Treatment strategies targeting the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin pathway against triple-negative breast cancer

Abstract

Triple negative breast cancer (TNBC) is an exceptionally aggressive subtype of breast cancer with a poor prognosis. TNBC patients have limited treatment options beyond conventional chemotherapy, and they face significant challenges associated with disease recurrence and resistance to chemotherapy. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway plays a pivotal role in cell proliferation, growth, metabolism, and survival. Its aberrant activation is closely linked to the development and progression of TNBC, as well as treatment response and drug resistance. Currently, numerous targeted drugs specifically inhibiting this signaling pathway are being developed and undergoing clinical trials. These include inhibitors targeting PI3K, AKT, or mTOR individually, as well as dual-target or multi-target inhibitors simultaneously targeting different components of this pathway. Encouragingly, some inhibitors have demonstrated promising potential in clinical trials. This review delves into the therapeutic potential of the PI3K/AKT/mTOR signaling pathway for TNBC and explores prospects for drug discovery.

Key Words: Triple negative breast cancer; Phosphoinositide 3-kinase; Protein kinase B; Mechanistic target of rapamycin; Biomarkers; Natural products; Inhibitors; Therapy

Core Tip: Triple-negative breast cancer is an aggressive subtype of breast cancer with a poor prognosis. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway regulates cell proliferation, growth, metabolism, and survival. This review analyzes biomarkers associated with this pathway in triple-negative breast cancer, including proteins, mRNA, non-coding RNA, and transcription factors. Furthermore, it systematically examines key natural products targeting the pathway, such as flavonoids, terpenoids, and alkaloids. Finally, it provides a comprehensive overview of inhibitors used to target the PI3K/AKT/mTOR pathway, including PI3K, mTOR, dual PI3K/mTOR, and AKT inhibitors.

INTRODUCTION

Breast cancer is one of the most prevalent malignancies among women worldwide, exhibiting diverse pathological and molecular subtypes[1], its incidence followed an upward trend, rising by 1% annually from 2012 to 2021, primarily in localized-stage and hormone receptor-positive cases. In contrast, the overall breast cancer mortality rate steadily declined by 44% overall from 1989 to 2022. The 5-year relative survival rate varies by stage, exceeding 99% for localized disease, 87% for regional disease, and 32% for distant stage disease[2]. Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (EGFR) 2 (HER2) expression in breast cancer cells[3,4], accounting for approximately 10% to 20% of all breast cancer cases[5]. It has a propensity for early-stage metastasis, frequent recurrence, high malignancy, and a poorer prognosis[6]. The average recurrence interval for patients with TNBC ranges from 19 to 40 months, with a staggering mortality rate of up to 75% within the initial three months after recurrence[7,8]. Following a diagnosis of cerebral metastasis, overall survival (OS) was reported to be 3 months with systemic therapy and 4 months without it[9]. Despite the complex and diverse treatment approaches currently available for TNBC, chemotherapy remains the primary treatment modality for patients. However, standard postoperative adjuvant radiotherapy and chemotherapy have demonstrated limited efficacy in preventing tumor recurrence[10], thereby contributing to increased TNBC-related mortality.

The activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway in TNBC is closely associated with the growth, proliferation, drug resistance, and other biological characteristics of the disease[11,12]. For instance, the activation of AKT can regulate the expression of downstream proteins such as cyclin A1, cyclin D1, Bax, Bcl-2, etc., thereby mediating the malignant biological behavior of various tumors[13]. The dysregulated activation of PI3K regulates the growth, proliferation, and metabolism of tumor cells. Various genomic alterations can trigger PI3K pathway activation including mutations in PI3K-alpha (PIK3CA) and AKT[14], which function as oncogenic drivers promoting tumor cell transformation, initiation, progression, and apoptosis[15]. Approximately 10% of TNBC patients have been reported to harbor PIK3CA mutations that are associated with enhanced chemotherapy resistance[5]. It has been reported that TNBC has four subtypes, namely intracellular androgen receptor, immunomodulatory type, basal-like immunosuppressive type and mesenchymal type, among which intracellular androgen receptor and mesenchymal subtypes have a higher mutation rate of PIK3CA[5].

The PI3K/AKT/mTOR signaling pathway is widely recognized as a crucial driver and potential therapeutic target for the treatment of TNBC[16,17]. Consequently, targeting this pathway may offer a promising strategy for the management of TNBC (Figure 1). In this review, we have critically analyzed the most recent advancements in research concerning the biomarkers linked to the PI3K/AKT/mTOR signaling pathway in TNBC (Table 1). Furthermore, we have systematically compiled and summarized key natural products that target the PI3K/AKT/mTOR signaling pathway in TNBC (Table 2), as well as we discussed the current inhibitors targeting this pathway (Table 3). Figure 2 summarizes the key points discussed in this study.

Figure 1 Representation of phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin sequential signaling pathway in breast cancer. RTKs: Receptor tyrosine kinases; PI3K: Phosphoinositide 3-kinase; PIP: Piperine; PTEN: Phosphatase and tensin homolog; ERK: Extracellular signal-regulated kinase; AKT: Protein kinase B; mTOR: Mechanistic target of rapamycin; PDK1: 3-phosphoinositide dependent protein kinase-1; GSK-3: Glycogen synthase kinase 3; PRAS40: 40-kDa proline-rich protein kinase B substrate; FOXO1: Forkhead box O1; BAD: Bcl-2 agonist of cell death; MDM2: Murine double minute 2.

Figure 2 The key points discussed in this study. TNBC: Triple negative breast cancer; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; mTOR: Mechanistic target of rapamycin; TYMS: Thymidylate synthase; GBP2: Guanylate-binding protein 2; ACTL8: Actin-like protein 8; OPN: Osteopontin; ROR2: Receptor tyrosine kinase-like orphan receptor 2; Nrf3: Nuclear factor erythroid-2-like factor 3; QC: Quercetin; PAB: Pseudolaric acid B; Tan IIA: Tanshinone IIA; BJE: Brucea javanica seed; RTEs: Radix tetrastigma extracts; PIP: Piperine; BBM: Berbamine; HHT: Homoharringtonine.

Table 1 Molecular biomarkers associated with the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin signaling pathway in triple negative breast cancer.

Biomarkers class	Biomarkers name	Mechanism of action/inhibition	Ref.
Protein biomarkers	TYMS	Knockdown of TYMS leads to downregulation of mTOR, p-PI3K, and p-AKT expressions, inhibits cell proliferation and migration, while promoting apoptosis	[20]
	GBP2	GBP2 overexpression collaborates with autophagy-related protein 2 to inhibit the PI3K/Akt/mTOR pathway, thereby enhances the sensitivity of TNBC cells to paclitaxel	[29]
	ACTL8	Facilitate the proliferation, migration, and invasion of TNBC cells by activating the PI3K/Akt/mTOR signaling pathway while suppressing apoptosis	[30,31]
	OPN	Activate PI3K/Akt/mTOR pathway to regulate anti-lipid peroxidation mediated by GPX4, thereby promoting tumor growth and metastasis in TNBC	[38]
mRNA biomarkers	ROR2	Downregulation of ROR2 augments chemical sensitivity towards adriamycin; upregulation of ROR2 activates the PI3K/Akt/mTOR signaling pathway to facilitate TNBC metastasis	[43]
Non-coding RNA markers	Linc00707	Competitively bind to miR-423-5p to up-regulate MARCH2 expression, promote TNBC progression and autophagy through the PI3K/Akt/mTOR pathway	[45]
	MiR-145	Decreasing in miR-145 attenuates PI3K activation by inhibiting the MAPK signaling pathway, thereby diminishing AKT and mTOR activity	[48]
Transcription factor	Nrf3	The overexpression of Nrf3 activates the PI3K/Akt/mTOR signaling pathway and regulates the expression of proteins associated with EMT	[23]

TYMS: Thymidylate synthase; GBP2: Guanylate-binding protein 2; ACTL8: Actin-like protein 8; OPN: Osteopontin; ROR2: Receptor tyrosine kinase-like orphan receptor 2; Nrf3: Nuclear factor erythroid-2-like factor 3; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; mTOR: Mechanistic target of rapamycin; TNBC: Triple negative breast cancer; GPX4: Glutathione peroxidase 4; MARCH2: Membrane-associated RING finger protein 2; MAPK: Mitogen-activated protein kinase; EMT: Epithelial-mesenchymal transition.

Table 2 Natural products targeting the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin signaling pathway in triple negative breast cancer.

Natural product class	Natural product name	Mechanism of action/inhibition	Ref.
Flavonoids	Prenylated xanthones	Decrease the levels of PI3K, AKT, and mTOR in both total protein and phosphorylated forms	[75]
	Ononin	Reverse EMT through down-regulation of EMT markers and matrix metalloproteinases, attenuate EGFR phosphorylation and inhibit the PI3K/Akt/mTOR pathway	[76]
	Myricetin	Inhibit the PI3K/Akt/mTOR pathway to enhance the anti-cancer efficacy of Myricetin	[77]
	Quercetin	Decrease cell viability, colony formation, angiogenesis, and increase apoptosis. Inhibit the activation of IGF1R and its downstream kinases AKT and ERK1/2. Suppress E-mediated proliferation and migration of TNBC cells by blocking β2-AR/ERK1/2 pathway	[78,80,81]
Terpenoids	PAB	Induces apoptosis through mitochondrial apoptosis and the PI3K/Akt/mTOR pathway	[87]
	Ezhu	Inhibit the proliferation, invasion, migration, and EMT of MDA-MB-231 cells through the PI3K/Akt/mTOR signaling pathway	[89]
	Tan IIA	Enhance the antitumor efficacy of doxorubicin by interfering with the PI3K/AKT/mTOR signaling pathway and inhibiting topoisomerase II	[79]
Alkaloids	RTEs	Enhance the sensitivity of resistant TNBC cells to Taxol through their inhibitory effect on PI3K/Akt/mTOR-mediated autophagy. Suppress MDA-MB-468/Taxol cells autophagy and reduce tumor growth	[91]
	BJE	Inhibit the proliferation, induce apoptosis, and promote the phosphorylation of mTOR, PI3K, and Akt in MDA-MB-231 TNBC cells	[68]
	PIP	Suppress the pro-survival AKT pathway and induce caspase-dependent apoptosis via the mitochondrial pathway. Impair the in vitro migratory capacity of TNBC cells and reduce MMP-2 and MMP-9 mRNA expression. The combination of doxorubicin and PIP can increase necrosis while downregulating PTEN, inhibiting PI3K levels, and the immunoreactivity of p-Akt, mTOR, and ALDH-1	[98,99]
	BBM	Impede TNBC development through regulation of the PI3K/Akt/MDM2/p53 and PI3K/Akt/mTOR signaling pathways	[100]
	HHT	Downregulate p-AKT, p-mTOR, and Bcl-2 expression, while upregulating TP53, Bax, cleaved caspase-3, and caspase-9 expression, and inhibiting the PI3K/AKT/mTOR pathway	[101]

PAB: Pseudolaric acid B; Tan IIA: Tanshinone IIA; RTEs: Radix tetrastigma extracts; BJE: Brucea javanica seed; PIP: Piperine; BBM: Berbamine; HHT: Homoharringtonine; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; mTOR: Mechanistic target of rapamycin; EMT: Epithelial-mesenchymal transition; EGFR: Epidermal growth factor receptor; IGF1R: Insulin-like growth factor-1 receptor; ERK: Extracellular signal-regulated kinase; TNBC: Triple negative breast cancer; MMP: Matrix metalloproteinase; PTEN: Phosphatase and tensin homolog; PIP: Piperine; MDM2: Murine double minute 2; ALDH-1: Aldehyde dehydrogenase-1.

Table 3 Existing inhibitors targeting the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin signaling pathway in triple negative breast cancer.

Inhibitors class		Inhibitors name	Mechanism of action/inhibition	Ref.
PI3K inhibitors	Pan-PI3K inhibitors	Buparlisib (BKM120)	Block hormone receptor-mediated repair through the PI3K/AKT/NF-κB/c-Myc signaling pathway as well as the PI3K/AKT/FOXM1/Exo1 signaling pathway	[102]
	Selective PI3K inhibitors	LY294002	Suppress the expression of PI3K, AKT, and mTOR as well as phosphorylated protein levels activation in TNBC cells while inducing alterations in multi-kinase phosphorylation	[104]
		Inavolisib (GDC-0077)	Inhibit PI3K signaling as well as reduce cell viability through p110α degradation	[106,107]
mTOR inhibitors		AZD8055	Inhibit the proliferation of breast cancer cells, inhibit glycolysis and reduce glucose consumption. Inhibit MEK1/2 activated AKT, cell cycle progression and cell proliferation	[116,117]
Dual PI3K/mTOR inhibitors		BEZ235	Inhibit HIF-1alpha expression and the PI3K/mTOR signaling pathways, induce autophagy, and prolong DNA damage repair. Suppress cell proliferation, migration, and colony formation; promote the degradation of mutant p53	[120,122]
		SF1126	Suppresses tumorigenesis and angiogenesis in vivo. Induce apoptosis by inhibiting the EGFR-PI3K/AKT/mTOR pathway	[123]
		GDC-0084	Reduce the activity of PIK3CA mutant BC BMS cell lines in vitro, induce apoptosis, and inhibit the phosphorylation of AKT and p70S6	[124]
		Gedatolisib	Stimulate the response of dendritic cells, CD8+ T cells, and natural killer cells	[125]
		GDC-0980	Induce DNA damage, inhibit proliferation signaling, upregulate the expression of apoptotic markers, and suppress tumor growth	[127]
AKT inhibitors		Ipatasertib (GDC-0068)	Bind to three subtypes of AKT, thereby inhibit AKT activity, obstruct the PI3K/AKT/mTOR signaling pathway, and diminish tumor cell proliferation and survival	[134,136]
		MK-2206	Inhibit Akt phosphorylation and induct DNA damage	[139]
		Capivasertib (AZD5363)	Reduce stemness and re-sensitize BCSCs to chemotherapeutic agents by modulating mitochondrial fusion	[129]

PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; mTOR: Mechanistic target of rapamycin; NF-κB: Nuclear factor-kappa B; FOXM1: Forkhead box M1; TNBC: Triple negative breast cancer; HIF: Hypoxia-inducible factor; EGFR: Epidermal growth factor receptor; BCSC: Breast cancer stem cell; PIK3CA: Phosphoinositide3 kinase-alpha.

MOLECULAR BIOMARKERS ASSOCIATED WITH THE PI3K/AKT/MTOR SIGNALING PATHWAY

Studies have confirmed that some biomarkers in TNBC are related to the activation of PI3K/AKT/mTOR signaling pathway, which is of great significance for the treatment and prognosis evaluation of TNBC. Therefore, it is imperative to identify molecular biomarkers and validate their utility as targets in personalized treatments and as indicators of clinical efficacy[18,19]. Currently, investigation on such biomarkers encompasses not only protein markers[20], mRNA expression markers[21], non-coding RNA markers[22], but also transcription factors (TFs)[23]. The subsequent elaboration focuses on the biomarkers associated with the PI3K/AKT/mTOR pathway.

Protein biomarkers associated with PI3K/AKT/mTOR signaling pathway

Thymidylate synthase: Thymidylate synthase (TYMS) is an enzyme that plays a pivotal role in the biosynthesis of thymidylate, an essential component necessary for DNA synthesis. It plays a regulatory role in controlling the availability of four precursor cells during DNA biosynthesis and repair processes, which are essential for organismal development. One study indicates that TYMS exhibits the highest expression levels in BT549 cells of TNBC, and its overexpression is associated with poor prognosis in patients[20]. Knockdown of TYMS leads to downregulation of mTOR, p-PI3K, and p-AKT expressions. TYMS knockdown effectively inhibits cell proliferation and migration while promoting apoptosis, ultimately impeding TNBC tumor growth.

Guanylate-binding protein 2: Guanylate-binding protein 2 (GBP2) is a member of the guanylate binding protein family and functions as an inducer of interferon. It plays a crucial role in host defense against protozoan, viral, and bacterial infections. In terms of anti-tumor effects, GBP2 can serve as both a regulatory factor and tumor marker during the process of tumor development[24]. GBP2 overexpression in breast carcinomas has been associated with better prognosis and serves as a predictor of a pathologically complete response to anthracycline-based chemotherapy[25]. Although some studies have provided evidence of GBP2 involvement in breast cancer progression[25-27], the specific mechanisms underlying its role require further investigation. A study has confirmed that GBP2 overexpression significantly influences the autophagy process in TNBC cells, which is critical in the development of paclitaxel (PTX) resistance[28]. Another study has demonstrated that GBP2 inhibits TNBC cell progression both in vitro and in vivo[29]. GBP2 overexpression activates pro-death autophagy in MDA-MB-231 cells by inhibiting the PI3K/AKT/mTOR pathway. Additionally, GBP2 and autophagy-related protein 2 exert synergistic effects on autophagy, thereby significantly enhancing the sensitivity of TNBC cells to PTX. Simultaneously, augmentation of autophagy impedes TNBC cell proliferation. Hence, targeting GBP2 holds great potential as a therapeutic strategy to improve the treatment of TNBC.

Actin-like protein 8: The overexpression of the protein encoded by actin-like protein 8 (ACTL8) gene is significantly correlated with tumor growth and prognosis in various cancer types. The biological function of ACTL8 involves promoting the proliferation, invasion, and metastasis of tumor cells, potentially through activation of the PI3K/AKT/mTOR signaling pathways[30]. ACTL8 is also upregulated on TNBC and demonstrates a significant correlation with an unfavorable prognosis. The findings of a single study have been demonstrated that ACTL8 may facilitate the proliferation, migration, and invasion of TNBC cells by activating the PI3K/AKT/mTOR signaling pathway while suppressing apoptosis[31].

Osteopontin: The secreted protein osteopontin (OPN) plays a pivotal role in various biological processes, encompassing bone remodeling, immune response, cell adhesion, migration, and survival[32]. The overexpression of OPN is observed in pathological conditions, such as cancer. Recent studies have demonstrated that OPN facilitates the initiation, progression, invasion, and metastasis of tumors by inducing the upregulation of proteins within the body to enhance the degradation of extracellular matrix surrounding tumor cells. Moreover, it is closely associated with the prognosis of various malignancies[32-36]. The correlation between the expression of OPN and lymph node metastasis in TNBC has been substantiated, indicating a pivotal role of OPN in the invasion process of TNBC[34]. The autocrine and paracrine role of OPN in driving tumor recurrence has been identified, suggesting its potential as a therapeutic target for breast cancer recurrence[37]. The activation of the PI3K/AKT/mTOR pathway by OPN has been confirmed by several scholars to regulate anti-lipid peroxidation mediated by glutathione peroxidase 4, thereby promoting tumor growth and metastasis in TNBC[38]. After the addition of LY294002 to MD-MB-436 cells, there was a significant enhancement in TNBC cell proliferation, invasion, migration, tumor pellet formation ability, and angiogenesis ability. Additionally, the expression of OPN in both TNBC tissues and cells was significantly increased[38]. Therefore, OPN may be regarded as a promising biomarker for TNBC.

mRNA biomarkers associated with PI3K/AKT/m-TOR signaling pathway

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a type I transmembrane protein and a member of the ROR subfamily of cell surface receptors. It functions as a receptor protein tyrosine kinase (PTK) that activates the non-classical Wnt/β-catenin pathway, thereby regulating various cellular processes including growth, proliferation, differentiation, and migration[39]. The expression of ROR2 is upregulated in several solid tumors such as metastatic melanoma, osteosarcoma, breast cancer, and renal cell carcinoma. Conversely, its expression is downregulated in liver cancer, colorectal cancer, and gastric cancer. Importantly, alterations in ROR2 expression levels are significantly associated with poor prognosis in malignant tumors[40]. It has demonstrated that ROR2 can induce epithelial-mesenchymal transition (EMT) by independently modulating the mitogen-activated protein kinase (MAPK)/p38 signaling pathway without relying on Wnt5a stimulation, and this leads to enhanced migratory capabilities of epithelial breast cancer cells towards distant sites[41]. Furthermore, overexpression of ROR2 along with Wnt5a stimulation synergistically enhances invasion ability in MCF-7 cells[42].

One study has demonstrated a significant upregulation of ROR2 in metastatic TNBC tissues, and knockdown of ROR2 has been shown to effectively impede the migratory, invasive, and chemoresistant properties of TNBC cells[43]. In HC1599 and MDA-MB-435 adriamycin-resistant cells, downregulation of ROR2 augments chemical sensitivity towards adriamycin, whereas upregulation of ROR2 activates the PI3K/AKT/mTOR signaling pathway to facilitate TNBC metastasis[43].

Non-coding RNA markers associated with PI3K/AKT/mTOR signaling pathway

Linc00707: The involvement of non-coding RNA, specifically long-stranded non-coding RNA (lncRNA) and microRNA (miRNA), has been demonstrated to be crucial in the development and progression of breast cancer. The expression of lncRNA HAGLROS has been demonstrated to be significantly elevated in breast cancer tissues and plasma exosomes[44]. This upregulation promotes the proliferation, migration, and angiogenesis of breast cancer cells by inducing M2 polarization of tumor-associated macrophages, moreover, its overexpression is closely associated with a poor prognosis in patients with breast cancer[44]. A study has confirmed that Linc00707 is upregulated in TNBC patients and cell lines, where it is mainly located in the cytoplasm of MDA-MB-231 and MDA-MB-468 cells[45]. Knockdown of Linc00707 influenced autophagy via the PI3K/AKT/mTOR signaling pathway in TNBC cells, and mechanistic studies have confirmed that Linc00707 competitively binds to miR-423-5p to up-regulate membrane-associated RING finger protein 2 expression, ultimately promoting TNBC progression and autophagy through the PI3K/AKT/mTOR pathway[45].

MiR-145: An increasing body of evidence suggests a robust association between miRNA and cancer development. Among these miRNAs, miR-145 has been identified as a tumor suppressor gene[46]. The downregulation of its expression is significantly correlated with increased tumor size, distant metastasis, elevated Ki67 expression levels, and reduced OS[47]. Studies have demonstrated that the expression level of miR-145 is frequently downregulated in TNBC, and this reduction is associated with aberrant activation of the PI3K/AKT/mTOR signaling pathway. The decrease in miR-145 can attenuate PI3K activation by targeting the inhibition of the MAPK signaling pathway, thereby diminishing AKT and mTOR activity, consequently indirectly influencing the PI3K/AKT/mTOR signaling pathway[48].

Transcription factor associated with PI3K/AKT/mTOR signaling pathway

TFs play a crucial role in various biological processes and disease states, encompassing cancer, autoimmune disorders, and viral infections[49]. Specific TFs can serve as prognostic indicators for certain types of cancer and novel targets for treatment. The mutation or dysregulation of TFs can promote aberrant gene expression, including the blocking of differentiation and cell death gene expression programs, as well as cancer’s hallmark features[50,51]. The eventual misalignment of TFs results in an imbalance between oncogenes and tumor suppressor genes[52].

Nuclear factor erythroid-2-like factor 3 (Nrf3) belongs to the Cap’n’collar basic leucine zipper family of TFs[52]. Increasing evidence suggests a physiological relationship between the TF Nrf3 and cancers[53]. Nrf3 has been shown to play a vital role in cancer progression-related processes such as cell proliferation[53], invasion[54], metastasis and angiogenesis[55]. Nrf3 activates the invasion and metastasis of thyroid cancer cells[56]. Moreover, a biological relationship between Nrf3 and angiogenesis in pancreatic adenocarcinoma has been reported[57]. Furthermore, elevated expression Nrf3 has been demonstrated across numerous cancer types[23,53,58], indicating a crucial role for Nrf3 in the progression of cancer. It has been confirmed that Nrf3 effectively inhibits the invasion and proliferation of breast cancer cells[54]. Meanwhile, the downregulation of Nrf3 in TNBC tumors has been demonstrated to be associated with an unfavorable prognosis, thereby establishing it as a potential therapeutic biomarker for TNBC[59].

The findings of a study indicate a significant upregulation of Nrf3 expression in TNBC tissue, with an observed inverse correlation between its expression and survival time. The overexpression of Nrf3 activates the PI3K/AKT/mTOR signaling pathway and regulates the expression of proteins associated with EMT, and PI3K inhibitors has been observed to partially reduce the proliferation and migration of TNBC cells that overexpress Nrf3[23], thereby presenting a promising therapeutic target for TNBC.

The aforementioned studies indicate that these biomarkers influence the PI3K/AKT/mTOR signaling pathway via distinct mechanisms, which have substantial clinical implications for the prognosis and treatment of TNBC. With further investigation, these molecules are anticipated to emerge as novel and efficacious therapeutic targets.

NATURAL PRODUCTS TARGETING THE PI3K/AKT/MTOR SIGNALING PATHWAY

Natural products exhibit a wide range of pharmacological and biological activities[60], making them valuable resources for the discovery and development of novel therapeutic agents[61]. Compounds derived from various plant sources, such as flavonoids, terpenoids, alkaloids, coumarins, and lignans, have exhibited remarkable in vitro and in vivo anticancer properties[62]. Plant extracts and natural products containing bioactive molecules like flavonoids, terpenoids, and alkaloids have become valuable resources in this field, demonstrating both in vitro and in vivo activity against various malignant tumor cells. For instance, betaine exhibits potential chemotherapeutic effects on human non-small cell lung cancer via modulating the PI3K/AKT/mTOR signaling pathway[63]. Berberine regulates the Notch1/phosphatase and tensin homolog (PTEN)/PI3K/AKT/mTOR pathway and exhibits synergistic effects with 17-AAG and SAHA in SW480 colon cancer cells[64]. Sinomenine ester derivative inhibits glioblastoma by inducing mitochondria-dependent apoptosis and autophagy through the PI3K/AKT/mTOR and AMP-activated protein kinase/mTOR pathway[65]. Sanguinarine inhibits melanoma invasion and migration by targeting the focal adhesion kinase/PI3K/AKT/mTOR signaling pathway[66]. Salidroside induces apoptosis and protective autophagy in human gastric cancer AGS cells through the PI3K/AKT/mTOR pathway[67].

These compounds can interfere with the PI3K/AKT/mTOR signaling pathway through various mechanisms including direct inhibition of PI3K kinase activity, blockade of AKT phosphorylation activation, or suppression of mTOR signaling[68], and this interference can be achieved by influencing the expression of cytokines and proteins associated with this pathway or regulating signaling through other unspecified mechanisms[69-71]. Ultimately, these interventions lead to an anti-tumor effect. By optimizing the chemical structure of these natural products, diverse PI3K/AKT/mTOR inhibitors with distinct structures and functions can be designed and synthesized to offer a novel therapeutic strategy for patients with TNBC. The following section will provide detailed explanations on each of the three categories of inhibitors derived from plants.

Flavonoids targeting the PI3K/Akt/mTOR signaling pathway

The flavonoid group, which can be further classified into six major subclasses: Flavones, isoflavones, flavonols, catechins, anthocyanins, and flavanones[72], exhibit a range of significant biological effects such as scavenging free radicals, antioxidation, anti-inflammation, anticancer activity, enzyme activity regulation, inhibition of cell proliferation and platelet aggregation as well as reduction in plasma low-density lipoprotein levels[72-74]. These effects may contribute to the potential benefits of flavonoids in tumor prevention. The following compilation consists of flavonoids that exhibit inhibitory properties against the PI3K/AKT/mTOR signaling pathway in TNBC.

Prenylated xanthones: Nguyen et al[75] isolated three prenylated xanthones from the leaves of Garcinia mckeaniana Graib and evaluated their anti-proliferative effects in various tumor cell lines. Cisplatin was used as a positive control, and the effects of compounds 1-3 were assessed using the MTT assay in MDA-MB-231, CNE-2, and A549 cancer cells. The results demonstrated that compounds 1-3 have stronger inhibitory effects than cisplatin in MDA-MB-231 cells. Further studies on the effects of compounds 1-3 in the TNBC cell lines, MDA-MB-231 and MDA-MB-468, were conducted using cell cycle and apoptosis assays. The findings demonstrated that compounds 1-3 effectively arrested the cell cycle at the G2/M phase and induced apoptosis. Compound 2 notably decreased the levels of PI3K, AKT, and mTOR in both total protein and phosphorylated forms. These studies suggest that triterpenoids hold potential as therapeutic agents for treating TNBC[75].

Ononin: Overexpression of EGFR is observed in approximately 70% of patients with TNBC. EGFR plays a crucial role in promoting tumor metastasis and is associated with poor prognosis[76]. One study demonstrated that treatment with ononin, an isoflavone derived from plants, significantly suppressed tumor growth and lung metastasis in TNBC xenograft models. Ononin exerted its effects by reversing EMT through down-regulation of EMT markers and matrix metalloproteinases[76]. Furthermore, ononin attenuated EGFR phosphorylation and inhibited the PI3K/AKT/mTOR pathway[76]. These findings suggest that ononin holds promise as a potential therapeutic option for metastatic TNBC.

Myricetin: Multiple studies have provided substantial evidence supporting the anti-cancer properties of the naturally occurring flavonoid “Myricetin” in various malignancies; however, its therapeutic application is impeded by its limited water solubility and low oral bioavailability. To overcome this limitation, Sharma et al[77] developed a nanoemulsion of myricetin and assessed its superiority over myricetin alone in TNBC cells. Mechanistic investigation revealed that the nanoemulsion enhanced the anti-cancer efficacy of myricetin, potentially through inhibition of the PI3K/AKT/mTOR pathway, ultimately leading to increased cell death in TNBC cells. The nanoemulsion of myricetin formulation holds promise as an effective therapeutic agent for TNBC treatment[77].

Quercetin: Quercetin (QC) is a natural flavonoid which is typically found in apples, berries, and red wine. Its anti-cancer and anti-inflammatory properties have been previously demonstrated, but its low bioavailability hampers its clinical use[78]. QC has structural similarity with LY294002, a selective PI3K inhibitor, in which both structures belong to the benzopyranone class[79].

Hatami et al[78] conducted a comprehensive investigation into the anti-cancer effects of QC-solid lipid nanoparticles on TNBC cell line MDA-MB231. The study demonstrated that the optimized QC-solid lipid nanoparticles formulation enhanced physicochemical properties and facilitated sustained QC release, leading to decreased cell viability, colony formation, and angiogenesis, as well as increased apoptosis in the MDA-MB231 cell line[78].

Chen et al[80] have demonstrated that QC inhibit the activation of insulin-like growth factor-1 receptor and its downstream kinases AKT and extracellular signal-regulated kinase (ERK)1/2 in a dose-dependent manner in human MDA-MB-231 breast cancer cells[80]. Zhang et al[81] evaluated the effect of QC on tumor growth and metastasis in TNBC xenograft mice undergoing stress and investigated its underlying mechanisms. The findings indicated that QC could inhibit chronic stress-induced TNBC growth and the incidence of lung metastasis. Further mechanistic studies revealed that QC could suppress E-mediated proliferation and migration of TNBC cells by blocking β2-AR/ERK1/2 pathway, and thereby reducing the potential for TNBC growth and metastasis[81].

To further enhance the bioavailability of QC, several researchers have endeavored to develop the drug in nanoparticle form. Research conducted by Hatami et al[82] demonstrated that solid lipid nanoparticles improve the cytotoxic effect of QC in MDA-MB231 cells by increasing its bioavailability and inhibiting EMT, thereby effectively inhibiting cancer stem cell (CSC) generation[82].

Terpenoids targeting the PI3K/AKT/mTOR signaling pathway

Terpenoids are a class of compounds composed of isoprene units, which are widely distributed in nature[83]. They exhibit structural diversity, encompassing monoterpenes, sesquiterpenes, diterpenes, triterpenes, and polyterpenes. Research has demonstrated that certain natural terpenoids possess potent anti-tumor activities. These compounds exert anticancer effects through various mechanisms such as modulation of multiple signaling pathways, induction of apoptosis, cell cycle arrest, inhibition of cell proliferation, and anti-angiogenic activity to impede tumor growth.

Pseudolaric acid B: Pseudolaric acid B (PAB), a diterpene acid derived from the root bark of pseudolarix kaempferi, exhibits potent antitumor properties. Specifically, PAB exerts an anti-tumor effect on MCF-7 human breast cancer cells and may induce autophagy in these cells, thereby enhancing their resistance to apoptosis[84]. It was demonstrated that PAB induced mitotic arrest in the MCF-7 cells and inhibited proliferation through apoptosis and senescence, and the apoptosis was independent of the death receptor pathway[85]. Mechanistic studies have demonstrated that PTK was upstream of ERK and c-Jun-N-terminal kinase, and PTK induced apoptosis through activating c-Jun-N-terminal kinase and inactivating ERK in PAB-treated MCF-7 cells[86].

Research conducted by Yang et al[87] demonstrated that PAB triggers apoptosis in a concentrationdependent manner through the mitochondrial apoptosis pathway. The migration and invasion ability of MDAMB231 cells were inhibited by decreasing the expression levels of the EMTrelated markers, Ncadherin and vimentin, and increasing the expression of Ecadherin. Moreover, the expression levels of PI3K (p110β), phosphorylated (p)AKT [ser(473)], and pmTOR [ser(2448)] were downregulated. LY294002 interacted with PAB to induce apoptosis of MDAMB231 cells. Overall, the results demonstrated that PAB induces apoptosis via mitochondrial apoptosis and the PI3K/AKT/mTOR pathway in TNBC. Additionally, PAB suppressed cellular proliferation, migration, and invasion, suggesting its potential as a valuable phytomedicine for treating TNBC.

Ezhu (Rhizoma Curcumae Phaeocaulis): Ezhu, a terpenoid plant, is classified as a sesquiterpenoid compound. Studies have demonstrated that Ezhu exhibits significant anti-tumor properties. In vivo and clinical studies have demonstrated that both crude extracts and seven primary bioactive phytochemicals (curcumol, beta-elemene, furanodiene, furanodienone, germacrone, curdione, and curcumin) isolated from Ezhu exhibit a range of anti-breast cancer pharmacological properties. These include the inhibition of cell proliferation, migration, invasion, and stemness, as well as the reversal of chemoresistance and the induction of cell apoptosis, cycle arrest, and ferroptosis. The underlying mechanisms involve the regulation of the MAPK, PI3K/AKT, and nuclear factor kappa B signaling pathways[88]. A study conducted by Zhu et al[89] confirmed that the combination of Ezhu and Biejia (Carapax, Trionycis) can effectively inhibit the proliferation, invasion, migration, and EMT of MDA-MB-231 cells through the PI3K/AKT/mTOR signaling pathway. Importantly, this inhibitory effect surpasses that observed with Biejia or Ezhu alone[89].

Tanshinone IIA: Tanshinone IIA (Tan IIA) is one of the main active compounds derived from the traditional Chinese herbal medicine Danshen. It exhibits a wide range of pharmacological effects, such as antioxidant, antitumor, and antiangiogenic effects[90]. Ghanem et al[79] conducted a study to investigate the potential of Tan IIA to enhance the antitumor activity of doxorubicin (Dox) in TNBC MDA-MB-231 cells. The study has demonstrated that Tan IIA synergistically enhances the antitumor efficacy of Dox by interfering with the PI3K/AKT/mTOR signaling pathway and inhibiting topoisomerase II. The combination of Tan IIA with Dox resulted in synergistic effects, wherein the combined action of the two compounds proved to be more efficacious than the administration of Dox alone. The reduction in cell viability and inhibition of TNBC cell growth underscored the therapeutic potential of this treatment regimen. Molecular docking studies suggest that Tan IIA may function as a DNA intercalator and topoisomerase II inhibitor, thereby contributing to its antitumor activity and synergy with Dox in the treatment of TNBC[79].

Alkaloids targeting the PI3K/Akt/mTOR signaling pathway

Radix tetrastigma extracts (RTEs): Radix tetrastigma refers to the dried root of tetrastigma hemsleyanum Diels et Gilg. This root or the entire herb is utilized in traditional medicine for its properties of clearing heat and detoxifying, expelling wind and phlegm, promoting blood circulation, and alleviating pain. Recent research has predominantly focused on the anti-tumor potential of this plant.

The antitumor properties of RTEs have been proven effective against different types of tumors. A study has provided evidence that RTEs enhance the sensitivity of resistant TNBC cells to Taxol through their inhibitory effect on PI3K/AKT/mTOR-mediated autophagy[91]. Moreover, RTEs suppress autophagy in MDA-MB-468/Taxol cells and resultantly reduce tumor growth. Conversely, blocking the PI3K/AKT/mTOR pathway promotes autophagy in MDA-MB-468/Taxol cells[91].

Brucea javanica seed: Brucea javanica seed (BJE) is a significant Chinese medicinal plant. Studies have demonstrated that brucea javanica extract possesses anti-tumor, antiviral, and immunostimulatory biological activities, and exhibits efficacy in the treatment of certain cancers and viral infections[92]. Additionally, it aids in the restoration of immune function following chemotherapy. These therapeutic properties have contributed to the extensive utilization of brucea javanica extract in both traditional and contemporary medical practices. The research conducted by Wang et al[93] demonstrated that brucea javanica oil (BJO) inhibits the proliferation of A549 (non-small cell lung cancer) and H446 (small cell lung cancer) cells. Further studies showed that BJO induces G0/G1 arrest in these cells partly through regulating p53 and cyclin D1. Additionally, BJO triggers apoptosis in both cell lines via a mitochondria/caspase-mediated pathway, initiated by increased intracellular reactive oxygen species. A study has confirmed that bruceine A inhibits glycolysis by inhibiting phosphofructokinase-fructose bisphosphatase 4, leading to cell cycle arrest and apoptosis in MIA PaCa-2 cells via phosphofructokinase-fructose bisphosphatase 4/glycogen synthase kinase-3β-mediated glycolysis[94].

A study demonstrated that brusatol significantly inhibits the proliferation, migration, and invasion of A498, ACHN, and OSRC-2 cells while increasing their apoptosis[92]. Additionally, brusatol upregulated PTEN mRNA and protein expression and downregulated p-PI3K and p-AKT expression. These findings confirm that brusatol suppresses the growth of these renal cancer cell lines likely through modulating the PTEN/PI3K/AKT signaling pathway. The research conducted by Ye et al[95] demonstrated that brusatol effectively inhibits proliferation and induces apoptosis in hepatocellular carcinoma through autophagy induction, likely via the PI3K/AKT/mTOR pathway. Brusatol also inhibited tumor invasion and migration in vivo and in vitro.

The research conducted by Chen et al[68] has demonstrated that BJE can effectively inhibit the proliferation of MDA-MB-231 TNBC cells and induce apoptosis in these cells. Additionally, BJE promotes the phosphorylation of mTOR, PI3K, and AKT in MDA-MB-231 cells, while simultaneously inhibiting tumor growth in vivo. The inhibitory effect of BJE is attributed to its capacity to restrain autophagy via the PI3K/AKT/mTOR signaling pathway. Given its significant anti-TNBC activity, which results from the inhibition of autophagy, BJE shows great potential as a natural therapeutic agent for TNBC treatment[68].

Piperine: Piperine (PIP) is an alkaloid derived from black pepper, possessing diverse pharmacological and biological properties. PIP shows promise as a P-gp inhibitor, capable of sensitizing chemotherapeutic drugs and exhibiting antitumor properties[96]. The combination of PTX and PIP may improve the bioavailability of PTX for cancer therapy[97].

The study by Greenshields et al[98] examined the impact of PIP on TNBC cell growth and motility. PIP inhibited in vitro proliferation of TNBC and hormone-dependent breast cancer cells, without affecting normal mammary epithelial cells. PIP exposure reduced the proportion of TNBC cells in the G2 phase and downregulated proteins associated with the G1 and G2 phases, while upregulating p21 (Waf1/Cip1). PIP also suppressed the pro-survival AKT pathway and induced caspase-dependent apoptosis via the mitochondrial pathway. The combination of PIP and gamma radiation showed enhanced cytotoxicity against TNBC cells compared to gamma radiation alone. PIP impaired the in vitro migratory capacity of TNBC cells and reduced matrix metalloproteinase 2 and matrix metalloproteinase 9 mRNA expression, indicating a potential antimetastatic effect. Intratumoral PIP administration inhibited TNBC xenograft growth in immunodeficient mice.

The research by Hakeem et al[99] demonstrates a synergistic effect of Dox and PIP on MDA-MB-231 cells. The study shows that the combination treatment enhances suppression of the PI3K/AKT/mTOR signaling pathway, associated with increased PTEN expression, a negative regulator of this pathway. Furthermore, aldehyde dehydrogenase-1 levels, a biomarker for CSCs, are reduced. In vivo studies confirm these findings, indicating that the combination therapy can increase necrosis while downregulating PTEN and inhibiting PI3K levels, as well as the immunoreactivity of p-Akt, mTOR, and aldehyde dehydrogenase-1. These results suggest that PIP may reduce resistance to Dox both in vitro and in vivo by modulating the PI3K/AKT/mTOR pathway and CSCs[99]. Collectively, these results suggest that PIP may have therapeutic potential in the treatment of TNBC.

Berbamine: The traditional Chinese medicine berbamine (BBM), derived from Coptis and other plants, is commonly utilized for the treatment of leukopenia. Recent reports have revealed the anti-cancer effects of BBM. The study conducted by Li et al[64] has indicated that BBM enhances the expression of other regulatory proteins, such as histone deacetylases, thereby positioning it as a promising natural compound for its anticancer properties.

The study conducted by Liu et al[100] has demonstrated that BBM significantly suppresses cell proliferation in MDA-MB-231 cells and MCF-7 cells. Moreover, BBM reduces the apoptosis ratio in both MDA-MB-231 cells and MCF-7 cells. Additionally, BBM inhibits cell migration and invasion in TNBC cells. Furthermore, BBM downregulates the expression levels of PI3K, p-Akt/Akt, cyclooxygenase-2, LOX, murine double minute 2, and mTOR while upregulating p53 expression. These results indicate that BBM may effectively impede TNBC development through regulation of the PI3K/AKT/murine double minute 2/p53 and PI3K/AKT/mTOR signaling pathways. Therefore, it is plausible to consider BBM as a potential drug candidate for future TNBC treatment[100].

Homoharringtonine: Homoharringtonine is a naturally occurring alkaloid that has demonstrated inhibitory effects on the growth of various human tumors[101]. It has been shown to markedly downregulate the expression of p-AKT, p-mTOR, and Bcl-2, while upregulating the expression of TP53, Bax, cleaved caspase-3, and caspase-9. Furthermore, homoharringtonine has been shown to effectively reduce the volume and weight of breast tumors in murine models, inhibit the PI3K/AKT/mTOR signaling pathway, and induce mitochondrial apoptosis in canine mammary tumors cells[101].

In conclusion, numerous plant-derived inhibitors targeting the PI3K/AKT/mTOR pathway have demonstrated significant potential in treating TNBC. However, several challenges persist, including the need to develop and optimize drug delivery systems for enhanced bioavailability and reduced side effects, as well as the requirement for rational design of clinical trial protocols to assess their safety and efficacy. In the future, advancements in molecular biology, medicinal chemistry, synthetic biology, and related fields are expected to facilitate the development of safer, more efficient plant-derived PI3K/AKT/mTOR pathway inhibitors that demonstrate excellent therapeutic effects when combined with traditional drug formulation concepts and modern drug design methods. This will provide novel ideas and strategies for treating TNBC.

EXISTING INHIBITORS TARGETING THE PI3K/AKT/MTOR SIGNALING PATHWAY

The dysregulation of the PI3K/AKT/mTOR pathway is intricately associated with the onset and progression of breast cancer[4]. In recent years, there has been significant progress in the development of various drugs targeting this signaling cascade, including PI3K inhibitors, mTOR inhibitors, and AKT inhibitors, with ongoing clinical trials.

PI3K inhibitors

The activation of the PI3K signaling pathway is closely related to the occurrence and development of a variety of cancers, especially in breast cancer. PIK3CA is one of the most frequently mutated oncogenes in breast cancer. The development of PI3K inhibitors targets these specific gene mutations, thus offering novel therapeutic approaches. PI3K inhibitors can be categorized into three classes: Pan-PI3K inhibitors, selective PI3K inhibitors, dual-target inhibitors of PI3K/mTOR.

Pan-PI3K inhibitors

Buparlisib (BKM120): Pan-PI3K inhibitors exhibit broad-spectrum inhibition across all subtypes of PI3K, including PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ. BKM120 is a potent and highly specific oral inhibitor of class I PI3K, which has been demonstrated to effectively block hormone receptor-mediated repair through the PI3K/AKT/nuclear factor kappa B/c-Myc signaling pathway as well as the PI3K/AKT/forkhead box M1/Exo1 signaling pathway. Additionally, it down-regulates the expression of BRCA1/2 and Rad51. In combination with olapalil, BKM120 exerts dual inhibition on both PI3K and poly-ADP ribose polymerase (PARP) leading to a significant reduction in proliferation of BRCA-mutated TNBC cell lines MDA-MB-231 and MDA231-LM2[102]. Garrido-Castro et al[103] discovered in a clinical trial investigating the application of BKM120 as a treatment for advanced TNBC that while its use alone could prolong stable disease in a small subset of patients, no clear objective response was observed. Notably, downregulation of key nodes within the PI3K pathway was observed among those with stable disease population. These findings suggest that targeting the PI3K pathway alone may not be sufficient as an effective therapeutic strategy for TNBC[103].

Selective PI3K inhibitors

LY294002: LY294002 is a selective inhibitor of PI3K. It primarily targets specific subtypes of PI3K, including PI3Kα, PI3Kβ, and PI3Kδ, while exhibiting reduced selectivity for PI3Kγ. This targeted selectivity enhances the specificity of LY294002 in inhibiting the relevant subtypes, thereby minimizing its impact on other subtypes and reducing potential side effects.

The synthetic PI3K inhibitor LY294002 was the first to be utilized in investigating the mechanism of apoptosis induced by AKT inhibitors. It has been demonstrated that the combination of the PARP inhibitor talzenna with LY294002 synergistically inhibits proliferation in BRCA1 mutant HCC1937 TNBC cells through apoptosis, G0/G1 cell cycle arrest, oxidative stress, and enhanced DNA damage. This approach significantly suppresses the expression of PI3K, AKT1, and mTOR as well as phosphorylated protein levels activation in TNBC cells while inducing alterations in multi-kinase phosphorylation. Such a combined strategy holds promise as an effective therapeutic option for TNBC and may overcome talzenna resistance[104].

Inavolisib (GDC-0077): The protein encoded by p110a plays a pivotal role in the proliferation of tumor cells. GDC-0077 is a highly potent and selective inhibitor of p110a catalytic subunit of PIK3CA complex, encoded by PIK3CA, which also facilitates the degradation of mutated p110α[105]. GDC-0077 was identified as a more potent inhibitor of PI3K signaling, reducing cell viability by promoting the degradation of p110α, making it more effective at prolonging PI3K pathway suppression than other PI3K inhibitors[106]. Small-molecule inhibitors that target the PI3K p110α catalytic subunit have advanced to clinical trials, with early-phase studies of GDC-0077 demonstrating antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer[107].

In a phase I/Ib clinical trial (NCT03006172) involving patients with locally advanced or metastatic breast cancer characterized by PIK3CA mutations and HR(+)/HER2(-), researchers assessed the safety, tolerability, pharmacokinetics, and preliminary antitumor efficacy of GDC-0077, and it is considered to be an effective and selective small molecule inhibitor of p110α, which can promote the degradation of mutant p110α[108]. In summary, the majority of studies on GDC-0077 have concentrated on breast cancer patients with HR(+)/HER2(-) status, and such studies are relatively scarce. To date, there has been no research conducted on GC007 in TNBC, thus the potential application of GC007 in TNBC remains to be validated.

mTOR inhibitors

The use of mTOR inhibitors can enhance PI3K/PDK1 signal targeting, resulting in better anticancer activity[109]. Currently, first-generation mTOR inhibitors, such as everolimus and sirolimus, have been approved for the treatment of HR(+)/HER2(-) breast cancer[110-114]. However, mTOR inhibitors targeting TNBC, including AZD8055, are still in the preclinical trial phase.

AZD8055 is a pyridinopyrimidine derivative that exhibits ATP-competitive inhibition of both mTORC1 and mTORC2, demonstrating significant antitumor activity. It was observed that the combination treatment of AZD8055 and AUY922 (an heat shock protein 90 inhibitor) exhibited a synergistic effect on triple-negative MDA-MB-468 xenografts without increasing toxicity[115].

Some researchers compared the anti-proliferative capacity of rapamycin and a novel mTORC1/2 dual inhibitor (AZD8055) in two TNBC cell lines (MDA-MB-231 and MDA-MB-453). Their findings indicated that AZD8055 exhibited a stronger inhibitory effect on breast cancer cell proliferation compared to rapamycin. In terms of inhibiting glycolysis and reducing glucose consumption, AZD8055 demonstrated greater potency than rapamycin. Regarding amino acid metabolism, while both AZD8055 and rapamycin exerted broad effects, the degree of these effects varied[116].

A study examined the effects of combining the mTOR1/2 inhibitor AZD8055 and the MEK1/2 inhibitor PD0325901 in one TNBC MDA-MB-435 cell line. Inhibiting MEK1/2 activated AKT, a downstream signaling protein of the PI3K pathway. The combination therapy effectively suppressed AKT phosphorylation, disrupting the feedback loop between the two pathways. Cell proliferation and DNA replication assays showed that the dual treatments significantly and synergistically inhibited cell cycle progression and cell proliferation[117].

Dual PI3K/mTOR inhibitors

Due to their structural similarities, both PI3K/mTOR dual inhibitors can target the kinase pockets of PI3K and mTOR[118]. Dual inhibitors of PI3K/mTOR exhibit potent anticancer effects. Dual inhibitors demonstrate a more robust inhibition of the signaling pathway compared to single-site inhibitors. Dactolisib (BEZ235) was the first dual PI3K/mTOR inhibitor to advance into clinical trials, and it has now progressed to phase II clinical studies. Additionally, SF1126, paxalisib (GDC-0084), and gedatolisib are currently being evaluated in preclinical trials for TNBC.

BEZ235: BEZ235 exerts its antitumor activity by arresting the cell cycle at the G1 phase, promoting apoptosis, inducing phagocytosis, and inhibiting tumor cell proliferation via DNA repair mechanisms. The drug response-related genes exemplified by BEZ235 are potential biomarkers for TNBC molecular typing, prognosis prediction and targeted therapy[119]. In the study conducted by Kuger et al[120], the researchers evaluated whether the novel dual PI3K/mTOR inhibitor NVP-BEZ235 could enhance the radiosensitivity of TNBC MDA-MB-231 and ER-positive MCF-7 cells to ionizing radiation under varying oxygen conditions. The results demonstrated that NVP-BEZ235 treatment effectively inhibited hypoxia-inducible factor-1alpha expression and PI3K/mTOR signaling pathways, induced autophagy, and prolonged DNA damage repair in both cell lines across all tested oxygen conditions[120]. It has been confirmed that the concurrent use of NVP-BEZ235 and caffeic acid phenyl ester can effectively reduce tumor metastasis and progression in TNBC cells[121]. In a recent study, researchers examined the impact of NVP-BEZ235 on two TNBC cell lines with mutant p53: MDA-MB-231 and MDA-MB-468. The results showed that BEZ235 effectively inhibited cell proliferation, migration, and colony formation. Additionally, BEZ235 induced the degradation of mutant p53. Researchers also noted that BEZ235 might stimulate autophagy by suppressing the AKT/mTOR signaling pathway[122].

SF1126: SF1126 is a chemically modified derivative of LY294002, as a novel pan-PI3K inhibitor that effectively suppresses tumorigenesis and angiogenesis in vivo. Deng et al[123] discovered that the combination of SF1126 with gefitinib induces apoptosis in TNBC cells by inhibiting the EGFR-PI3K-AKT-mTOR pathway.

GDC-0084: GDC-0084 a dual inhibitor of PI3K/mTOR that is capable of crossing the blood-brain barrier, has been shown to reduce the activity of PIK3CA mutant BC BMS cell lines in vitro, induce apoptosis, and inhibit the phosphorylation of AKT and p70S6[124].

Gedatolisib: Gedatolisib is a potent inhibitor of the PI3K/mTOR pathway. Preclinical studies have demonstrated that combining gedatolisib with immune checkpoint inhibitors effectively suppresses the growth of breast cancer cells. Furthermore, this combination treatment continuously stimulates the response of dendritic cells, CD8⁺ T cells, and natural killer cells, suggesting that targeting the PI3K/AKT pathway may enhance the efficacy of immune checkpoint inhibitor in breast cancer[125].

In a phase I clinical trial (NCT01920061), 22 patients with TNBC were enrolled and treated with a combination of gedatolisib, docetaxel, cisplatin, and dacomitinib. The results showed an objective response rate of 40.0% (4/10) in first-line patients and a complete response rate of 10% (1/10). Second- or third-line patients had an objective response rate of 33.3% (4/12) and a complete response rate of 8.3% (1/12)[126].

GDC-0980: It was demonstrated that the combination of ABT-888 and cardoplatin with GDC-0980, a dual inhibitor of PI3K-mTOR, can induce DNA damage, inhibit proliferation signaling, upregulate the expression of apoptotic markers, and effectively suppress tumor growth in MDA-MB231 and xenograft models harboring normal BRCA[127]. This finding underscores the critical role of the PI3K/AKT/mTOR pathway in mediating the antitumor effects of PARP inhibitors in TNBC.

AKT inhibitors

AKT plays a crucial role in regulating tumor cell survival and proliferation. Aberrant activation of AKT has been implicated in the initiation and progression of tumors. The AKT signaling pathway is hypothesized to play a pivotal role in regulating breast cancer stemness and metastasis[128]. Furthermore, this pathway is also posited to be essential in the modulation of mitochondrial dynamics, encompassing both fusion and fission processes[129]. Currently, there are several AKT inhibitors undergoing clinical trials for potential treatment of breast cancer, including GDC-0068, MK-2206, and AZD5363 that exhibit enhanced efficacy in PTEN-altered tumors[130-133].

Ipatasertib (GDC-0068): Ipatasertib is capable of binding to three subtypes of AKT, thereby inhibiting AKT activity, obstructing the PI3K/AKT/mTOR signaling pathway, and diminishing tumor cell proliferation and survival. The tolerability and antitumor efficacy of a combination therapy comprising the allosteric MEK1/2 inhibitor cobimetinib and the AKT inhibitor GDC-0068 were evaluated in a phase Ib study involving patients with advanced solid tumors, including an extended cohort of PTEN-deficient TNBC, ovarian cancer, and endometrial cancer. However, it has been observed that the combination of MEK and AKT inhibitors exhibits limited tolerability and efficacy[134]. The AKT inhibitor GDC-0068 has also been utilized as a monotherapy for patients with TNBC[135]. The FAIRLANE study assessed the efficacy of neoadjuvant GDC-0068 in combination with PTX for early-stage TNBC treatment, revealing that the pathological complete response rate was higher in the GDC-0068 group compared to the placebo group among patients harboring mutations in the PI3K/AKT/mTOR signaling pathway[136].

MK-2206: MK-2206 is a highly selective inhibitor targeting Akt1, Akt2, and Akt3. However, one study has confirmed that MK-2206 monotherapy exhibited limited clinical efficacy in advanced breast cancer patients with PIK3CA/AKT1 mutations or PTEN loss[137]. A combination of MK-2206 and standard neoadjuvant chemotherapy has been demonstrated to enhance response rates in patients with HER2-positive and/or HR-negative breast cancer[138]. Ruxolitinib serves as an inhibitor of Janus kinase (JAK)1 and JAK2. A study investigated the impact of combining ruxolitinib with MK-2206 on apoptosis and the JAK2/signal transducer and activator of transcription 5 and PI3K/AKT signaling pathways in MDA-MB-231 cells. The findings indicated that concurrently inhibiting the JAK2/signal transducer and activator of transcription 5 and PI3K/AKT pathways may reduce metastasis by lowering tumor cell viability[133]. It was found that MK-2206 combined with WZB117 (a glucose transporter type 1 inhibitor), showed a synergistic effect on growth inhibition and apoptosis induction in MCF-7 and MDA-MB-231 cells. Mechanism studies have demonstrated that MK-2206 and WZB117 exert a synergistic cytotoxic effect on both MCF-7 and MDA-MB-231 breast cancer cells through the inhibition of AKT phosphorylation and the induction of DNA damage. This combination may further impair DNA damage repair mechanisms, ultimately leading to apoptosis[139].

Capivasertib (AZD5363): Capivasertib has shown preclinical activity in TNBC models, and drug sensitivity has been associated with the activation of PI3K, AKT and/or PTEN deletions[140]. The efficacy and safety of combining capivasertib with PTX in TNBC patients has been further evaluated in the PAKT trial (NCT03997123). Part of the study indicated that adding AZD5363 to first-line PTX therapy significantly prolonged progression-free survival and OS, particularly demonstrating a more pronounced clinical benefit rate in tumor patients exhibiting alterations in PIK3CA/AKT1/PTEN[140]. Previous research has demonstrated that the activation of AKT contributes to the characteristics of CSCs, such as self-renewal, migration, and chemoresistance[141]. A preliminary investigation into TNBC has confirmed that AZD5363 effectively suppresses the expression of mitofusin1 in breast CSCs (BCSCs). This inhibition has the potential to reduce stemness and re-sensitize BCSCs to chemotherapeutic agents by modulating mitochondrial fusion[129]. These findings support the further exploration of AZD5363 as a therapeutic target for BCSCs.

In conclusion, these studies focused on existing PI3K/AKT/mTOR inhibitors in TNBC that remain largely preclinical, with a few progressing to clinical trials. The therapeutic potential of these inhibitors is still under exploration. It is anticipated that a series of clinical trials evaluating the efficacy of these inhibitors in TNBC are currently ongoing (Table 4). As research progresses, the efficacy of these inhibitors in TNBC is expected to be further validated and confirmed. Suggest future studies that could validate these findings in patient-derived xenograft models.

Table 4 Ongoing clinical trials associated with the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin pathway in triple negative breast cancer.

Inhibitors class	Trial code	Trial content	Trial period
mTOR inhibitors	NCT02616848	A phase I clinical trial to evaluate the efficacy and safety of everolimus in combination with arebuline for previously treated mTNBC	I
Pan-PI3K inhibitors	NCT01790932	A phase II clinical trial evaluating the efficacy of monotherapy with buparlisib (BKM120) in patients with TNBC	II
Pan-PI3K inhibitors	NCT01629615	A phase II clinical trial evaluating the efficacy of monotherapy with buparlisib in patients with TNBC	II
PI3K inhibitors	NCT02389842	A phase Ib clinical trial assessing the efficacy and safety of taselisib, a beta-preserving PI3K inhibitor, in combination with palbociclib for metastatic breast cancer, including those with TNBC	Ib
PI3K inhibitors	NCT03207529	A phase I clinical trial evaluating the combination of alpelisib, an alpha-specific PI3K inhibitor, with enzalutamide in patients with AR+ and PTEN+ breast cancer, including those with TNBC	I
AKT inhibitors	NCT02576444	A phase II clinical trial investigated various combinations of olaparib, specifically focusing on patients with breast cancer who received both olaparib and capivasertib	II
AKT inhibitors	CAPITELLO290	A clinical trial of the capivasertib in combination with paclitaxel for first-line treatment in patients with locally advanced or metastatic TNBC, included evaluating OS in subgroups whose tumors harbored PIK3CA, AKT1, or PTEN biomarker alterations	III

PI3K: Phosphoinositide 3-kinase; AKT: Protein kinase B; mTOR: Mechanistic target of rapamycin; mTNBC: Metastatic triple-negative breast cancer; TNBC: Triple negative breast cancer; PTEN: Phosphatase and tensin homolog; AR: Androgen receptor; OS: Overall survival; PIK3CA: Phosphoinositide3 kinase-alpha.

CONCLUSION

In summary, the occurrence of breast cancer is associated with the sequential activation of the PI3K/AKT/mTOR signaling pathway and multiple other signaling pathways, which can drive tumor progression and contribute to treatment resistance. Currently, most studies investigating the role of the PI3K/AKT/mTOR signaling pathway in TNBC are at a preclinical stage, and their efficacy remains controversial. In conclusion, TNBC treatment still encounters numerous challenges; however, advancements in novel therapeutics and treatment strategies are guiding us towards more personalized and efficient approaches. Immunotherapy combination strategies (such as the integration of immune checkpoint inhibitors with chemotherapy or targeted therapies), precision medicine approaches targeting specific genetic mutations (such as BRCA1/2 and PIK3CA), tumor vaccines, optimization of combination therapy regimens, liquid biopsy technologies, and applications of artificial intelligence have recently emerged as key areas of focus. Future research should prioritize a comprehensive understanding of TNBC biological characteristics and molecular mechanisms, develop more efficacious treatments, rigorously validate safety and efficacy through well-designed clinical trials, and enhance translation of basic research findings into clinical practice to facilitate application of novel therapeutic concepts and technologies.

ACKNOWLEDGEMENTS

The authors would like to acknowledge all the authors whose work has been reviewed in the preparation of the manuscript.