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Folate receptors: a therapeutic target for Anti – tumor drug delivery and cancer therapy

Updated: Nov 17, 2022

Cancer has always been a major health issue and economic burden for both patients & society. More significantly, the burden of this disease is increasing dramatically; finally, cancer cases have increased to 19.3 million and 10 million cancer deaths in 2020 (updated byThe International Agency for Research on Cancer), and the figures are anticipated to increase in future. The only way to save millions of lives is to develop a treatment method that will specifically kill the cancer cell without harming non-malignant cells. One such approach gaining popularity is internalising an anti-tumor drug by cancer cells through folate receptors as it binds with folic acid, which is conjugated with the drug. These folate receptors have numerous applications, but here in this article, we will focus on folate receptors and the types of folate receptors; overexpression in humans and folate receptor as a target for drug delivery.

Folate receptor and Its types

The cell surface receptor for folic acid (FA), the folate receptor (FR) can selectively deliver folate-conjugated radiopharmaceuticals as means of tumour imaging and therapeutic agents to the target cells, such as cancer cells. The compelling reason for extensive use of folic acid is mainly due to its properties to the fewer undetectable expression of its receptor on normal cells, high affinity to its target (Folate receptors) even with various therapeutic and imaging conjugation and efficacy conjugate with diagnostic agents.

Moreover, In humans, there are three glycosylphosphatidylinositol-anchored membrane-bound isoforms of folate (FRα, FRβ, and FRδ), and one isoform FRγ as soluble protein constitutively secreted by Lymphoid cells. The FOLR1- FOLR4, which codes for FR, is located on the long arm of chromosome 11 (q11.3-q13.5), have a molecular weight range from 38 to 45 kDa molecular weight, binds with high affinity (Kd ∼ 0.1 to 1 nM for folic acid). FRα (FOLR1) and FRβ (FOLR2) are considered essential in cancers, as FRγ (FOLR3) secreted at low levels from Hematopoietic tissues (spleen, bone marrow, thymus) and demonstrated to have a lower affinity for FA than FOLR1 whereas, FRδ (FOLR4) suggested to have highly restricted spatial/temporal expression pattern. Although all the subunits of FR can bind with Folic acid, FRα has the strongest affinity and is more closely related to human disease. It is overexpressed in several cancers, i.e. in brain tumours (appx. 90% expression), ovarian, lung Etc., thus used as a prognostic marker for cancers. FR-β is extensively expressed on activated tumour-associated macrophages and the surfaces of malignant hematopoietic cells (TAMs). Furthermore, immune cells such as CD163+, CD68+, CD14+, and IL-10 responsible for tumour progression are also known to carry FRα and FRβ.

After processing in the secretory pathway, mature FR isomers capable of transporting folate share 68-79% amino acid sequence identity consists of 220-237 residues with posttranslational modifications, including the attachment of N-linked glycan and stabilization of 16 conserved cysteines by eight disulphide bonds. Thus, FR-α globular tertiary structure is stabilized by eight disulphide bonds and has three predicted N-glycosylation sites (N47, N139, and N179). In contrast, FR-β has only two N-glycosylation sites, which could explain differences in their expression level. This correlation with the number of N-glycosylation sites illustrated in mutagenesis studies where only 2% of FR-α and 8% of FR-β were expressed on the cell surface in the absence of N-glycosylation. The FR-α has a long open negatively charged binding pocket (residues conserved in all receptor subtypes) formed by helices tied together by four sulphide bridges and the amino-terminal loop (β1 and β2), where folic acid docks its basic pteroate moiety. Whereas the carboxyl group of FA glutamate moiety sticks out of the positively charged ligand-binding entrance, leading to its conjugation of drugs unaffecting the binding with FR-α.

Overexpression of folate receptors in Cancer cells

Extensive studies on Folate receptors revealed that overexpression of FR on the surface is the hallmark of most cancer cells, including those of ovary, brain, kidney, breast, colon, and lung cancers, whereas non-malignant cells are deficient in folate receptors. The qRT-PCR illustrated the hike in FRα mRNA and increased protein expression in Endometrial carcinoma (EC) tissue, possibly because of the role and requirement of FA in DNA synthesis of rapidly dividing cancer cells. Elevation of FRα has a functional role in Ras/Raf/MEK/ERK (main components of the ERK pathway), promoting cancer cell proliferation and suppressing apoptosis, thus involved in the pathogenesis of EC. Cheung et al., 2018, studies revealed that aggressive high-grade Triple-negative breast cancers (TNBCs) overexpress the cell surface tumour-associated FR-α that that lacks suitable therapeutic receptors, progesterone receptor (PR), estrogen receptor (ER), and HER2 expression. Notably, FR-α offers suitable means to direct antibody immunotherapy that primes an Fc-mediated antitumour immune response and immune cell-mediated killing with MOv18-IgG1 in vitro and in vivo in human breast cancer with the expression in post-neoadjuvant chemotherapy residual disease. Most importantly, FR-α overexpression at transcriptomic and protein levels plays a role in activating the Src-family, cancer cell signalling through the Src/ERK pathway and associated with STAT3/JAK signalling.

Folate receptor as a target for cancer therapy

Overexpression of FR by tumour cells, high-affinity of Folic acid for folate receptor and easy conjugation with anticancer drug vehicles drive FR as a prime candidate for tumour-specific targeting in cancer therapy. According to meeting reports and findings presented in honour of late Dr Kamen (a significant contributor to folate receptor biology for many decades) by Professor Joseph Bertino (Cancer Institute of New Jersey, Rutgers University, USA) stated that the mitochondrial folate enzyme (MTHFD2), a bifunctional enzyme (function of MTHFD and cyclohydrolase activities) profoundly express in fast replicating tumour cells. However, a superficial level in normal tissue provides a substantial rationale for knocking down this enzyme as potential anticancer therapy.

Looking at the deleterious effects in normal tissues of widely-used antitumour agent Doxorubicin (DOX), the delivery confirmed by some of the nano-carrier such as new folate (FA)-decorated amphiphilic bifunctional pullulan-based copolymer (named as FPDP) and near-infrared-sensitive polydopamine nanoparticles (NPs) of Fe3O4/Au core coated with polydopamine by conjugating Folic acid, a high-affinity ligand of folate receptors. In vitro, cell-culture experiments confirmed the enhanced chemotherapeutic cytotoxicity and cell targeting efficiency in overexpressing FR+ cells (Hela cell line) compared to FR- cells (HepG2 cells). DOX covalently attached to nanoparticles via hydrazone bond comparatively showed lower antitumour activity than DOX loaded glucose/gluconic acid-coated magnetic nanoparticles (DGNP)- loaded and folate-attached erythrocyte vesicles (FVDGNP). Furthermore, FVDGNP is contrary safer than DOX when compared to the parameters of liver damage (albumin (ALB) and alanine aminotransferase, aspartate aminotransferase and globulin levels) and kidney injury (inorganic phosphorus (PHOS), creatinine (CRE)). The mice receiving FVDGNP showed biochemical results close to the reference, whereas, in contrast, DOX receiving mice revealed traces of kidney, liver and cardiac damage.

The results of Yu et al., 2019, evinced the potential of i-ExNC (i-Extract nano complex of Withania somnifera leaves) as suitable natural source-based nanomedicine and folate conjugation improved the potency of i-ExNC. The folate receptor-targeting i-Extract nanocomplex (FRi-ExNC) in a preclinical trial could induce more potent cytotoxicity (3-fold), more substantial growth arrest (28% of cells in G2/M phase) as compared to i-ExNC (20% of cells in G2/M phase). Similarly, in vivo, there was more robust tumour suppression of FR-α-positive xenografts.

In brief, these results presented by different researchers cue the potential of folate receptor for cancer therapy and a ray of hope that it will be used for target-based drug delivery and cancer therapy.

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