Autophagy and cancer research in Iran

ABSTRACT

In August 2018, three events were held in Iran on clinical biochemistry, molecular biology, cancer/autophagy, laboratory management, and proteomics. On August 25–28 at the Isfahan University of Medical Sciences, the 15th National Biochemistry Congress and the 6th International Congress on Biochemistry and Molecular Biology were held, gathering together international professors from Canada, USA, Germany, Australia, Italy, France, and Sweden, as well as Iran to discuss mainly the roles of autophagy in cancer therapy. On August 29, a one-day ‘Autophagy’ symposium was held at the Shiraz University of Medical Sciences. The symposium was a place for specialist talks and discussions on the double-edged role of autophagy in cancer biology, which brought together approximately 200 participants, from basic and clinical fields who are interested in the autophagy field. Furthermore, the opening ceremony for the Autophagy Research Center was held on the same day, and the establishment of the center was announced in Shiraz.

The 15th National Biochemistry Congress & the 6th International Congress on Biochemistry and Molecular Biology in Isfahan

The 15th Iranian National Congress of Biochemistry & 6th International Congress of Biochemistry and Molecular Biology were held on August 25–28 in Isfahan University of Medical Sciences, Isfahan, Iran. The main topics were clinical biochemistry, molecular biology, cancer and autophagy, laboratory management, and proteomics. A summary of key discussions and presentations are as follows:

Dr. Marek J. Łos (Director, Biotechnology Center, Silesian University of Technology, Poland, Senior Scientific Adviser at Linkocare Life Sciences AB, Sweden, and an affiliated member of the Autophagy Research Center, Shiraz University of Medical Sciences, Iran) presented the role of autophagy in the regulation of iPS differentiation: Tissue stem cells are located in every tissue and they help in maintaining tissue homeostasis. The corneal epithelium is maintained by a small pool of tissue stem cells located at the limbus. Through certain injuries or diseases, this pool of stem cells may get depleted. This leads to visual impairment. Standard treatment options include autologous or allogeneic limbal stem cell (LSC) transplantation; however, graft rejection and chronic inflammation lower the success rate over a long time. Induced pluripotent stem (iPS) cells have opened new possibilities for treating various diseases with patient-specific cells, eliminating the risk of immune rejection. In recent years, several protocols have been developed, aimed at the differentiation of iPS cells into the corneal epithelial lineage by mimicking the environmental niche of limbal stem cells. However, the risk of teratoma formation associated with the use of iPS cells hinders most applications from the lab into clinics. Dr. Łos and his group have presented an alternative technology of direct transdifferentioation of human fibroblasts into human limbal cells. The resulting cells, obtained by direct transdifferentiation of fibroblasts into limbal cells, exhibited corneal epithelial cell morphology and expressed corneal epithelial markers. Dr. Łos has also broadly discussed the role of AKT-mTOR pathway (and other AKT-regulated pathways) in stemness maintenance. Hence Dr. Łos’s group showed for the first time a direct transdifferentiation of human dermal fibroblasts into the corneal epithelial lineage that may serve as the source for corneal epithelial cells for transplantation approaches and proved autophagy is a determining step in this process.

Dr. Maryam Mehrpour (French Institute of Health and Medical Research, Institut Necker Enfants-Malades, France, and an affiliated member of the Autophagy Research Center) began her talk by pioneer data, about the role of autophagy in breast cancer stem cells (CSCs): CSCs are a sub-set of cells within tumors that exhibit self-renewal properties and the capacity to seed tumors. CSCs are typically refractory to conventional treatments and have been associated with metastasis and relapse. Using primary cells from patients and a cell culture model, she reported that autophagy is critical for self-renewal and tumorigenicity of CSCs. She then reported that the anti-CSC activity of salinomycin, an antibacterial and coccidiostat ionophore drug, is associated with autophagic flux inhibition. Notably, as part of collaborative work, she showed that a synthetic derivative of salinomycin, named ironomycin, exhibited more potent and selective activity against breast CSCs both in vitro and in vivo compared to the parent drug. The last part of her talk was focused on the mechanism of action of salinomycin and ironomycin and how these compounds kill the CSCs. She reported that these compounds accumulate and sequester iron in lysosomes. In response to the resulting cytoplasmic depletion of iron, cells trigger the degradation of ferritin in lysosomes. This process leads to further iron loading in lysosomes and increases the production of reactive oxygen species (ROS). Iron-mediated ROS production promotes lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These recent findings indicate the prevalence of iron homeostasis in breast CSCs, pointing towards iron-mediated processes as potential targets to kill these cells.

Dr. Mojgan Djavaheri-Mergny (French Institute of Health and Medical Research, France and affiliated member of the Autophagy Research Center) gave a talk on her recent findings on the roles of autophagy in cancer cell growth, survival, and death. She focused her talk especially on two anti-proliferative agents, ATRA, a well-established differentiating molecule that is effectively used in the treatment of acute promyelocytic leukemia (APL), and a new G-quadruplex DNA ligand that promotes cell growth arrest through induction of senescence: She reported that ATRA promotes autophagy and upregulation of the SQSTM1/p62 autophagy receptor in APL. Furthermore, she pointed out that autophagy activation and SQSTM1 upregulation are both impaired in APL cells that are resistant to ATRA-induced maturation, indicating a possible role of autophagy in resistance to ATRA. In the last part of her talk, she presented their recent data related to the G-quadruplex ligand 20A. Her group demonstrated that 20A promotes the activation of the ATM-autophagy axis and this response operates as a linchpin between senescence and apoptosis. She suggested that targeting the ATM-autophagy pathway might be a promising strategy to achieve the maximal anticancer effect of G-quadruplex ligands.

Dr. Saeid Ghavami (University of Manitoba, Canada, and Honorary Professor at the Shiraz University of Medical Sciences, Iran and an affiliated member of the Autophagy Research Center) gave a talk on his recent achievements on Glioblastoma multiforme (GBM) brain cancer therapy. He reported an interesting approach for enhancing temozolomide (TMZ) anticancer drug efficacy in GBM therapy. The approach is based on the inhibition of autophagy flux induced by the anticancer drug. He pointed out that although TMZ can induce tumor cell apoptosis through DNA damage, it also induces autophagy, resulting in drug-resistant tumor cells, and therefore its efficacy in GBM therapy is suppressed. In the newly suggested approach, the autophagy flux induced by TMZ can be inhibited through targeting autophagosome and lysosome fusion, which could consequently improve the patients’ survival rate. In the experimental part, he stated that a statin family cholesterol-lowering drug (i.e., simvastatin) was utilized to inhibit autophagy flux in TMZ-treated 2D and 3D human GBM cell culture media. The results of the experimental part showed that the statin drug sensitizes cancer cell to TMZ and induces cell apoptosis by possibly the inhibition of the induced autophagy flux via a mevalonate cascade independent mechanism. In sum, he pointed out that the inhibition of autophagosome and lysosome fusion can be regarded as an alternative approach to GBM therapy, which can be further utilized in the treatment of other cancer types.

The first symposium of autophagy in Shiraz and Autophagy Research Center Establishment Ceremony

Since 2016, we have initiated an international effort to establish the first Autophagy Research Center in the Middle East. Dr. Saeid Ghavami has taken the lead in this initiative. On 29 August 2018, after the Isfahan meeting, in collaboration with the Department of Biochemistry, School of Advanced Medical Sciences and Technology, Shiraz Transplant Research Center and Health Policy Research Center in the Shiraz University of Medical Sciences a one day ‘Autophagy Symposium’ was held and the establishment of the Autophagy Research Center was announced. A summary of key discussions and lectures in the ‘Autophagy Symposium’ is summarized as follows:

Autophagy and stem cell biology was presented by Dr. Negar Azarpira (Distinguished Professor and an affiliated member of the Autophagy Research Center). Her discussion centered mainly on four groups of hematopoietic stem cells, neural stem, induced pluripotent stem cell, and cancer stem cells. Autophagy in these cells is high at rest, which leads in particular to decreased mitochondrial numbers and decreases aerobic metabolism. By reducing stress or moving toward differentiation, autophagy decreases in these cells. Precise knowledge of the autophagy mechanism will help to produce and maintain these cells more efficiently and lead to the production of anticancer drugs.

Dr. Saeid Ghavami shared his recent findings on the double-edged role of autophagy in cancer biology. He pointed out that autophagy sometimes activates an oncogenesis and invasion system in tumor cells, and occasionally makes tumor cells susceptible to apoptosis-dependent chemotherapy. Recently, investigations have been focused on this sequence and the processes and cellular prototypes have been regulated through autophagy pathways. He stated that one of the most important phenotypic changes that occur in the cell is the epithelial-to-mesenchymal transition (EMT) that results in epithelial cells having more mesenchymal features, which can be induced by autophagy. He presented the results of his extensive research in non-small cell lung cancer (NSCLC) therapy proving the role of autophagy in inducing EMT. As an example, he shared a summary of his recently published paper on the double-edged role of autophagy in cancer biology [1]. He stated that their results showed that TGF-β1 (transforming growth factor beta 1) induces both autophagy and EMT in NSCLC simultaneously, indicating the potentially positive role of autophagy as a regulator of TGF-β1-induced EMT in NSCLC cells (A549 and H1975 cell lines). He pointed out that the inhibition of autophagy can be considered as an alternative approach in the clinical progression of NSCLC.

Dr. Marek J. Łos presented the role of autophagy in salinomycin-induced toxicity. He discussed initially that the pro- or anti-survival effects of autophagy are particularly important if one tests anticancer drug candidates that induce autophagy. He continued that they had recently been investigating the molecular mechanism of action of salinomycin, which preferentially kills cancer stem cells; the molecular mechanism of salinomycin’s toxicity is not fully understood. Various studies reported that Ca²⁺, CYCS/cytochrome c, and cascade activation play a role in salinomycin-induced cytotoxicity. Furthermore, salinomycin may target the WNT-CTNNB1//β-catenin signaling pathway to promote differentiation and thus elimination of cancer stem cells. They had observed that salinomycin induced a massive autophagic response in treated cells. Salinomycin’s pro-autophagy effect was substantially stronger than the effect of the commonly used autophagic inducer rapamycin. The strong/massive autophagy induction was cell-type independent and it was observed in prostate, breast cancer, other cancer cell types, and to a lesser degree in human normal dermal fibroblasts. Upon in-depth investigation, they determined that autophagy induced by salinomycin was a cell protective mechanism in all tested cancer cell lines. Furthermore, salinomycin induced mitophagy, mitoptosis and increased mitochondrial membrane potential (∆Ψ) in a subpopulation of cells. The pro-autophagic effect of salinomycin may be in part due to a strong decrease of cellular ATP level, which may be the consequence of salinomycin-triggered mitochondrial damage (low ATP-level is one of the key cellular autophagy inducers). Interestingly, human normal dermal fibroblasts treated with salinomycin show some initial decrease in mitochondrial mass; however, they are largely resistant to salinomycin-triggered ATP-depletion, which explains salinomycin’s lower toxicity towards primary fibroblasts. Their data provided new insight into the molecular mechanism of preferential toxicity of salinomycin towards cancer cells and suggests possible clinical application of salinomycin in combination with autophagy inhibitors (such as the clinically used chloroquine).

Dr. Mojgan Djavaheri-Mergny is studying how autophagy regulates the balance between cell growth arrest, survival, and death in cancer cells. She presented her published and unpublished data on the regulation of autophagy during ATRA-induced granulocytic differentiation of acute promyelocytic leukemia. Dr. Djavaheri’s group has found that autophagy is upregulated during ATRA-induced granulocyte differentiation of APL cells through a mechanism that involves AMPK activation. Her group also investigated the role of selective autophagy, especially the autophagy receptor SQSTM1 during the differentiation of APL cells by ATRA. Knockdown of SQSTM1 expression enhanced both cell death and the accumulation of ubiquitinated protein aggregates in APL cells exposed to ATRA suggesting a critical role of SQSTM1 in maintaining the viability of APL. Moreover, she point out that autophagy and SQSTM1 upregulation also occurred during differentiation of myeloid leukemia cells into either monocytes or megakaryocytes, supporting the idea that this phenomenon represents a general response that takes place during terminal differentiation of myeloid leukemia towards a specific cell lineage.

At the end of the meeting, Miss. Sanaz Dastgheib (PhD student of the Autophagy Research Center), described the molecular interplay between the unfolded proteins response (UPR), autophagy and apoptosis. Under physiological conditions, the endoplasmic network regulates the protein folding in the cell; however, stress produces an accumulation of misfolded proteins in the ER, leading to the activation of the UPR, a process which is called ER stress. The activation of this pathway is primarily an adaptive response to compensate for the abnormalities or restore normal cellular conditions, but the prolonged activation of the endoplasmic stress promotes oxidative stress and inflammation, and results in cell death. The UPR has three main arms and is initiated by the activation of three proteins: (1) EIF2AK3/PERK (eukaryotic translation initiation factor 2 alpha kinase 3); (2) ERN1/IRE1α (endoplasmic reticulum to nucleus signaling 1); and (3) ATF6 (activating transcription factor 6). In recent years, the molecules regulating the response to endoplasmic stress have been considered by the researchers as a powerful and effective alternative for drug and therapeutic purposes in many diseases, including cancer, neurodegenerative disease, diabetes, heart and liver diseases, and allergies.

Summary

In these events, the scientists showed the possibility of taking effective steps in treating cancer by adjusting the cross talk of autophagy and apoptosis signals. Furthermore, the researchers showed that autophagy is involved in the regulation of cellular phenotype in lung epithelial cells and iPS cells. Moreover, they looked at how the drug could reduce the size and incidence of the tumor, along with standard chemotherapy treatments. In this case, salinomycin has been identified as a therapeutic approach that has received a lot of attention especially with regard to CSCs. During these scientific meetings the scientists have emphasized that autophagy is overally involved in the regulation of cell homeostasis during stress conditions but under certain long-term cellular stress, autophagy is capable of activating apoptotic and non-apoptotic cell death pathways through several distinct mechanisms.

Funding Statement

This work was supported by the Manitoba Health Research Council [21345].

Abbreviations

APL

acute promyelocytic leukemia

ATP

adenosine triphosphate

CSCs

cancer stem cells

ER

endoplasmic reticulum

EMT

epithelial-to-mesenchymal transition

GBM

glioblastoma multiforme

iPS

induced pluripotent stem

LSC

limbal stem cell

NSCLC

non-small cell lung cancer

ROS

reactive oxygen species

SQSTM1

sequestosome 1

TMZ

temozolomide

TGF-β1

transforming growth factor beta 1

UPR

unfolded proteins response

Disclosure statement

No potential conflict of interest was reported by the authors.