking between cancer cells and fibroblasts. All these events avoided the formation of stroma fibrosis, allowing paclitaxel to easily reach the tumor site [104]. The synergism results are essential for HCQ activity in pancreatic cancer. In monotherapy, indeed, HCQ (800–1200 mg/day) did not achieve significant autophagy. This resulted in negligible therapeutic effects in patients with already-treated metastatic pancreatic cancer, of which only 10% were without the progressive disease after two months of therapy [163].
Therefore, the use of modified formulations, such as liposomes, nanogels, etc., may be a precious tools for drug codelivery at the tumor site, enhancing efficacy and reducing side effects. Moreover, the availability of HCQ in those formulations encouraged the use of this molecule in brain tumors, as this formulation highly improved the penetration of this drug in the brain–blood barrier. The co-encapsulation of HCQ with a tyrosine kinase inhibitor, ZD6474, exhibited a synergistic effect, increasing the survival of glioma-bearing mice by two-times with respect to free ZD6474. Those synergistic effects are attributable to significant inhibition of autophagy exerted by HCQ and might provide a valuable therapeutic tool in glioma treatment [164].
The anticancer effect of free HCQ at 200–800 mg/day p.o. has been evaluated in two similar clinical trials on glioblastoma patients in concomitant temozolomide drugs and radiotherapy. Although a dose-dependent significant increase of autophagy markers, no significant effects on tumor suppression were recorded in both studies, as the dose-limiting toxicity was not allowed to achieve higher doses of HCQ [162,165]. The maximum tolerated dose is the dose over which at least one patient from six experienced dose-limiting toxicity, including myelosuppression, anorexia, fatigue, or nausea. Moreover, it has been proved that HCQ severely altered the organization of the Golgi apparatus and the endolysosomal system in C57BL/6JolaHsd mice under 60 mg/kg/day of HCQ i.p. [166]. However, different from Rosenfeld’s studies, no maximum tolerated dose was reached for HCQ in combination with chemotherapic temsirolimus, allowing us to perform a dose-escalation study on 27 patients with solid tumors and 12 with a melanoma diagnosis. In both cases, the standard intravenous dose of temsirolimus with 1200 mg of oral HCQ was considered safe and tolerated and inhibited tumor growth [167]. The same authors further assessed the HCQ anticancer properties and dose-limiting toxicity on 40 patients with metastatic melanoma, by administering a dose intense regimen of temozolomide and escalating doses of HCQ (200–1200 mg/day p.o.). Patients well tolerated the treatment, showing a positive response in the 14% of cases and stability of disease in 27%, due to the modulation of autophagy. No maximum tolerated dose was reached, although common toxicities were manifested [168]. According to the results of Rangwala, a phase I trial on 25 patients with myeloma demonstrated that the recommended dose of HCQ for a phase II trial is 600 mg twice a day. Among eligible patients, 14% experienced a very good response, 14% minor responses, and 45% a period of stability in the disease when the association of HCQ and bortezomib were provided. The synergic effect on myeloma was probably due to the combination of inhibition of HCQ on autophagy and bortezomib on proteasomal degradation, leading to the accumulation of misfolded proteins and autophagic vacuoles in cancer cells [169]. Likewise, doses of 600 mg of HCQ twice a day are not associated with toxicity and its usage as adjuvant therapy with everolimus was well tolerated and produced disease control in 67% of the metastatic clear-cell renal cell carcinoma patients and achieved the rate of six month progression-free survival in 45% of patients [170].