Tackling breast cancer chemoresistance with nano-formulated siRNA

Abstract

Breast cancer is the leading cancer diagnosed in women and the second leading cause of cancer-related deaths in women. Current limitations to standard chemotherapy in the clinic are extensively researched, including problems arising from repeated treatments with the same drugs. The phenomenon that cancer cells become resistant toward certain chemo drugs is called chemotherapy resistance. In this review, we are focusing on nanoformulation of siRNA for the fight against breast cancer chemoresistance.

INTRODUCTION

Breast cancer is the leading cancer diagnosed in women and the second leading cause of cancer-related deaths in women. It is estimated that 1 in 8 women will be diagnosed with breast cancer in their lifetime and over 40 000 women will die each year due to it.¹ Early detection is key for patients; with a >90% 5-year survival rate for patients diagnosed at stages 0, I or II. Approximately 10% of patients present with stages III and IV, where treatment options are determined on an individual patient basis.^2,3 Neoadjuvent treatment can be used for localized invasive breast cancer; such as, but not limited to: Herceptin (Her-2-positive tumors only), a combination of an anthracycline-based and taxane-based chemotherapy, hormone-replacement therapy with an aromatase inhibitor, or inhibitors of the cyclin-dependent kinase 4/6.⁴ For advanced breast cancer, traditional chemotherapy and radiation can be used. However, these patients may never be ‘cured’ after the cancer has spread into distant organs. Chemotherapy, radiation, bisphosphonates, herceptin, and other treatments have all been used in these cases to treat the disease but do not cure it.

Current limitations to standard chemotherapy in the clinic are extensively researched. Typical problems with chemotherapy include their systemic treatment, a lack of tumor targeting and side effects in off-target tissues and organs, insufficient tumor deposition and penetration which would be necessary to achieve cell killing. In addition, chemotherapy drugs can be very hydrophobic and poorly soluble which can limit their possible administration. Finally, repeated treatments with the same drugs can give rise to tumors that are comprised of cancer cells which have become resistant to the drug. This phenomenon is called chemotherapy resistance. Resistance can arise through several factors, however, it is most commonly associated with increased translation of anti-apoptotic proteins such as Bcl-2 and of ATP-binding cassette (ABC) transporters—more specifically, a family member known as p-glycoprotein (p-gp) drug-efflux pumps.^5–8

Although the discovery of RNA interference, or small-interfering RNA (siRNA), researchers have been able to selectively inhibit the expression of proteins within the cell. Considering that individual proteins have been described to give rise to multi-drug resistance (MDR) in breast cancer, many researchers have pursued the idea of targeted delivery of siRNA to combat and overcome chemotherapy resistance in cancer, and specifically in the context of breast cancer. However, delivering siRNA intracellularly and specifically to cancer cells to knock down the target gene can be difficult. Non-formulated, ‘naked’ siRNA is easily degradable by ubiquitous RNases, it is a macromolecule that does not readily cross membranes, and siRNA is negatively charged and hydrophilic. Ongoing research has been evaluating the encapsulation of siRNA inside various delivery vehicles, its delivery and targeting to cancer cells in order to knock down oncogenes, genes associated with cell survival and anti-apoptosis, genes associated with chemoresistance to re-sensitize resistant cells, and many others. The reader is referred to several excellent reviews which cover the current state of viral, non-viral, lipid, as well as other creative delivery systems for siRNA therapy.^9–11 Various mechanisms of chemotherapy resistance and approaches to combat this problem with nanodelivery of siRNA have been described.¹² One important approach is depicted in Figure 1, showing how siRNA can be co-encapsulated with a chemotherapy drug to achieve maximum re-sensitization toward the drugs. The siRNA delivered can be used to target known pathways which give rise to chemotherapy resistance such as efflux pumps or anti-apoptotic protein blc-2. In this review, we are focusing on nanoformulation of siRNA for the fight against breast cancer chemoresistance.

Figure 1.

Combination approach of targeted delivery of siRNA and chemo drugs to the cytoplasm of a cancer cell to treat chemotherapy-resistant breast cancer. By encapsulating both payloads within one nanoparticle, each drug is deposited inside the cell at the same time to achieve synergistic cytotoxic effects, for example, gene silencing of p-gp pumps.

SIRNA DELIVERY CHALLENGES AND INNOVATIVE CARRIERS

Multiple challenges oppose effective siRNA delivery. Owing to its negatively charged and hydrophilic nature and susceptibility to degradation by nucleases, for successful delivery, siRNA needs to be packaged inside a carrier in order to mediate its potent effects. Ideally, a carrier needs to be stable in circulation, have good cellular uptake and endosomal escape profiles, biocompatible and biodegradable, as well as being inherently non-toxic to the healthy cells.¹² With a variety of ‘smart’ vehicles being created and studied, these hurdles can be overcome. Successful siRNA delivery is reflected by efficient downregulation of specific proteins which give rise to MDR using nano-formulated siRNA.

Considering all the hurdles that need to be addressed in order to achieve safe and effective siRNA therapy, an abundant and wide range of carriers is being studied. Effective siRNA carriers for breast cancer therapy should have target specificity to the cancer cells which can be achieved with a targeting ligand to a receptor specifically expressed or overexpressed on the cell surface of breast cancer cells. In addition, the final formulations of siRNA-bearing nanocarriers should have suitable sizes, charge, solubility profiles, encapsulation efficiency, as well as translational relevance in order to be potential candidates for clinical translation.^13,14 Wu et al.¹⁵ and Wong et al.¹⁶ were able to successfully co-encapsulate doxorubicin and siRNA against p-gp. However, Wu et al.¹⁵ used a folic acid-targeting ligand on a PEG-b-(PCL-g-PEI)-b-PCL triblock copolymers which self-assembled into nanoparticles under 100 nm for delivery to MCF-7 cells, whereas Wong et al.¹⁶ utilized a polymer-lipid hybrid nanoparticle. These dox loaded polymer-lipid hybrid nanoparticles had an average size and zeta potential of 290 nm and −23.1 mV, respectively. The delivery systems of both groups were able to knock down p-gp whereas using a targeted versus a non-targeted carrier system with different hydrodynamic diameters and zeta potentials. Considering that optimal sizes are often reported to be below 260 nm in order to avoid macrophage detection and phagocytosis, and optimal zeta potentials are described to be slightly positive to have an inherent interaction with the cell surface,^17–20 the polymer-lipid hybrid nanoparticle characteristics could pose a problem for in vivo delivery and efficacy. The use of folic acid on the surface of nanoparticles offers several benefits. The binding affinity of folic acid to folate receptor is 1 nm. This allows for a very strong and specific binding to the receptor. Coincidently, folate receptor alpha is primarily expressed on the epithelial surface, whereas several cancers show a significant overexpression of the receptor.²¹ This allows for folate receptor targeted nanoparticles to achieve a higher selectivity in cellular uptake along with a higher binding affinity or avidity. Another example of targeting a receptor on the cell surface was reported by Dou et al.²² Their group targeted the HER-2 receptor on breast cancer cells by using positively charged protamine (F5-P) and attaching an anti-Her-2 single-chain antibody fragment on the surface. The Her-2 targeting ligand displayed effective delivery to Her-2-positive cells BT474, but not to Her-2-null cells MDA-MB-231.²² Utilizing a targeted approach with a single-chain antibody offers the advantages of a smaller size targeting ligand compared with a complete antibody, of a cheaper and easier to produce targeting ligand, and the ability to functionalize in order to increase circulation half-life.²³

Modifications to widely known polymers have been used to increase the efficacy of the latter or to increase their biocompatibility. Numerous groups have used polyethylene imine (PEI) as the cationic portion of their respective nanocarriers to condense siRNA for delivery. However, each group modified PEI differently in order to increase their carrier systems’ biocompatibility. Navarro et al.²⁴ modified PEI with the phospholipid dioleoylphosphatidylethanolamine which resulted in particles of 127–187 nm in size along with the ability to co-encapsulate p-gp siRNA and doxorubicin.²⁴ In attempts to make PEI more biocompatible and to evade detection by the immune system, a common modification is the PEGylation of PEI. Meng et al.²⁶ and Essex et al.²⁵ both PEGylated PEI in order to functionalize either a mesoporous silica nanoparticle with PEI-PEG, or a DOPE-modified phospholipid PEI, respectively. In the study by Meng et al.²⁶ silica-based nanoparticle achieved >70% reduction of p-gp mRNA in vivo, as well as decreasing off-target cardiotoxicity after systemic doxorubicin treatment. On the other hand, by modifying a low molecular weight PEI with PEG, Essex et al.²⁵ exhibited an increase in circulation half-life of the nanocarriers which lead to a deposition of 8% of the injected dose of siRNA in the tumor.²⁶ Finally, low molecular weight PEI was designed by Lin et al.²⁷ with different alkylation groups and formed nanocarriers with clustered iron oxide nanoparticles. These nanocarriers were 80–130 nm in size with a zeta potential of +44 mV and were able to be imaged in vivo. In the study by Lin et al.²⁷ iron oxide nanocarriers achieved a 50–60% downregulation of MDR1 in vivo after local administration.²⁷

Novel delivery devices and polymers are also engineered and tested. Yhee et al,²⁸ for example, developed 270 nm thiolated glycol chitosan nanoparticles for anti-MDR1 siRNA delivery. Thiolating the nanoparticles helped increase their binding affinity and cross linking to form nano-complexes. This complex can be intravenously administered and shows 2.7 × higher tumor-targeting efficiency compared with non chitosan-based nanoparticles.²⁸ A unique approach to increasing doxorubicin’s efficiency in treating triple-negative breast cancer is the use of a layer-by-layer nanoparticle film formation by alternately depositing siRNA and poly-l-arginine in layers atop a doxorubicin-loaded liposome. These nanoparticles were 120 nm in size with a zeta potential of −56 mV and could impressively hold 3500 siRNA molecules per particle. In addition, the LbL nanoparticles had a circulation half-life of 28 h, reduced MDR1 levels in the tumor by 80%, and increased doxorubicin’s cell killing efficacy fourfold.²⁹ This approach, along with Segovia et al.³⁰ approach of oligo-peptide terminated pBAE nanoparticles embedded within a hydrogel scaffold, can lead to an increased half-life and a controlled release mechanism of siRNA to achieve a more sustained knockdown.³⁰ Ultimately, several types of carriers, ranging from well-established building blocks to novel compounds, have been researched and shown promise. With all of the sophisticated carrier systems that have been developed, there has been a movement away from single payload delivery of siRNA to a co-delivery approach of multiple payloads to the cancer cell.

COMMON TARGETS TO OVERCOME RESISTANCE

In breast cancer research, several types of proteins are reported to lead to MDR. However, the most commonly studied form of MDR is caused by the overexpression of ABC transporters.³¹ In total, over 45 ABC transporter genes have been identified.³² These transporters actively transport various drugs through the plasma membrane and outside the cell. The most widely studied ABC transporter is ABCB1/MDR1. Overexpression of MDR1 can lead to an increase in the cells’ resistance to certain drugs that are a natural substrates to this efflux pump. The natural substrates for MDR1 include, but are not limited to, generic chemotherapeutic agents such as anthracyclines, taxanes and vinca alkaloids. However, even newer cancer drugs such as Gleevec (imatinib) can be a substrate.³² Numerous research groups around the globe have extensively studied the role of MDR1 in breast cancer.^33–36 Many of these groups have shown the effectiveness of inhibiting the overexpression of MDR1 with siRNA in order to re-sensitize breast cancer cells to various chemotherapeutic substrates of the protein pump. Other ABC family member transporters have been linked to giving rise to resistance in breast cancer. As demonstrated by Liang et al.³⁷ the inhibition of MRP-1/ABCC1 renders MCF-7 cells sensitive to doxorubicin. Furthermore, breast cancer-resistance protein BCRP/ABC2 was suppressed with siRNA in order to confer an increase in sensitivity to drugs such as methotrexate, doxorubicin, flavopiridol and anthracyclines.^38–40

In addition, several prosurvival and anti-apoptotic proteins have been linked to chemoresistance in breast cancer. Besides drug-efflux pumps such as MDR1, prosurvival and anti-apoptotic proteins are the second most studied area in the breast cancer-resistance field.⁵ Survivin, a small anti-apoptotic protein, can cause cells to avoid apoptosis when treated with paclitaxel. Salzano et al.⁴¹ described that when survivin is downregulated via siRNA treatment, cells became strongly resensitized to paclitaxel treatment and underwent significant inhibition of cell growth. Similar results were found with doxorubicin by Tang et al.⁴² By the same token, BCL-2, an anti-apoptotic protooncogene is overexpressed in at least 70% of breast cancers. By silencing >85% of BCL-2 expression in MCF-7 cells, Akar et al.⁴³ achieved efficient inhibition of cell growth and increased cell death. Other groups such as Li et al.⁴⁴ have studied NF-κB and its role in breast cancer chemoresistance. By co-delivering doxorubicin and siRNA against NF-κB, a significant decrease in doxorubicin’s IC₅₀ value was observed. Specifically, 80% of cells underwent apoptosis, and a >95% positive synergy between the treatment with dox and anti-NF-κB siRNA was observed.⁴⁴

Besides the inhibition of MDR1 and anti-apoptotic proteins with siRNA, numerous other proteins have been published and linked to MDR within breast cancer. Liu et al.⁴⁵ revealed that fatty acid synthase was overexpressed in breast cancers and gave rise to palmitic acid production which resulted in a decrease in apoptotic levels. Their work elucidates a potential new target for siRNA therapy to overcome chemotherapy resistance.⁴⁵ In addition, Gouazé et al.⁴⁶ provided a link to MDR through an overexpression of glucosylceramide synthase. By knocking down glucosylceramide synthase, MCF-7 cells exhibited a restored sensitivity to doxorubicin, vinblastine and paclitaxel. Members of the kinesin family have been linked to chemoresistance by Singel et al.⁴⁷ Two independent groups have shown that by knocking down the expression of Kif11 an Kif14, resistance can be overcome in triple-negative breast cancer.^47,48 This increase in kinesin family member proteins can be considered a potential biomarker for high-risk breast cancer tissue, according to Singel et al.⁴⁷ Finally, inhibition of angiogenesis in the context of breast cancer has been studied by knocking down vascular endothelial growth factor with nanoformulated siRNA. Successful inhibition of vascular endothelial growth factor was shown to inhibit the growth of tumor spheroids in vitro, whereas also showing efficacy in vivo. When combined with low-dose doxorubicin, tumor microvessel density was inhibited, along with an increase in overall survival.⁴⁹ Although less studied compared with MDR1 or BCL-2, these alternative targets hold promise in the battle to overcome breast cancer resistance.

EPIGENETIC TARGETS

In an approach to better understand the development of chemoresistance, histone-modifying and DNA-methylating enzymes, so-called ‘epigenetic enzymes’ have been reported to have important roles not only in cancer development,^50,51 but also in cancer chemoresistance.⁵² Calcagno et al.⁵² described that histone hyperacetylation is the reason for upregulation of ABCG2 in doxorubicin-selected cancer cell lines, including MCF-7 breast cancer cells, and mediates their resistance. The authors used Oligofectamine, a commercially available transfection reagent, to deliver siRNA against ABCG2 and observed that a 40-fold decrease in the ABCG2 levels led to 85% restored sensitivity compared with the parental MCF-7 cells.⁵² But epigenetic changes can also cause chemoresistance via pathways independent of p-gp. As described above, prosurvival signaling can prevent the effectiveness of chemotherapy also. Accordingly, Almeida et al.⁵³ reported that NF-κB signaling can cause histone modifications which in turn mediate chemoresistance in head and neck squamous cell carcinoma (HNSCC) via histone deacetylation. The authors showed that knockdown of IKKα and IKKβ, which represses NF-κB, resulted in induced acetylation of tumor histones and reduced chemoresistance against cisplatin.⁵³ Even though this study was conducted in head and neck squamous cell carcinoma cells, similar pathways may be found in breast cancer cells as well. In breast cancer, however, other epigenetic changes have already been described. Mungamuri et al.⁵⁴ investigated epigenetic changes that lead to overexpression of Her-2/neu, an EGFR family receptor. The authors observed that methylation of H3K4me3 mediates resistance toward trastuzumab and that silencing of Wdr5 with shRNA, one of the four structural components of the H3K4 methyltransferase complex, decreased Her-2/neu levels and chemoresistance.⁵⁴ shRNA was also used to silence DNA methyltransferase 3b in hypermethylator breast cancer cell lines BT549 and Hs578T, and caused sensitization toward doxorubicin, paclitaxel and 5-fluorouracil.⁵⁵ DNA methyltransferase 3b and DNMT1 were also the subject of other studies that used commercially available transfection reagents to deliver siRNA.^56,57 However, to our knowledge, so far no studies have been published using nanoformulated siRNA to silence epigenetic targets.

CO-DELIVERY OF PAYLOADS AND ALTERNATIVE APPROACHES

Recent literature has stated that simultaneous delivery of siRNA and a chemotherapeutic agent yields more synergistic results and more cell killing than separate or standalone treatment.⁵ This technique of encapsulating chemotherapeutic drugs within carriers offers the advantages to encapsulate poorly soluble drugs, eliminate off-target effects caused by harmful organic solvents needed to dissolve hydrophobic drugs and replaces the use of viscous emulsions. Encapsulating chemotherapeutic agents within the core of a micelle or liposome allows for the opportunity to add a targeting ligand to change the delivery profile from systemic non-targeted to targeted therapy. In addition, delivering both payloads at the same time ensures that both agents reach the tumor at the same time instead of relying on the pharmacokinetic circulation profiles, and targeting efficiency of each separate drug. Several groups have used this approach to their advantage to overcome resistance in breast cancer.

Owing to the frequent overexpression of p-gp in breast cancer, several groups have co-encapsulated anti-siRNA p-gp with doxorubicin. Examples of this strategy were described by Wu et al.¹⁵ and Wong et al.¹⁶ Both groups demonstrate that co-delivery of both payloads can reduce off-target toxicity and re-sensitize MCF-7 and MDA-435 cell lines.^15,16 Using a different carrier, Jiang et al.⁵⁸ synthesized a modified RGD-targeted peptide liposome encapsulating p-gp siRNA and doxorubicin. These liposomes were <200 nm in size and ex vivo imaging studies showed the accumulation of siRNA and dox within the tumors at the same site. Furthermore, co-delivery of these two agents showed significant inhibitory effects on tumor growth.⁵⁸ Peptide-based targeting moieties, such as integrin targeting RGD (arginine–glycine–aspartic acid), can bind to their respective receptors throughout the body. Peptides inherently are easy to synthesize, biocompatible, smaller than antibodies and have a wide variety of targeting receptors.⁵⁹ A tabulated summary of co-delivery approaches is shown in Table 1, adapted from Gandhi et al..¹² This table depicts various approaches utilizing nanoparticles to co-deliver a chemotherapeutic drug along with a nucleic acid-based payload in order to treat a variety of cancers. These results emphasize the potent synergy between co-administration versus single dosing.

Table 1.

Tabulated form of co-delivery approaches to treat cancer using nucleic acid therapeutic and chemotherapeutic drugs

siRNA/miRNA	Drug	Type of nanocarrier	Cell lines	In vivo model	Targeting	Targeting moiety/ peptide	Reference (s)
Co-delivery of siRNA in combination with chemotherapeutic drug and/or nucleic acid-based reagent for the treatment of cancer
siRNA targeting BCL-2 and MRP-1	DOX/CIS	Mesoporous silica nanoparticle	A549 human lung adenocarcinoma	Murine A549 lung cancer orthotopic model	Active	LHRH peptide	12
siRNA targeting P-gp	DOX	Mesoporous silica nanoparticles	MDR KB-V1 human cervical carcinoma	—	Passive	—	62
siRNA targeting P-gp	DOX	PEI-PEG functionalized mesoporous silica nanoparticles	MCF-7/MDR—breast cancer	Murine MCF-7/MDR breast cancer xenograft model	Passive	—	26
siRNA targeting mTERT	PTX	HTCC nanoparticles	LLC—lewis lung carcinoma	—	Passive	—	79
siRNA targeting GFP	DOX	G(4)-PAMAM-PEG-DOPE dendrimers	C166 cells—yolk sac endothelial	—	Passive	—	80
siRNA targeting Luc gene	DOX	(G3) poly (l-lysine) OAS dendrimer	U-87 glioblastoma	—	Active	RGD peptide	12
siRNA targeting BCL-2	Docetaxel	PEG-PLL-PLLeu cationic micelles	—	Murine MCF-7 breast cancer xenograft model	Passive	—	81
siRNA targeting MCL-1 and GL2	SAHA	TLO cationic liposomes	KB epithelial cancer	Murine KB epithelial cancer xenograft model	Passive	—	12
siRNA targeting VEGF	PTX	PDMAEMA–PCL–PDMAEMA cationic micelles	PC-3 human prostate cancer and MDA-MB-435-GFP breast cancer	—	Passive	—	82
siRNA targeting VEGF and c-Myc	DOX	Lipid polycation DNA nanoparticles	MDR NCI/ADR-RES ovarian tumor	Murine NCI/ADR-RES ovarian cancer xenograft model	Passive	—	83
siRNA targeting c-Myc	DOX	Liposome-polycation-DNA nanoparticles	HT-1080 fibrosarcoma	Murine HT-1080 fibrosarcoma xenograft model	Active	PEGylated NGR (aspargine-glycinearginine)	84
siRNA targeting BCL-2 and MRP-1	DOX	DOTAP cationic lipid nanoparticles	MDR lung cancerMDR A2780/AD ovarian cancer	—	Passive	—	12
siRNA targeting MCl-1	MEK inhibitor PD032590	Cationic liposomes	KB epithelial cancer	Murine KB epithelial cancer xenograft model	Passive	—	85
siRNA targeting VEGFR and EGFR	CIS	PEI complexes	—	Murine A549 NSCLC xenograft model	Passive	—	12
siRNA targeting X linked inhibitor of apoptosis	PTX	Deoxycholic acid-PEI complexes	HCT-116 colorectal cancer	Murine HCT-116 xenograft model	Passive	—	86
siRNA targeting BCL-2	DOX	Cationic PEI-PCl nanoparticles	C6 Glioma Bel-7402 human hepatoma	Murine C6 glioma xenograft model	Active	Folic acid	87
siRNA targeting P-gp	PTX	PLGA-PEI nanoparticles	JC mouse mammary cancer	Murine BALB/c JC breast cancer xenograft model	Active	Biotin	88
siRNA targeting MCL-1	PTX	Cationic solid–lipid nanoparticles	KB epithelial cancer	Murine KB epithelial cancer xenograft model	Passive	—	89
siRNA targeting Plk1	PTX	PEG-b-PCL-b-PPEEA micelleplex	MDA-MB-435 breast cancer	Murine MDA-MB-435 s breast cancer xenograft model	Passive	—	90
siRNA targeting BCl-2	S-1	Lipoplexes	DLD-1 colorectal adenocarcinoma	Murine DLD-1 colorectal adenocarcinoma xenograft model	Passive	—	76
iMdr-1-shRNA iSurvivin-shRNA	DOX	Poly (b-amino esters)-based nanoparticles	MCF-7 human breast adenocarcinoma	Murine BALB/c MDR MCF-7 breast adenocarcinoma xenograft model	Passive	—	42
siRNA targeting HMD2<comma4c-Myc>	VEGF siRNA	Lipid-coated calcium nanoparticles	A549 adenocarcinoma and H460 lung carcinoma	Murine A549 and H460 NSCLC xenograft model	Passive	—	77
siRNA targeting c-Myc and MDM2	VEGFR mir-24a	Liposome-polycation-hyaluronic acid	—	Murine B16F10 melanoma xenograft model	Active	scFv	78

An alternative approach to overcoming resistance is packaging two separate siRNA sequences for different targets, as well as encapsulating selenium. Zheng et al.⁶⁰ prepared layered double hydroxide nanoparticles that were 116 nm in size and were able to selectively deliver siRNA against p-gp and b-tubulin III. This approach was shown to inhibit cell mitosis, spindle formation but also induced apoptosis in MCF-7/ADR cells.⁶⁰ In addition, other groups have described the approach of using two siRNA sequences to silence multiple ABC transporters in MCF-7 cells. In a study by Li et al.⁶¹ ABCG2 an ABCB1 sequences were simultaneously delivered inside a pH-sensitive carbonate apatite nanoparticle. The dual targeted siRNA approach led to an enhanced toxicity (above 45–50% cell killing) when treated with cisplatin, paclitaxel and doxorubicin. Although the single delivery of siRNA did slightly re-sensitize the cells, the dual targeted siRNA approach had a greater cytotoxicity.⁶¹

ANIMAL MODELS

In order to move the various delivery systems, siRNA targets and disease states closer to the clinic, numerous animal models have been utilized to investigate overcoming MDR in a more complex in vivo setting. For the past four decades, several previous models have been utilized in vivo for breast cancer research. Current breast cancer models which are applied can be spontaneously forming tumors, mainly in larger animals, genetically modified models and xenografts. In addition, all of the various subtypes of breast cancer such as triple-negative, invasive ductile carcinoma or inflammatory breast cancer are studied. However, new models needed to be developed in order to specifically analyze the re-sensitization of chemotherapy-resistant cells in laboratory animals, mainly in mice.

Numerous research groups have created isogenic cell lines that are sensitive and resistant to various chemotherapeutic agents. Aliabadi et al.⁶² used a xenograft nu/nu nude mouse model with MDA-435 sensitive and resistance cells injected subcutaneously into the right flank of the mice.⁶³ This model, along with the authors’ work delivering vascular endothelial growth factor siRNA and doxorubicin to mice helped elucidate a decrease in tumor blood vessels which allowed for an increase in life span of the tumor-bearing mice. Commonly, MCF-7 cells are used in breast cancer in vivo xenograft models due to being extensively researched, being easily available and for having resistance to commonly used agents such as doxorubicin. A multitude of independent groups have all utilized MCF-7 cells to confer chemotherapy resistance and demonstrate their treatments efficacy on overcoming MDR in vivo.^{27,28,49,64,65} Similarly, triple-negative breast cancer cell lines such as MDA-MB-231 and MDA-MB-468 can be injected into immune-suppressed mice in order to study the response of triple-negative breast cancer to siRNA therapy.^14,66–68 Besides varying cell lines in order to study a wide variety of breast cancer subtypes, different injection sites have been described. Although the most common injection site for studies using nano-formulated siRNA to tackle chemoresistance in breast cancer is the subcutaneous area at the flank,^28,63,67 other models mimic metastases in the axilla region,^49,65 or primary orthotopic tumors in the mammary fat pad of the mice.^14,27,66 So far, no spontaneous tumors or genetic models have been used for siRNA delivery to breast cancer in the fight against chemoresistance. The lack of more relevant models may explain the large amount of pre-clinical but the very small amount of clinical studies.

CLINICAL TRIALS OF SIRNA UTILIZED IN BREAST CANCER TREATMENT

As the discovery of siRNA, researchers have been trying to transition this mechanism to the clinic. Advances have been made since the discovery of the RNA interference mechanism, however, the transition into clinical trials and into the clinic has remained challenging. There have only been a handful of clinical trials that have been translated into the clinic for solid tumors, and hardly any for breast cancer. In 2008, Calando Pharmaceuticals (Pasadena, CA, USA) started a clinical trial with their drug CALAA-01 for solid tumors, including breast cancer. Their study used a transferrin-targeted cyclodextrin-containing polymer which carried an anti-R2 siRNA sequence. Transferrin targeting utilizes a recycling pathway involving a clathrin-coated pits-mediated method of internalization which can be exploited to help delivery payloads into the cell while also achieving a tumor-targeting approach. Several cancers such as breast, pancreas, colon, lung and bladder have demonstrated an increased expression of transferring receptors, including several drug-resistant tumors.⁶⁹ This transferrin-targeted clinic trial was performed to study the safety and tolerability of a nanoparticle and siRNA-based injection in patients and has been subsequently terminated due to not meeting their primary or secondary outcome measures.⁷⁰ Lately, M.D. Anderson Cancer Center (Houston, TX, USA) has been recruiting participants for their EphA2 gene-targeting study using a liposomal siRNA delivery agent. This study also assesses the safety of their liposomal formulation. Data such as dose-limiting toxicity and hematologic toxicity are being recorded.⁷¹ On the other hand, ever since the discovery of ABC transporters, such as MDR1, several clinical trial studies have investigated inhibitors of ABC transporters. These clinical trials range from the early 1990s until recently. Although these trials do not include siRNA, but rather small-molecule inhibitors of the transporter pumps, they have been studied in several cancers, including breast cancer, and shown to increase overall survival in patients.⁷ The knowledge obtained through these clinical trials could in fact be a promising basis for subsequent trials with siRNA for the inhibition of ABC transporters. Overall, siRNA-based therapies have not yet reached the clinic, but with further development of multiple targets, sophisticated delivery systems and combination treatments, hopefully a breakthrough can be achieved.

CONCLUSION AND OUTLOOK

Resistance to chemotherapy is a challenging obstacle that needs to be addressed and overcome in the clinic. One mechanism that has been used to re-sensitize cells has been targeted delivery of siRNA. Since the discovery of RNA interference, researchers have been trying to exploit its benefits in order to provide therapeutic gene knockdown of target proteins. This approach yields several advantages, especially in combination with standard chemotherapy. For years it has been known that several proteins (namely ABC transporters and anti-apoptotic factors) are overexpressed in breast cancer leading to resistance toward chemotherapy drugs such as doxorubicin or paclitaxel. Effective siRNA delivery can selectively knock down the overexpression of such proteins, thus resensitizing the cells to treatment. Although this review focused on resistance mechanisms derived from MDR expression, antiapoptotic factors, angiogenesis and epigenetic factors, there are a variety of alternative pathways and factors that can give rise to MDR, which—so far—have not been addressed using nanoformulated siRNA, however. Owing to the scope of this review, those factors such as tumor microenvironment-mediated drug resistance will not be addressed here even if they may offer great potential for future siRNA-based approaches in the fight against chemotherapy-resistant breast cancer. However, in order to effectively deliver siRNA, a carrier needs to be used. One of the major advantages is that these carriers can encapsulate multiple payloads for a combination treatment. It has been shown that combination treatment of drugs such as doxorubicin and siRNA have a greater therapeutic efficacy than the delivery of single agents. This approach has shown significant promise both in vitro and in vivo. Albeit multiple studies have been shown to achieve significant therapeutic efficacy with nano-formulated siRNA therapies, there are hurdles that need to be addressed in the future. For a more in depth analysis on the toxicity and off-target effects of siRNA and nanoparticles, the reader is referred to several in depth reviews on the matter.^72–75

The transition of siRNA therapy into the clinic has yet to be achieved. Only a handful of clinical trials have used siRNA, and only a small fraction included breast cancer patients. It is expected that with newer targeted delivery agents, the most common hurdles for specific and efficient siRNA delivery can be overcome. If successful, siRNA treatment has a promising future in the clinic, especially for chemoresistant breast cancer patients.