Bioengineered siRNA-Based Nanoplatforms Targeting Molecular Signaling Pathways for the Treatment of Triple Negative Breast Cancer: Preclinical and Clinical Advancements

Triple negative breast cancer (TNBC) is one of the most aggressive types of breast cancer. Owing to the absenteeism of hormonal receptors expressed at the cancerous breast cells, hormonal therapies and other medications targeting human epidermal growth factor receptor 2 (HER2) are ineffective in TNBC patients, making traditional chemotherapeutic agents the only current appropriate regimen. Patients’ predisposition to relapse and metastasis, chemotherapeutics’ cytotoxicity and resistance and poor prognosis of TNBC necessitates researchers to investigate different novel-targeted therapeutics. The role of small interfering RNA (siRNA) in silencing the genes/proteins that are aberrantly overexpressed in carcinoma cells showed great potential as part of TNBC therapeutic regimen. However, targeting specificity, siRNA stability, and delivery efficiency cause challenges in the progression of this application clinically. Nanotechnology was highlighted as a promising approach for encapsulating and transporting siRNA with high efficiency-low toxicity profile. Advances in preclinical and clinical studies utilizing engineered siRNA-loaded nanotherapeutics for treatment of TNBC were discussed. Specific and selective targeting of diverse signaling molecules/pathways at the level of tumor proliferation and cell cycle, tumor invasion and metastasis, angiogenesis and tumor microenvironment, and chemotherapeutics’ resistance demonstrated greater activity via integration of siRNA-complexed nanoparticles.


Introduction
Cancer is heterogeneous complex disease characterized by rapid and uncontrollable cell proliferation resulting from genetic and epigenetic mutations [1,2]. Cancer burden is greatly significant; it is the second leading cause of morbidity and mortality in the word and is the first in low-and middle-income countries [3,4]. Breast cancer is one of the most common cancers in women [5]. Globally, breast cancer is the leading cause of mortality among females [6]. The overall breast cancer-related mortality rate increased in the past decade, although the overall death rate has declined significantly from 1990 to 2017. The early detection of breast cancer and the advances in cancer treatments and diagnostic tools are the major contributor to decreasing the overall cancer-related mortality [7].

Pre-Clinical Activity of Engineered siRNA-Mediated Therapies for Treatment of TNBC
TNBC is characterized by inter-and intra-individual diversity of gene mutations. TNBC genes mutations that are concomitant with poor TNBC prognosis are involved in substantial signaling pathways including: cell proliferation and disease progression, cell survival and death, angiogenesis, cancer metastasis and drug resistance ( Figure 1). Growing body of evidence revealed promising results concerning the effectiveness of targeted gene suppression using engineered siRNA mediated nanocarriers in the following major areas: Tumor cell proliferation and cell cycle regulation, tumor invasion and metastasis, angiogenesis, and tumor microenvironment and chemotherapeutics resistance ( Table 2).
Pharmaceutics 2020, 12, 929 5 of 24 pathways including: cell proliferation and disease progression, cell survival and death, angiogenesis, cancer metastasis and drug resistance ( Figure 1). Growing body of evidence revealed promising results concerning the effectiveness of targeted gene suppression using engineered siRNA mediated nanocarriers in the following major areas: Tumor cell proliferation and cell cycle regulation, tumor invasion and metastasis, angiogenesis, and tumor microenvironment and chemotherapeutics resistance ( Table 2).

Tumor Cell Proliferation and Cell Cycle Regulation
Forkhead box protein M1 (FOXM1) is a key oncogenic transcription factor, which plays a crucial role in regulating the expression of genes involved in cancerous cell proliferation and cell cycle progression. It was found that FOXM1 is significantly overexpressed in patients with TNBC. Hamurcu et al. examined the effectiveness of liposomal FOXM1 siRNA in mice implanted with MDA-MB-231 cells and found that these gene therapeutics cause a pronounced downregulation of FOXM1 expression and consequently suppress TNBC cells growth significantly [66]. Wang et al. further revealed incorporation of anti FOXM1 siRNA into polyethylimine (PEI)-based cationic polymers enhances cellular uptake of the targeted therapy within the tumor cells for 24 h after its administration. Both in vitro and in vivo studies indicated that PEI-based anti-FOXM1 siRNA knockdown the expression of FOXM1 causing reduction in the protein levels associated with cell proliferation and disease progression [67].
Eukaryotic Elongation Factor 2 Kinase (eEF2K) is a principal regulator of tumor growth and progression. Patients with TNBC had significant elevation in eEF2K expression. As FOXM1 and eEF2K are involved in the same functions in TNBC cells, it was expected that FOXM1 regulates the expression of eEF2K. Hamurcu et al. demonstrated that the engineered siRNA liposomal nanoparticles were able to bind to the promoter region of eEF2K and regulates its expression [66]. Modified gold anti-eEF2K siRNA nanoparticles are highly effective for treating TNBC cells on xenografted mice resulting in a remarkable down-regulation of eEF2K expression and, thus, inhibiting tumor growth and progression [68].
MDM2 is an oncogene that inactivates the tumor suppressor p53, which are greatly overexpressed in TNBC patients. Overexpression of MDM2 leads to suppression of p53 affecting tumor cell proliferation and apoptosis. Single-walled carbon nanotubes (SWNTs) were functionalized with PEG for efficient delivery of siRNA for targeting MDM2. It was concluded that PEG-modified SWNTs are effective drug delivery system promoting major MDM2 silencing effect causing tumor cell proliferation suppression and induced apoptosis in breast cancer cells [69].

Tumor Cell Proliferation and Cell Cycle Regulation
Forkhead box protein M1 (FOXM1) is a key oncogenic transcription factor, which plays a crucial role in regulating the expression of genes involved in cancerous cell proliferation and cell cycle progression. It was found that FOXM1 is significantly overexpressed in patients with TNBC. Hamurcu et al. examined the effectiveness of liposomal FOXM1 siRNA in mice implanted with MDA-MB-231 cells and found that these gene therapeutics cause a pronounced downregulation of FOXM1 expression and consequently suppress TNBC cells growth significantly [66]. Wang et al. further revealed incorporation of anti FOXM1 siRNA into polyethylimine (PEI)-based cationic polymers enhances cellular uptake of the targeted therapy within the tumor cells for 24 h after its administration. Both in vitro and in vivo studies indicated that PEI-based anti-FOXM1 siRNA knockdown the expression of FOXM1 causing reduction in the protein levels associated with cell proliferation and disease progression [67].
Eukaryotic Elongation Factor 2 Kinase (eEF2K) is a principal regulator of tumor growth and progression. Patients with TNBC had significant elevation in eEF2K expression. As FOXM1 and eEF2K are involved in the same functions in TNBC cells, it was expected that FOXM1 regulates the expression of eEF2K. Hamurcu et al. demonstrated that the engineered siRNA liposomal nanoparticles were able to bind to the promoter region of eEF2K and regulates its expression [66]. Modified gold anti-eEF2K siRNA nanoparticles are highly effective for treating TNBC cells on xenografted mice resulting in a remarkable down-regulation of eEF2K expression and, thus, inhibiting tumor growth and progression [68].
MDM2 is an oncogene that inactivates the tumor suppressor p53, which are greatly overexpressed in TNBC patients. Overexpression of MDM2 leads to suppression of p53 affecting tumor cell proliferation and apoptosis. Single-walled carbon nanotubes (SWNTs) were functionalized with PEG for efficient delivery of siRNA for targeting MDM2. It was concluded that PEG-modified SWNTs are effective drug delivery system promoting major MDM2 silencing effect causing tumor cell proliferation suppression and induced apoptosis in breast cancer cells [69].
Cyclin dependent kinase (CDK) 11 and casein kinase 2 (CK2) are important protein kinases for cancerous cell survival, which are also highly expressed in TNBC cells. Kren et al. prepared novel Tenfibgen-coated nanocapsules (TBG nanocapsules) containing anti-CDK11 siRNA or anti-CK2 siRNA targeting Tenascein-C receptors that are found abundantly within the breast cancer stroma. Intravenous administration of these gene therapeutics suppresses mRNA and protein expression causing significant reduction in tumor cell proliferation and growth [70].
Several studies demonstrating molecular targeting of v-Myc myelocytomatosis viral oncogene homolog (c-Myc) that is highly expressed in TNBC. The significance of c-Myc overexpression arises when its suppression is concomitantly associated with inhibition of CDK1 expression. Downregulation of these two genes results in specific targeting and synthetic lethality in TNBC cells. As c-Myc, CDK1 plays a key role in cell death and apoptosis. Administration of anti-c-Myc siRNA-encapsulated nanoparticles causes significant c-Myc gene silencing that contributes to a slight therapeutic effect on TNBC cells whereas the delivery of siRNA targeting CDK1 results in silencing of CDK1 expression only in TNBC cells containing high levels of c-Myc expression. The combination is ideal not only for its pronounced decreased cell proliferation and cell apoptosis, but also for its selective and specific targeting of TNBC cells with no adverse events on normal breast cells [71].
Tumor suppression (TP53) mutation is one of the most frequent oncogenic mutations in TNBC. Remarkable preclinical studies were conducted to reestablish TP53's effect but none of them were transformed into human clinical studies. Hemizygous loss of TP53 is the most recurrent TNBC mediated TP53 mutations and is associated with deletion of neighboring genes, most importantly, hemizygous deletion of POLR2A [69]. Antitumor-based silencing of POLR2A expression was investigated by Xu and his coworkers. A pH-sensitive nano-bomb was developed incorporating antiPOL siRNA and guanidine-CO 2 functionalized chitosan (CG) at neutral pH. siRNA-nanoplatforms-containing CG-CO 2 were not only efficaciously internalized into TNBC cancerous cells harboring hemizygous TP53 deletion, but also able to escape the intracellular trafficking and release siRNA at low pH. TNBC cell lines embracing hemizygous loss of POLR2A are more sensitive than those harboring the wild type for siRNA nanotherapeutics, as a result, knockdown of POLR2A expression cause selective TNBC cell death with no cytotoxicity. Inhibition of tumor growth by targeting POLR2A was not influenced with the status of TP53 loss [70].
Ras homologous A (RhoA) and Ras homologous C (RhoC) are low molecular weight GTPases of the Ras family. Significant high levels of RhoA and RhoC are reported in all subtypes of breast cancer especially TNBC. Overexpression of RhoA enhances tumor cell proliferation, and induces angiogenesis and tumor invasion and metastasis [72]. Li et al. studied the antitumor effect of anti-RhoA siRNA nanoparticles in vivo where chitosan-coated polyisohexylcyanoacrylate (PIHCA) nanoparticles encapsulating anti-RhoA siRNA were fabricated and given to MDA-MB-231 xenografted mice intravenously. Complete suppression and more than 90% inhibition of tumor growth were perceived after intravenous administration of anti-RhoA siRNA [73]. As RhoA and RhoC are expressed exclusively in cancerous cells only, specific and selective targeting of RhoA is a promising candidate for treatment patients with TNBC [72].
Polo-like kinase 1 (PLK1) is extensively upregulated in patients with either primary or metastatic TNBC. Inhibition of PLK1 expression is associated with ameliorating tumor cell death in a wide variety of metastatic cancer [74,75]. Targeted nanoplatforms incorporating anti-PLK1 siRNA encapsulated within antibody-conjugated bioreducible cross-linked with PEI and polyethylene glycol (PEG) layer-by-layer with coated mesoporous silica nanoparticles (MSNP) showed the following characteristics: PEI is highly effective for siRNA binding and escapes the intracellular trafficking. PEG is incorporated for siRNA protection by enzymatic degradation, reducing PEI toxicity, circumventing the immune system, and decreasing nanoparticles' aggregation. In vitro and in vivo studies revealed that these nanoparticles were efficiently internalized into targeted TNBC metastatic cells overwhelming both cancer hallmarks: tumor cell proliferation and tumor metastasis. It is worth noting that reactive oxygen species' (ROS) scavenging ability of MSNP contributes to the antimetastatic effect, whereas the internalized anti-PLK1 siRNA exhibits apoptotic cellular death activity [76].

Tumor Invasiveness and Metastasis
Tumor metastasis is the primary factor associated with cancer related deaths in patients with TNBC. Epithelial mesenchymal transition (EMT) is the driving force for tumor invasiveness, metastasis, drug resistance, and remodeling of TME [96]. The morphological alterations of the normal epithelial cells' characteristics into the mesenchymal phenotype allow the cancerous cells to disseminate out into distant organs [97,98]. Central nervous system metastasis and lung metastasis are among the utmost fatal metastatic repercussions in patients with TNBC [20]. Upregulation of numerous transcriptional factors is among the foremost causes to induce EMT. Epidermal growth factor (EGF), fibroblast growth factor, hepatocyte growth factor, transforming growth factor (TGF)-β, and bone morphogenetic proteins are EMT master regulators that govern the development and progression of breast cancer metastasis [99,100].
Breast cancer metastasis and EMT are governed by the transmembrane protein β3 integrin. Upregulation of TGF-β in cancerous cells persuades overexpression of β3 integrin, therefore, it is expected that silencing β3 integrin expression inhibits TGF-β induced EMT, tumor invasion, and metastasis [101]. Parvani et al. prepared a cationic lipid nanocarrier (ECO) containing siRNA that are delivered specifically to the targeted β3 integrin gene. Treatment of TGF-β pre-stimulated TNBC cells with ECO/β3-siRNA nanoparticles reduces TGF-β-mediated EMT, tumor proliferation, and invasion as these engineered nanoparticles prompt inhibition of more than 70% of β3 integrin expression for approximately seven days. Downregulation of mesenchymal markers (N-cad and PAI-1) and upregulation of epithelial markers (E-cad and CK-19) attributes to inhibition of TGF-β induced EMT. Cellular uptake of β3-siRNA based nanoparticles into targeted cells was improved by surface modifications of the engineered nanoparticles with RGD peptide. In vivo analysis of the modified ECO/β3-siRNA nanoparticles decreased the primary tumor burden and tumor recurrence, designating that systemic administration of modified siRNA nanoparticles demonstrated significant suppression of tumor metastasis and invasion [102].
Patients with TNBC and ovarian cancers are characterized by elevated levels of TWIST. TWIST is a major transcription factor activates EMT, promotes stem like phenotyping, and inhibits apoptosis, increasing the risk of tumor recurrence and poor prognosis [103]. Inhibition of TWIST overexpression in TNBC cells was examined by the administration of poly(amidoamine) dendrimer (PAMAM) loaded with TWIST-siRNA. The modified siRNA-based dendrimers were efficiently internalized into TNBC cells causing significant reduction in TWIST expression accompanied by phenotyping changes associated with decreasing cancerous cells motility, decreasing TWIST-mediated upregulation mesenchymal markers (Vimentin and N-cad) and attenuating the dissemination and invasion of TNBC cells for approximately one week after dendrimer treatment. As TWIST protein is expressed only in cancerous cells and not in normal breast cells, silencing TWIST expression using siRNA-based dendrimer is a promising safe and biocompatible approach for treating breast cancer-associated metastasis [104].
A novel class of RNA consisting of more than 200 nucleotides, so-called long noncoding RNAs (lncRNAs), are vital regulators of gene expression associated with cellular differentiation and development [105]. The oncogenic lncRNAs have a pivotal role in inducing malignant cell proliferation, invasion, and metastasis [106]. BMP/OP-Responsive Gene (BORG) [107], HOX Antisense intergenic RNA (HOTAIR) [108] and Differentiation Antagonizing Non-Coding RNA (DANCR) [109] Pharmaceutics 2020, 12, 929 8 of 24 are lncRNAs concerned in tumor recurrence and progression, indicating poor cancer prognosis. As DANCR is highly expressed in TNBC cells, Vaidya et al. revealed targeting DANCR lncRNAs causes suppression of TNBC metastasis, and inhibition of cell proliferation and survival. Vaidya and his colleagues prepared RGD-PEG-ECO nanoparticles loaded with anti-DANCR siRNA. In vitro transfection of MDA-MB-231 and BT549 cells with anti-DANCR siRNA showed significant reduction of cancer cell proliferation and inhibition of tumor cell migration and invasion. Targeting DANCR in MDA-MB-231 xenografted mice using RGD-PEG-ECO/siRNA-engineered nanoparticles results in decreasing tumor volume and the suppression of tumor growth [109].
A recent study examined the silencing effect of EF2 kinase in tumor proliferation, apoptosis, invasion, and metastasis. EF2K is a typical kinase that is overexpressed in BRCA1-mutated breast cancer cells. Silencing EF2K was performed by treatment of HCC-1937 and MDA-MB-436 cells with cobalt-ferric anti-EF2K siRNA nanoparticles where internalization of anti-EF2k-mediated siRNA into TNBC cells were enhanced in comparison to normal breast cells. Cobalt-ferric anti-EF2K siRNA nanoparticles knocked-down the expression of EF2K in transfected TNBC cells and, hence, inhibited TNBC cells proliferation and colony formation and impaired cell migration and invasion. Targeting of EF2K in BRCA1-mutated MDA-MB-436 and HCC-1937 cell-xenografted mice inhibited tumor growth, reduced cancer cell proliferation, angiogenesis, and induced apoptosis. Remarkably, COFe-siRNA nanoparticles showed no obvious cytotoxicity [110].

Angiogenesis and Tumor Microenvironment
Both tumor growth and metastasis are driven by angiogenesis [111]. Tumor angiogenesis is characterized by high neovascular density, high lymphatic density, and vascular invasion. Numerous angiogenic factors are secreted in the tumor microenvironment stimulating new blood vessels sprout. Angiogenic factors overexpression is correlated with cancer development as vascular endothelial growth factors (VEGF) especially VEGF-A subtype, attributed for cancer progression such insulin like growth factors (IGF) and TGF-β1 and concerned with tumor invasion and metastasis like vascular endothelial growth factor receptors (VEGFRs) [112]. These proangiogenic factors are more pronounced in TNBC compared to other subtypes of breast cancer [111,113].
It was reported that inhibition of VEGF using monoclonal antibodies is not effective for treating patients with TNBC as theses anti-VEGF antibodies hinder VEGF interaction with their receptors at the endothelial cells and the plasma membrane of the tumor cells without affecting the internal VEGFR expressed at the nuclear envelope [114][115][116][117][118][119][120][121][122]. Salva et al. prepared chitosan nanoplexes encapsulating siRNA targeting VEGF-A, VEGFR-1, VEGFR-2, and neuropilin-1 (co-receptors for VEGF, NPR-1). Intratumoral administration of VEGF/siRNA nanoplexes into TNBC induced rats declined VEGF expression by more than 80% and reduced the tumor volume by more than 90%. Chitosan/siVEGF-mediated nanoplexes transfection did not increase interferon mediated immune response. Salve et al. demonstrated the silencing effect of the combination of VEGF genes cause potent and specific inhibition of breast cancer growth [117]. In another study, PLEGP nanocomplex containing anti-VEGF siRNA was developed. Different PLEGP nanocomplexes were investigated for their anti-tumor and cytotoxic activity. PLGEP 1800 was shown to have a modest downregulation of VEGF expression with no significant cytotoxicity. In vitro studies revealed that transfection of MDA-MB-231 cells with PLGEP 1800 nanocomplex suppressed cancerous cell migration and invasion. Vascular endothelial cell proliferation and tube formation were repressed in PLEGP 1800 /VEGF siRNA transfected HUVECs cells. Knock down of VEGF expression and subsequent inhibition of CD31 level in siVEGF treated MDA-MB-231 xenografted mice indicated suppression of tumor growth and impairment of angiogenesis [118].
Many identifiable factors attribute for lymphangiogenesis in breast cancer. Among them is VEGF-C that activates the lymph vascular cells and lymphatic cells growth. Upregulation of VEGF-C level indicates tumor metastasis particularly for the lymph nodes. The silencing of VEGF-C expression was studied by Chen and his colleagues using siRNA mammalian vectors. Anti-VEGF-C siRNA knocked down VEGF-C mRNA and VEGF-C protein production in C166 cells. Downregulation of VEGF-C expression leads to suppression lymphangiogenesis but not angiogenesis, suppression of regional and distant metastasis implicated by inhibition of lymph node metastasis and spontaneous lung metastasis, immunomodulating effect, and improving mice survival [119].
Lipocalin 2 (Lcn2), a subtype of lipocalin protein family, is upregulated in TNBC contributing to tumor progression, invasion and metastasis through its interaction with matrix metalloproteinase-9 (MMP-9) [119]. Lipocalin 2 play a striking role in regulating of angiogenesis, its overexpression in TNBC is accompanied by increased VEGF expression. It is suggested that Lcn 2 induced VEGF production through the Erk/HIF-1α signaling pathway [120]. Elevated urinary levels of Lcn 2 in patients with TNBC correlated with its poor progression representing that Lcn 2 is biomarker for both diagnosis and prognosis of breast cancer [119]. Anti-angiogenic engineered liposomes encapsulating both ICAM-1 and  [121].
Angiogenesis is a key driver for tumor growth and metastasis. Selective targeting, accessibility and low drug resistance liability are major advantages for targeting vascular endothelial cells in addition to cancerous cells [122]. Receptor mediated endocytosis is an innovative tactic targeting receptors available at many cell populations. Nucleolin is a receptor that is highly expressed at both cancerous cells and endothelial cells originating from blood vessels [91]. Novel fabrication of nucleolin targeted siRNA based nanoparticles is a promising approach employing gene delivery for dual therapy. Gomes-da-Silva et al. developed F3 targeted liposomes incorporating enhanced green fluorescent protein (eGFP) and transfected them into MDA-MB-231 cells and HMEC-1 cells. It was reported that F3 targeted liposomes enhanced the cellular uptake by both cancerous cells and endothelial cells but not by non-cancerous cells. Additionally, improved targeted siRNA liposomes internalization by MDA-MB-231 and HMEC-1 cells was accompanied by knock down eGFP expression in both cells [91].
Most TNBC cells exhibits mutation in the tumor suppressor p53 gene. p53 is an oncogene that is overexpressed in patients with TNBC and involved in multiple molecular pathways including tumorigenesis, apoptosis, metastasis and angiogenesis [123,124]. Braicu et al. demonstrated anti-angiogenic impact of silencing mutated p53 gene using p53-siRNA based approach where transfection of Hs578T cells with anti-p53 siRNA induced apoptosis, decreased cell survival and reduced angiogenesis. Treatment of TNBC cells with a combination of p53 siRNA and epigallocatechingallate (EGCG) demonstrated more pronounced anti-apoptotic and anti-angiogenic as compared for each treatment alone [125].
cAMP and its two receptors protein kinase A (PKA) cAMP dependent and exchange protein activated cAMP (EPAC1). EPAC1 is a cAMP activated guanine nucleotide exchange factor. cAMP and its receptors are exposed to be overexpressed in patients with breast cancer; associated with cell differentiation, cell migration and cell survival and death [126]. Cell culture studies revealed that suppression of EPAC1 expression in anti-cAMP siRNA treated MDA-MB-231 cells co-cultured HUVEC cells exhibited activities include cessation in the formation of the nodes and tubes in HUVEC cells, upregulation of the anti-angiogenic proteins involved in adhesion and angiogenesis, downregulation of pro-angiogenic proteins such as fibroblast growth factor (FGF), transforming growth factors (TGF) and many others attributed to cell migration and locomotion, inhibited vascular cells invasion mediated TNBC cells and downregulation of Paxillin and MENA molecules that are concerned with focal adhesion. It is suggested that EPAC1 have a remarkable role in regulating tumor microenvironment, neovascularization and invasiveness of TNBC cells contributing to vascular metastasis [127].

Chemotherapeutic Resistance
Cancerous breast cells are highly sensitive to chemotherapeutic agents with a response rate extent approximately 80% but unfortunately most of the cancerous cells develop resistance contributing to treatment failure and poor TNBC prognosis [128]. Emerged chemotherapeutics' resistance is incongruent with the type; structure and the biological activity of the chemotherapeutics therefore substitution between different agents is no longer effective [129,130].
Though 90% of the initially chemo-responsive TNBC cells develop resistance, advanced research exploring the impact of siRNA-based nanotherapeutics as a targeted platform to overcome doxorubicin-based [131][132][133] and taxane-based resistance [134][135][136] exploiting non-TNBC cell lines, most commonly MCF-7 cells. Extensive research are exploring the mechanisms of multidrug resistance (MDR) evolved in breast cancerous cells including ATP-binding cassette (ABC) transporter, activation of anti-apoptotic pathways, enhancement chemotherapeutics detoxification, overexpression of β-tubulin III, KIF14, and many other signaling factors, alterations of DNA repair enzymes, and chemoresistance-induced hypoxia and NF-κB signaling pathway [137]. Among these different mechanisms, ABC transporter, alterations of the antiapoptotic genes and the role of hypoxia are major mechanisms of chemoresistance ( Figure 2). downregulation of pro-angiogenic proteins such as fibroblast growth factor (FGF), transforming growth factors (TGF) and many others attributed to cell migration and locomotion, inhibited vascular cells invasion mediated TNBC cells and downregulation of Paxillin and MENA molecules that are concerned with focal adhesion. It is suggested that EPAC1 have a remarkable role in regulating tumor microenvironment, neovascularization and invasiveness of TNBC cells contributing to vascular metastasis [127].

Chemotherapeutic Resistance
Cancerous breast cells are highly sensitive to chemotherapeutic agents with a response rate extent approximately 80% but unfortunately most of the cancerous cells develop resistance contributing to treatment failure and poor TNBC prognosis [128]. Emerged chemotherapeutics' resistance is incongruent with the type; structure and the biological activity of the chemotherapeutics therefore substitution between different agents is no longer effective [129,130].
Though 90% of the initially chemo-responsive TNBC cells develop resistance, advanced research exploring the impact of siRNA-based nanotherapeutics as a targeted platform to overcome doxorubicin-based [131][132][133] and taxane-based resistance [134][135][136] exploiting non-TNBC cell lines, most commonly MCF-7 cells. Extensive research are exploring the mechanisms of multidrug resistance (MDR) evolved in breast cancerous cells including ATP-binding cassette (ABC) transporter, activation of anti-apoptotic pathways, enhancement chemotherapeutics detoxification, overexpression of β-tubulin III, KIF14, and many other signaling factors, alterations of DNA repair enzymes, and chemoresistance-induced hypoxia and NF-κB signaling pathway [137]. Among these different mechanisms, ABC transporter, alterations of the antiapoptotic genes and the role of hypoxia are major mechanisms of chemoresistance ( Figure 2). Extruding the internalized chemotherapeutics out of the cancerous cells is governed by ATPbinding cassette (ABC) transporter family. Two efflux pump members namely ABCB1 (Pglycoprotein) and ABCG2 (BCRP) transporters are identified [130]. ABCB1 and ABCG2 transporters are overexpressed in patients with TNBC [138]. Clinical studies failed to verify MDR phenotype modulated therapeutics either for their cytotoxicity or for their unwanted pharmacokinetics interactions, thus silencing gene expression mediating thwarting the synthesis of ABC transporters is the most accessible approach to circumvent MDR clinically [139,140]. It was earlier reported that Extruding the internalized chemotherapeutics out of the cancerous cells is governed by ATP-binding cassette (ABC) transporter family. Two efflux pump members namely ABCB1 (P-glycoprotein) and ABCG2 (BCRP) transporters are identified [130]. ABCB1 and ABCG2 transporters are overexpressed in patients with TNBC [138]. Clinical studies failed to verify MDR phenotype modulated therapeutics either for their cytotoxicity or for their unwanted pharmacokinetics interactions, thus silencing gene expression mediating thwarting the synthesis of ABC transporters is the most accessible approach to circumvent MDR clinically [139,140]. It was earlier reported that multidrug resistance genes (lung resistance-related protein (LRP), multidrug resistance associated proteins 1 and 2 (MRP1, MRP2), multidrug resistance gene 1 (MDR1), and breast cancer associated proteins (BCRP)) mediated mRNA expressions in metastatic breast tumor cells specimens of patients receiving first line chemotherapy based regimens correlates inversely with treatment efficacious with elevated genes expression predicting poor prognosis in those patients [141].
Growing bodies of evidence revealed that knockdown of ABCB1 and ABCG2 expression mediated siRNA-based nanoparticles increased intracellular concentration and cancerous cells chemosensitivity of many cytotoxic drugs such as doxorubicin and paclitaxel [142][143][144]. Deng et al. prepared multi-layers nanoparticles constituting of doxorubicin-based nanoparticles encircled with alternating layers of siRNA targeted MDR1 and poly-l-arginine enclosed by an outer shell of hyaluronic acid (HA). The engineered drug delivery platform posed high siRNA loading capacity, stability and cellular internalization with high efficacy-toxicity profile. Administration of doxorubicin-siRNA nanoparticle complexes decreases the tumor volume significantly in comparison to the treatment with either anti-MDR1 siRNA or doxorubicin alone [145].
Targeting the anti-apoptotic genes/proteins is another major mechanism-induced chemoresistance. Survivin is one of the most attractive inhibitors of apoptosis (IOP) controlling tumor growth, angiogenesis, apoptosis, and recently drug resistance, in particular, paclitaxel (Figure 3) [146]. Therapeutic effectiveness of survivin to enhance TNBC cells sensitivity to chemotherapeutics agents were investigated using polymeric micelles of PEG 2000 -PE incorporating survivin siRNA. Treatment of anti-survivin siRNA PM transfected MDA-MB-231 cells with paclitaxel showed that knockdown survivin expression in resistant cancerous cell enhance paclitaxel cytotoxicity significantly and cause destabilization of microtubules. In addition, co-encapsulation of paclitaxel and anti-survivin siRNA into PM exhibited a significant suppression of tumor growth in comparison to the paclitaxel free anti-survivin siRNA PM [147]. Triple therapies of chemotherapy, gene therapy, and photothermal therapy revealed outstanding antitumor effect in vitro and in vivo in comparison to mono or dual therapies [148].
Pharmaceutics 2020, 12, x 11 of 24 multidrug resistance genes (lung resistance-related protein (LRP), multidrug resistance associated proteins 1 and 2 (MRP1, MRP2), multidrug resistance gene 1 (MDR1), and breast cancer associated proteins (BCRP)) mediated mRNA expressions in metastatic breast tumor cells specimens of patients receiving first line chemotherapy based regimens correlates inversely with treatment efficacious with elevated genes expression predicting poor prognosis in those patients [141]. Growing bodies of evidence revealed that knockdown of ABCB1 and ABCG2 expression mediated siRNA-based nanoparticles increased intracellular concentration and cancerous cells chemosensitivity of many cytotoxic drugs such as doxorubicin and paclitaxel [142][143][144]. Deng et al. prepared multi-layers nanoparticles constituting of doxorubicin-based nanoparticles encircled with alternating layers of siRNA targeted MDR1 and poly-L-arginine enclosed by an outer shell of hyaluronic acid (HA). The engineered drug delivery platform posed high siRNA loading capacity, stability and cellular internalization with high efficacy-toxicity profile. Administration of doxorubicin-siRNA nanoparticle complexes decreases the tumor volume significantly in comparison to the treatment with either anti-MDR1 siRNA or doxorubicin alone [145].
Targeting the anti-apoptotic genes/proteins is another major mechanism-induced chemoresistance. Survivin is one of the most attractive inhibitors of apoptosis (IOP) controlling tumor growth, angiogenesis, apoptosis, and recently drug resistance, in particular, paclitaxel ( Figure 3) [146]. Therapeutic effectiveness of survivin to enhance TNBC cells sensitivity to chemotherapeutics agents were investigated using polymeric micelles of PEG2000-PE incorporating survivin siRNA. Treatment of anti-survivin siRNA PM transfected MDA-MB-231 cells with paclitaxel showed that knockdown survivin expression in resistant cancerous cell enhance paclitaxel cytotoxicity significantly and cause destabilization of microtubules. In addition, coencapsulation of paclitaxel and anti-survivin siRNA into PM exhibited a significant suppression of tumor growth in comparison to the paclitaxel free anti-survivin siRNA PM [147]. Triple therapies of chemotherapy, gene therapy, and photothermal therapy revealed outstanding antitumor effect in vitro and in vivo in comparison to mono or dual therapies [148].

MDR1
Layer by layer nanoparticles In vitro and in vivo/MDA-MB-468 cells [145] Survivin PEG 2000 -PE PM In vitro and in vivo/MDA-MB-231 and paclitaxel resistant SKOV3 cells [147] Survivin Nanocopolymer In vitro and in vivo/MDA-MB-231 human breast cells [148] Abbreviations and Hs578T) but also in non-TNBC cells (MCF-7 and T47D). Src kinase was highly expressed in TNBC cells rather than in normal breast cells. Src silenced MDA-MB-231 cells showed low protein levels of STAT3 and AKT proposing that Src/AKT/STAT3 signaling pathway play a role in chemoresistance [149]. These results are consistent with Moreira et al. findings that confirmed overexpression of STAT3 protein in doxorubicin-treated TNBC BT-549 cells suggesting that STAT3 is an essential EGF gene involved in doxorubicin-based resistance [150].

Clinical Trials of Anticancer siRNA-Mediated Nanotherapeutics
Current pre-clinical advances exploiting siRNA-based nanotherapeutics for treatment of cancer, in general, and TNBC, in particular, established the effectiveness of these therapeutics in inhibition cell proliferation and tumor growth, suppression tumor invasion and metastasis, pronounced anti-angiogenic effect, and enhancement of chemotherapeutics efficacy. Remarkably, several siRNA nanoparticles succeeded in being investigated in clinical trials. To date, eight siRNA-mediated nanoparticles have commenced phase I and II clinical trials (Figure 4) for treatment of a wide variety of solid cancers that could be employed for patients with TNBC (Table 3). The phase I trial of siRNA-EphA2-DOPC has recruited but not completed yet, whereas the mesenchymal stem cell-derived exosomes with KRAS G12D trial has not yet recruited. ALN-VSP02, Atu027, TKM-PLK1, and siG12D LODER passed phase I successfully. Four siRNA nanotherapeutics moved to phase II; Atu027 and TKM-PLK1 completed phase II while siG12D LODER is currently being recruited. The phase II trial of DCR-MYC was terminated.    EphA2-siRNA-DOPC is the most recent anticancer siRNA-mediated nanotherapeutics investigated clinically. EphA2 is a protein member of tyrosine kinases receptors that are expressed abundantly in the embryos and expressed to a lesser extent in adults mainly at the surface of epithelial cells. Several studies reported that EphA2 is selectively upregulated in diverse arrays of cancer including, but not limited to, breast, pancreas, prostate, lung, and most importantly ovarian cancer affecting cancerous cells growth, invasion, and metastasis and angiogenesis and increasing the tumor burden. EphA2-siRNA was incorporated into liposomal nanoparticles (DOPC) called EPHARNA (EphA2-siRNA-DOPC) specifically targeting EphA2 expression in the epithelial mediated tumor [164][165][166]. In vitro and in vivo studies demonstrated that EPHARNA has anti-angiogenic effect and reduce tumor growth dramatically. Moreover, remarkable tumor growth inhibition was found with simultaneous administration of EphA2-siRNA-DOPC and paclitaxel [167]. In vivo toxicological studies showed that DOPC nanoliposomes exhibit no observed adverse events at dose range of 75-225 mcg/kg after single administration and twice administration results in no major toxicities [168]. In 2015, EphA2-siRNA-DOPC entered phase I trial recruiting patients with advanced metastatic solid cancer receiving intravenous EPHARNA twice weekly over two hours [169].

Conclusions
Despite comprehensive knowledge and advanced research on breast carcinoma, the cancer burden remains significantly high. High diversification of genomic transmutations and enormous phenotypic alterations attributed to traditional anticancer therapeutics' failure, which contributes to relapse, recurrence, and cancer-related mortality, especially on TNBC. Most patients with TNBC exhibit drug resistance, invasion, and metastasis causing poor disease prognosis and poor overall patient survival rate. Inter-and intra-individual heterogeneity in TNBC limits intervention and efficacious treatment for TNBC. RNAi interference and, in particular, siRNA-based therapeutics technology attracted attention via salient physicochemical characteristics, and pharmacokinetics properties along with the systemic biodegradation liability mitigate the systemic administration of naked siRNA. Targeted delivery systems are needed to overcome the aforementioned obstacles attaining specific and selective gene-based therapeutic products' internalization in the tumor cells, sufficient intracellular retention, and efficacious antitumor activity with no off-target side effects. Nanosized platforms aid in encapsulating siRNA protecting it from the external environment, targeting the tumor cells and evading the immune-stimulating effect. Most importantly, multiple drug modalities (chemotherapeutics, photothermal agent and siRNA-based therapeutics) may be incorporated in these nano-formulations. Integrations of different drug modalities in one nanocarrier posed significant antitumor effectiveness compared to the delivery of one agent or sequential administration of multiple agents. Several anticancer-based gene nanotherapies have commenced phase I and II clinical trials and have shown promising results. To date, there are no siRNA-anticancer therapies on the market, but the progression of siRNA-based therapy from the bench to the clinic will open the horizon for these advanced anticancer therapeutics to evolve.