Manipulation of the mitochondrial networks as a therapeutic approach to treat multi-drug resistant Triple Negative Breast Cancer using peptide-loaded liposomes

Abstract

Cancer, a highly complex and heterogenous disease involving abnormal growth and multiplication of cells, is one of the leading causes of death in humans worldwide1 . Triple negative breast cancer (TNBC), the most belligerent sub-types of breast cancer (BC) in women, is widely defined and characterized by the negative expression of three important receptors namely - estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth hormone receptor (HER2/neu)2 . TNBC significantly accounts for about 10 - 15% of all breast cancers and is most often diagnosed in women associated with breast cancer type 1 (BRCA1) mutations2 . TNBC tumors are known to be large and highly proliferative in nature thus exhibiting chemo-resistance3 . This sub-type exhibits the poorest prognosis out of all breast cancer sub-types and is generally associated with hereditary conditions when compared to other breast cancer sub-types, such as Luminal A and Luminal B breast cancer4 . There exists an intricate interplay between the tumor micro-environment, cancer stem cells and drug efflux mechanisms which contributes to the development of chemoresistance in TNBC3 . One of the major hurdles that has come across while treating such a molecularly diverse disease is its resistance to more than one drug, a phenomenon routinely termed as the multidrug resistance (MDR)5 . A widely known refractory outcome of chemotherapy, multi-drug resistance is majorly caused due to the over-expression of drug efflux transporters such as the p-glycoproteins (p-gps) and breast cancer receptor proteins (BCRPs). The development of such a defiant state against structurally and/or functionally unrelated drugs often leads to metastasis6 . P-glycoprotein, also known as multidrug resistance - 1 (MDR1) protein, belong to a family of ABC transporters and positions to be one of several important proteins associated with MDR5 . This protein is encoded by the ABCB1 (ATP Binding Cassette subfamily B member 1) gene that possess an ability to actively capture and exude many foreign 3 substances, majorly drugs, out of the cells thus welcoming a significant challenge which is yet to overcome7 . In TNBCs, an intra-tumoral cellular heterogeneity has appeared to rise as a hallmark of malignant state and accounts for therapeutic resistance, persistent tumor growth and metastasis. Multidrug resistance is a standard phenomenon in cancer and emerges from its hallmarks8 . Recognized as an exceptionally dynamic organelle, mitochondria have emanated to be one of the prime pharmacological targets in triple negative breast cancer (TNBC) therapeutics in recent years9 . These power plants of eukaryotic cells play a vital role in cellular energetics as its function is not limited to providing adequate energy to the cells via oxidative phosphorylation (OXPHOS), but also rendering the highly aggressive TNBC cells resistant to an increased levels of reactive oxygen species (ROS), the morphology of which is dictated by the balance of fission and fusion10. Mitochondrial fusion is known to be mediated by two dynamin-related GTPase mito-fusins (MFNs) namely, MFN1 and MFN2 along with optic atrophy-1 (Opa1). The cornerstone of the project, mitofusin-2 (MFN2), is present on the outer mitochondrial membrane (OMM) that assists the organelle in fusion activities (mitochondrial network formation)10. Several studies have demonstrated that an imbalance in fission and fusion activity in addition to an elevated fission and/or decreased fusion activity often result in fragmented mitochondrial network leading to mitochondrial dysfunction11 . The aim of this project was to potentially target the mitofusin-2 (MFN-2) protein on the OMM using a liposomal formulation loaded with anti-MFN2 peptide that would inhibit mitochondrial network formation (mitochondria - mitochondria networks; mitochondria - ER networks) in BT-549 cells in normal conditions as well as when subjected to hypoxia-like stress conditions. Cationic liposomes with an optimized lipid composition were prepared and used as delivery vehicle to potentially target the intended site. This cell line was exposed to 4 stress conditions to validate the use of hypoxia in inducing multidrug resistance and observing the activation of hypoxia-inducible factors - indicating the expression of drugefflux transporters. This project also focuses on the changes in mitochondrial function / activity post-treatment with cationic liposomes (Blank, anti-MFN and Pro-MFN liposomes) such as - caspase-3 activation, LDH release, mitochondrial permeability transition and changes in oxidative phosphorylation--Author's abstract