Abstract
Breast cancer (BC) is a common malignancy in women, with hormone receptor (HR)-positive subtype responsible for approximately 70% of cases. Currently, patients with metastatic HR-positive BC rely on endocrine therapy and cyclin-dependent kinase (CDK)-4/6 inhibitors for treatment. Currently, approved CDK4/6 inhibitors include palbociclib, ribociclib, and abemaciclib. However, clinical evidence of CDK-4/6 inhibitor resistance is emerging, suggesting that the gap in the knowledge of its resistance mechanism requires further investigation. This review discusses the mechanisms of CDK4/6 inhibitor resistance in BC, including both intrinsic and extrinsic mechanisms. We also discuss possible alternative strategies to overcome CDK4/6 inhibitor resistance in future clinical applications.
Breast cancer (BC) is a commonly diagnosed malignancy accounting for 25% of cancer cases among women worldwide (1). Currently, BC can be divided into different subtypes based on the presence of three hormone receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Different combinations of these hormone receptors characterize three main subtypes of breast cancer: luminal type, also known as hormone receptor (HR)-positive subtype, HER2-positive subtype, and triple-negative subtype. Among these, HR-positive BC is the most common subtype, accounting for 70% of all BC malignancies (2).
Clinically, HR-positive BC relies on endocrine therapy as its first-line treatment (3). These include tamoxifen, used in premenopausal women, and aromatase inhibitors, used in postmenopausal women (4). However, many patients develop resistance to hormonal blockade, resulting in distant recurrence (5). Because cyclin-dependent kinases (CDKs) play an essential role in initiating the cell cycle in cancers (6), CDKs inhibitors have been developed over the past two decades for the treatment of BC. CDK4/6 inhibitors combined with endocrine therapy for HR-positive BC have recently become a new treatment standard (7) (Figure 1). However, clinical evidence of CDKs inhibitor resistance (8, 9) suggests the need for alternative approaches for HR-positive BC patients. In this review, we will discuss the mechanism of CDK inhibitor resistance in HR-positive BC. We will also discuss possible alternative strategies to overcome CDKs inhibitor resistance for future clinical application.
Regulation of cell cycle in hormone receptor-positive breast cancer. In breast cancer cells, mechanisms involved in cell cycle regulation can be broadly classified as cell cycle-specific mechanisms and cell cycle non-specific mechanisms. In cell cycle-specific mechanisms, cyclin-dependent kinases (CDKs) and cyclins remain the major regulatory factors. Several CDKs including CDK1, CDK2, CDK4, CDK6, and CDK7 bind with different cyclins (cyclin-A, -B, -D, and -E). CDKs-cyclin complexes then work together to facilitate cell cycle progression, but these complexes can also be inhibited by cyclin inhibitors, such as p16 and p21. Other cell cycle non-specific mechanisms, such as PI3K signaling, MAPK signaling, or DNA repair systems may also indirectly affect cell cycle progression. Many current drugs target these pathways to combat hormone receptor-positive breast cancer. Arrows suggest positive regulation; blunt arrows indicate inhibitory regulation. The red box shows current therapeutic targets and corresponding drugs.
CDK4/6 Inhibitors
Cyclins, including cyclin G1, cyclin G1/S, cyclin S, and cyclin M, are the most critical cell cycle regulators that drive cells in a particular cell cycle phase, which helps to regulate their growth and metabolism (10). Cyclins are activated or inactivated by cyclin-dependent kinases (CDKs) (11). However, aberrant CDK activation is usually observed in cancers. Overactivation of CDKs, such as CDK4 and CDK6 has been shown to positively contribute to tumor progression (12). The CDK4/6 complex, after interacting with cyclin D1, a direct transcriptional target of estrogen-receptor signaling, can facilitate the inhibition of retinoblastoma (Rb) protein, which leads cells to enter the G1 checkpoint to the S phase of the cell cycle (13). In BC, the dysregulated activity of cyclin D1 and CDK4/6 is considered to play a significant role in estrogen-driven tumor proliferation (14). CDK4/6 up-regulation and cyclin D1 amplification have also been found in HR-positive BC (15).
Due to the importance of CDK4/6 activity in BC progression, CDK4/6 inhibitors have been considered a promising target (13, 16, 17). Three U.S. Food and Drug Administration (FDA)-approved CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib are currently used for BC treatment (18). Combining these CDK4/6 inhibitors with endocrine therapy has been shown to prolong the progression-free survival of patients with advanced HR-positive BC (19-24) (Table I). In addition, newer CDK4/6 inhibitors, such as trilaciclib and dalpiciclib (also known as SHR6390) have recently been developed and have shown favorable pharmaceutical properties in in vivo models (25-27). These drugs have recently entered phase III clinical trials (ClinicalTrials.gov identifier: NCT04799249 and NCT03927456).
Current usage of CDK4/6 inhibitors with hormone therapy.
Although CDK4/6 inhibitors hold great promise against BC, tumor resistance to CDK4/6 inhibitors has been reported worldwide (28, 29), and there is an urgent need to investigate its mechanism.
CDK4/6 Inhibitor Resistance
Approximately 20% of patients with BC are not responsive to CDK4/6 inhibitors (30). The tumors of these patients were shown to process intrinsic mutations that allowed them to avoid the action of CDK4/6 inhibitors and proliferate in the presence of the drug. These intrinsic mutations include but are not limited to CDK4/6 over-expression, loss of Rb function, and p16 amplification (Figure 2).
Mechanisms of CDK4/6 inhibitor resistance. Multiple mechanisms, directly or indirectly, involved in cell cycle regulation may be associated with CDK4/6 inhibitor resistance. Current mechanisms implicated in developing resistance to CDK4/6 inhibitors are highlighted in the figure. The red box shows intrinsic mechanisms, whereas the blue box shows extrinsic mechanisms. CDK4/6 over-expression remains a primary reason for intrinsic mechanisms. Intrinsic mechanisms that lead to CDK4/6 inhibitor resistance also include loss of Rb or ER and over-expression of p16 or CDK-2. Some of the miRNAs can also affect the sensitivity of tumor cells to CDK4/6 inhibitors. Resistance to CDK4/6 inhibitors can also occur through an acquired (extrinsic) mechanism. These mechanisms include over-activation of oncogenic signaling pathways, such as PI3K, MAPK, and TGFR signaling. PI3K mutation can also lead to overactivation of its signaling cascade. Defects in DNA damage repair mechanisms, such as ATR Chk1 and ATM Chk2 pathways, result in un-arrested cell cycles. Other mechanisms, such as alternation in the Hippo pathway, HDAC over-expression, and ER over-activation may also lead to CDK4/6 inhibitor resistance.
The phase III clinical trial, PALOMA-2 (ClinicalTrials.gov identifier: NCT01740427) (20) showed that more than 30% of BC patients experienced a recurrence within two years of palbociclib treatment, and over 70% of patients developed resistance to palbociclib and letrozole treatment 40 months later, suggesting that even the combined use of CDK4/6 inhibitor with other therapeutic drugs may lead to insensitivity to CDK inhibitors and allow tumor cells to return to a proliferative phenotype. Several underlying mechanisms have been proposed to explain this acquired resistance, such as epigenetic alternations, overactivation of mitogenic signaling, such as PI3K-AKT-mTOR signaling, or mutations in the component of the signaling pathway (Figure 2).
Intrinsic Resistance Mechanism
CDK4/6 over-expression. Over-expression of CDK4 or CDK6 has been suggested as the primary resistance mechanism to CDK4/6 inhibitors. Higher CDK4 expression has been detected in CDK4/6 inhibitor-resistant cancer cells (31), indicating that CDK4 over-expression may be correlated with resistance to CDK4/6 inhibitors. In addition, over-expression of CDK6 suppressed the sensitivity of CDK4/6 inhibitors in luminal BC cells (12). Although there are reports that amplification of CDK6 promotes BC resistance to CDK4/6 inhibitors, and CDK6 knockdown rescued therapy sensitivity, this phenomenon may be independent of CDK4 (12). In support of this, CDK4 amplification has not been detected in a CDK inhibitor-resistant BC model, although amplification of CDK4 has been reported in other resistant cancers (32). Cyclin D1, D2, and D3 up-regulation has also been observed in CDK4/6 inhibitors-resistant BCs (33-35). These results suggested that resistance could occur by either non-canonical activation of CDKs or by forming cyclin D-CDK4/6 complexes.
Although no reports have so far described mutations in CDK4 or CDK6 that could possibly reduce the affinity of CDK4/6 inhibitors, this remains a possible mechanism which cannot be overlooked.
Loss of Rb Function. Rb is a tumor suppressor that prevents G1/S transition during cell cycle progression (36). One of the major mechanisms of resistance to CDK4/6 inhibitors is the loss of functional Rb, which has been observed in many tumor types. In a case report in which HR-positive BC patients were treated with a combination of palbociclib and fulvestrant or ribociclib and letrozole, acquired Rb mutations were detected (28). In the phase III PALOMA-3 trial (ClinicalTrials.gov identifier: NCT0192135), whole-exome sequencing of circulating tumor DNA (ctDNA) confirmed CDK4/6-induced, acquired Rb mutations in patients receiving palbociclib and fulvestrant (29). Loss of functional Rb was subsequently identified in other studies where patients resisted CDK4/6 inhibitors (37-39). Notably, the loss of Rb is correlated with the increased expression of E2F (40), resulting in the constitutive activation of its downstream target proteins. However, the presence of Rb does not usually guarantee resistance to CDK4/6 inhibitors, as seen in Rb-mutated bladder cancers where palbociclib was effective (41). Furthermore, putative attenuation of Rb functions through Rb mutations, identified in ctDNA, has been observed in CDK4/6 inhibitor-resistant patients (28, 42).
p16 over-expression. p16 is a CDK4 inhibitor that prevents Rb phosphorylation and promotes G1 cell cycle arrest, leading to cell senescence (10, 43). Therefore, p16 generally serves as a tumor suppressor. However, p16 over-expression may suppress the activity of CDK4 and expression of cyclin D1 (44), which are the main targets of CDK4/6 inhibitors, therefore reducing the effects of CDK4/6 inhibitors (45). Furthermore, resistance to CDK4/6 inhibitors is related more to the over-expression of p16 than Rb expression, as the expression level of p16 largely correlates to the effectiveness of CDK4/6 inhibition (44). However, low expression of p16 did not improve the therapeutic benefit of palbociclib in Rb-positive or HR-positive BC patients (46). However, p16 over-expression in the absence of Rb in CDK4/6 inhibitor-resistant patients (47) suggests that p16 over-expression may mediate CDK4/6 inhibitor resistance through other mechanisms, such as Rb loss, to introduce.
Overactivation of cyclin E-CDK2 signaling. In normal cells, phosphorylated cyclin D-CDK4/6 and cyclin E1/2-CDK2 complexes work together to inactivate Rb through phosphorylation, which releases E2F transcription factors. Therefore, without cyclin D1-CDK4/6, endogenous cyclin E1/2-CDK2 cannot effectively phosphorylate Rb to release E2F. Although cyclin E2 is also a transcriptional target of E2F, cyclin E2-CDK2 complexes are thought to be suppressed by CDK4/6 inhibition (48). However, over-expression of cyclins E or CDK2 may allow cells to overturn CDK4/6 inhibition.
Expression of cyclin E1, cyclin E2, and CDK2 has been shown to be up-regulated in several CDK4/6 inhibitor resistance models (12, 34, 49). The up-regulation can occur through the amplification of CCNE1 (which encodes cyclin E1) (49) or CCNE2 (34). Cyclin E1 or CDK2 ablation also re-sensitized palbociclib-resistant cells to cell cycle arrest (49). In the PALOMA-3 (ClinicalTrials.gov identifier: NCT01942135) trial, higher CCNE1 gene expression was associated with poor response to palbociclib (50). Similar unsatisfactory responses to palbociclib were also observed in patients with higher CCNE2 gene expression in preoperative-palbociclib (POP) trial (ClinicalTrials.gov identifier: NCT02008734) (51). In this context, CDK2 inhibitors may benefit CDK4/6 inhibitor-resistant tumors (52).
Regulation of microRNAs. MicroRNAs (miRNAs) regulate approximately 30% of human genes. More than 50% of these miRNAs are tumor-associated or located in fragile sites of chromosomes (53). Changes in miRNAs in tumor cells revealed an essential role in tumor development (53). While miRNAs could have been seen as a hallmark in BC (54), they are also related to resistance to chemotherapy and endocrine therapy (53).
Currently, six miRNAs, miR-126, miR-326, miR3613-3p, miR29b-3p, miR-497, and miR-17-92, have been suggested to be associated with sensitivity to CDK4/6 inhibitors (55-57). miR-126 was also found to be a modulator of CDK4/6 inhibitors, as its transfection increased anti-tumor activity of CDK 4/6 inhibitor (58). Conversely, six miRNAs, miR-193b, miR-432-5p, miR-200a, miR-223, Let-7a and miR-21, are associated with resistance to CDK4/6 inhibitors (55, 59). CDK4/6 inhibitor resistance was mediated by miR-432-5p through the suppression of the TGF-β pathway and induction of CDK6 expression (60). Down-regulation of miR-223 contributed to HR-positive BC resistance to CDK4/6 inhibitors (59). Other miRNAs, such as miR-124a, miR9, miR200b, and miR-106b mediate cellular response to CDK4/6 inhibitors without affecting sensitivity to treatment (55).
The long noncoding RNA TROJAN has been found to promote HR-positive BC proliferation by regulating the G1/S transition. An antisense oligonucleotide of TROJAN has been shown to impair tumor cell proliferation. Moreover, combining an anti-TROJAN antisense oligonucleotide with palbociclib significantly enhanced the efficacy of palbociclib in HR-positive BC (61).
Loss of ER expression. In HR-positive BC, ER signaling predominantly induces the activation of cyclin D1 and CDK4/6 (16). Therefore, CDK4/6 inhibitors have been widely used with ER-related endocrine drugs, such as fulvestrant, tamoxifen, and aromatase inhibitors. The current understanding is that resistance to CDK4/6 inhibitors may be related to the down-regulation of cyclin D1 due to the loss of ER (62). Resistance to abemaciclib has been reported to be associated with the loss of cyclin D1 and ER/PR expression (12). Additionally, CDK6 over-expression may reduce the effectiveness of ER antagonists and lead to CDK4/6 inhibitor resistance by reducing ER expression (12, 63).
Acquired Resistance Mechanism
Overactivation of PI3K–AKT–mTOR signaling. The phosphatidylinositol 3-kinase (PI3K; also known as PIK3CA)–AKT–mammalian target of rapamycin (mTOR) signaling pathway is involved in tumor cell growth and progression and is activated in almost 40% of BC cases (64, 65). In HR-positive BC, ER expression can be induced by activating PI3K–AKT–mTOR signaling, which drives endocrine therapy resistance (66). Furthermore, activation of the PI3K–AKT–mTOR pathway can stabilize CDK4/6, leading to resistance to CD4/6 inhibitors (33, 49, 66). The loss of phosphatase and tensin homolog (PTEN) expression can increase AKT activation and decrease p27 expression, leading to excessive activation of CDK4 and inducing CDK4/6 inhibitor resistance (38). However, CDK4 can phosphorylate the tumor suppressor folliculin (FLCN) and activate the mammalian target of rapamycin complex 1 (mTORC1), which is associated with cancer progression (67). Furthermore, CDK4/6 inhibitors tend to activate PI3K–AKT–mTOR pathway by phosphoinositide-dependent kinase-1 (PDK-1) (33, 68), further leading to drug resistance.
PI3K mutation. Because overactivation of the PI3K–AKT–mTOR pathway has been linked to BC oncogenesis, pharmacological targeting of PI3K by PI3K inhibitors, such as alpelisib, has shown significant benefits in HR-positive, endocrine therapy-resistant BC patients (64). Treatment with alpelisib-fulvestrant has also prolonged progression-free survival among HR-positive BC patients with PI3K mutation and prior endocrine therapy failure (69). While inhibition of PI3K and mTOR has been shown to restore treatment sensitivity of CDK4/6 inhibitor-resistant BC cells (70-72), PI3K inhibitors have been implicated in the prevention of early CDK4/6 inhibitor resistance by down-regulating cyclin D1 expression (49). However, an assessment of the genetic aberrations of CDK4/6 inhibitor-resistant BC showed that PI3K mutations are more frequent than Rb mutations (29). Therefore, the combination of PI3K inhibitors or mTOR inhibitors with CDK4/6 inhibitors may require more clinical trials before it can be used as a standard strategy in the future. Currently, triple combination therapy with CDK4/6 inhibitors, PI3K inhibitors, and endocrine therapy is being investigated in patients whose tumors progress after CDK4/6 inhibitor treatment. Results from this trial suggested that such a treatment strategy is a promising strategy for patients with pre-treated, PI3K-mutated, HR-positive BC (73).
Defects of the ATR-Chk1 and ATM–Chk2 pathway. The ataxia telangiectasia mutated (ATM) and checkpoint kinase-2 (Chk2) pathways are critical in DNA damage repair (74). The ATM recognizes double-strand breaks, whereas the ataxia telangiectasia and Rad3-related protein (ATR) recognize single-strand breaks. Upon recruitment to DNA damage sites, ATM and ATR activate the checkpoint kinase 2 (Chk2) and Chk1. Both pathways lead to the inhibition of CDC25A, a phosphatase that hampers the inhibition of CDK4/6 and CDK2, leading to the blockage of cell entry into cell cycle checkpoints (74). A recent study showed that defects in the ATM-Chk2 pathway and single-strand break repair in HR-positive BC can drive endocrine therapy resistance (75). Moreover, the efficacy of endocrine agents in HR-positive BC depends on both the ATM and Chk2 expression; inactivation of either ATM or Chk2 prevents ER inhibition-mediated cell cycle arrest (76). Because the ATM-Chk2 pathway is highly associated with CDK4/6 (74), a defect of the ATM-Chk2 pathway may also be associated with CDK4/6 inhibitor resistance. In addition, Chk1 also stimulates WEE1 G2 checkpoint kinase (WEE1), a serine/threonine protein kinase that suppresses the function of CDK1; therefore, defects in the ATR-Chk1 pathway or over-expression of WEE1 may allow the cells to undergo cell cycle, lowering the efficiency of CDK4/6 inhibitors (77).
Alterations of Hippo pathway. In human breast cells, the Hippo pathway has been linked with ER to regulate breast cell development (78, 79). In the Hippo pathway, mammalian sterile 20-like kinase (MST)-1/2 and large tumor suppressor kinase (LATS)-1/2, phosphorylate the yes-associated protein 1 (YAP1) and WW domain containing transcription regulator 1 (WWTR1; also known as TAZ), resulting in the inhibition of cell growth (80). Similarly, tumors that lack MST-1/2 and LATS-1/2 fail to inhibit YAP1 and TAZ (79, 81). The Hippo pathway is associated with the development and progression of BC and has emerged as a keystone in therapy resistance (82, 83). It has been suggested, directly or indirectly, that alternations in the Hippo pathway are associated with CDK4/6 inhibitor resistance. Transcriptional enhanced associate domain (TEAD), a regulatory transcription factor in the Hippo pathway, has been shown to induce CDK6 expression (84). The loss of the FAT atypical cadherin 1 (FAT1) gene, which encodes a proto-cadherin, is associated with CDK4/6 inhibitor resistance mediated by YAP/TAZ nuclear localization and CDK6 over-expression in ER-positive breast cancer (37).
FGFR1 activation. The fibroblast growth factor receptor (FGFR) activation is involved in the proliferation and survival of HR-positive BC (85). Similar to other mitogenic signalings, FGFR links cyclin D and CDK4/6. Of the five FGFRs, FGFR1 is associated with resistance to CDK4/6 inhibitor. FGFR1 activates the PI3K–AKT–mTOR and RAS–MEK–ERK signaling pathways (86). FGFR1 over-expression was shown to mediate resistance to palbociclib or ribociclib (87, 88), which can be reversed by the FGFR tyrosine kinase inhibitor (TKI), lucitanib (89). In addition, treatment of tumors with increased FGFR2 expression with the combination of palbociclib and letrozole has been associated with higher progression-free survival (90). FGFR1/2 amplification or activating mutations were also identified in ctDNA in patients with progression after CDK4/6 inhibitors treatment (67).
Overactivation of MAPK signaling pathway. The mitogen-activated protein kinase (MAPK) pathway is one of the signaling pathways downstream of FGFR activation. The MAPK is composed of six distinct kinase groups: extracellular signal-regulated kinase (ERK)-1/2, ERK-3/4, ERK-5, ERK-7/8, Jun N-terminal kinase (JNK)-1/2/3 and the p38; therefore, the MAPK pathway is also known as the RAS/RAF/MEK/ERK pathway (91). These pathways interfere with the cell cycle machinery at many stages. Among these, ERK signaling is the best-studied MAPK pathway. ERK signaling is activated by extracellular signals, such as growth factors and mitogens, which are particularly relevant to cancer. These signals phosphorylate RAS and subsequently lead to RAF phosphorylation. Activated RAF, in turn, phosphorylates the kinases MEK-1/2, which then phosphorylates ERK-1/2 (91).
Transcriptional regulation of cell cycle components by ERK links CDKs to the ERK signaling. Whereas ERK signaling stimulates cyclin D1 expression upon growth factor exposure, p38 signaling suppresses cyclin D1 expression (91). Therefore, the alternation of cyclin D levels largely affects their cell cycle partners, CDK4 and CDK6, leading to acquired resistance to CDK4/6 inhibitor (92).
Immune evasion. CDK4/6 inhibitors promote anti-tumor immunity, while stimulating the interferon-lambda (IFN-λ) and increasing the presentation of tumor antigens (93, 94). CDK4/6 inhibitors also inhibit the proliferation of regulatory T cells, which hinders immune surveillance against tumor cells (94). In addition, CDK4/6 inhibitors promote cytotoxic T-cell-mediated clearance of tumor cells (94).
However, immune evasion or alterations in the tumor microenvironment eventually leads to resistance to CDK4/6 inhibitor (94). Cytokine array analysis suggested that chemokine (C-C motif) ligand 5 (CCL-5) was up-regulated in palbociclib-treated cancer cells (95), which may be related to immune invasion (96). Moreover, RNA sequencing analysis suggested enriched IFN-α and IFN-β expression in CDK4/6 inhibitor-resistant BC cells (97). Recently, transforming growth factor-beta (TGF-β) has also been suggested to induce the expression of epithelial-mesenchymal transition (EMT)-related genes via the suppressor of mothers against decapentaplegic (SMAD) and the PI3K/AKT/mTOR pathway (98, 99), which may therefore lead to the insensitivity to CDK4/6 inhibitors.
Other Possible Mechanisms
A study has revealed that the loss of CDK inhibitors p21 and p27, which targeted CDK2, could lead to palbociclib insensitivity (100). Furthermore, p21 can be inhibited by histone deacetylase (HDAC), leading to cell cycle progression; therefore, over-expression of HDAC could also lead to CDK4/6 inhibitor resistance (101). The NeoPalAna trial (ClinicalTrials.gov Identifier: NCT01723774) has identified that p19, which is transcribed by E2F after phosphorylation of Rb, was over-expressed in patients with palbociclib resistance (35, 102). In addition to CDK4/6 over-expression, as mentioned earlier, CDK7 over-expression may also contribute to drug resistance, as CDK7 may activate CDK4/6, promoting G1 progression (103). This has also been observed in a mouse model, showing up-regulation of CDK7 after palbociclib treatment (104). Thymidine kinase and CDK9 levels in plasma-derived exosomes have been shown to play a role in reducing effectiveness of palbociclib (105). Overactivation of androgen receptor (AR) has been suggested in palbociclib-resistant BC cells (106), where dual inhibition of AR could reverse palbociclib resistance.
Potential Strategies to Overcome CDK4/6 Inhibitor Resistance in the Future
Use of CDK4/6 inhibitor with other clinically available drugs or other target therapeutic drugs. Although CDK4/6 inhibitors are currently used with endocrine therapy for HR-positive BC (107), their persistent use may induce acquired-tumor drug resistance. Yet, interestingly, combination therapies with other drugs have been shown to inhibit this adaptive response.
Currently, several clinical trials have investigated combination treatment in BC patients with CDK4/6 inhibitor resistance, and most of them have resulted in a promising result (Table II). For example, the MAINTAIN trial (ClinicalTrials.gov identifier: NCT02632045) has suggested a prolonged progression-free survival in CDK4/6 inhibitor-resistance patients receiving ribociclib combined with endocrine therapy, compared to ribociclib monotherapy, therefore offering a potential therapeutic benefit of continuing on CDK 4/6 inhibitors with endocrine therapy (108). Although the elderly population with BC remains excluded from most clinical trials, the beneficial effects of combined therapy can still be observed in these patients in some studies (109). The EMERALD trial (ClinicalTrials.gov identifier: NCT03778931) showed that elacestrant was more effective than aromatase inhibitor in BC patients who had advanced following prior co-therapy with CDK4/6 inhibitors and endocrine therapy (110, 111). The TRINITI-1 trial (ClinicalTrials.gov identifier: NCT02732119) with aromatase inhibitors-resistant patients following progression after treatment with CDK4/6 inhibitors showed clinical benefits of triplet treatment combination of ribociclib, everolimus, and exemestane; further supporting the idea that therapy targeting both CDK4/6, PI3K, and mTOR may show therapeutic benefits to CDK4/6 inhibitor resistant patients (112).
On-going clinical trials on patients with CDK4/6 inhibitors resistance.
Current target therapeutic drugs for BC focus mainly on the PI3K–AKT–mTOR signaling. In cell models, the use of PI3K inhibitor with palbociclib delayed the recommencement of S phase entry and abolished the accumulation of cyclin D1, consistent with the role of PI3K signaling in promoting cyclin D1 expression (49). Another study showed that the mTOR inhibitors synergized with CDK4/6 inhibitors to prevent the resumption of breast cancer proliferation, and this regimen induced a significant suppression of E2F genes (71). In addition, activated AKT recruits and activates more PI3K on the cell membrane. In turn, activated PI3K phosphorylates AKT, leading to a feedback amplification loop (113). Therefore, AKT inhibitors may become one of the treatment choices, especially when patients are PI3K-mutated or unresponsive to PI3K inhibitors. In the ongoing TAKTIC trial (ClinicalTrials.gov Identifier: NCT03959891), the efficacy of ipatasertib, an AKT-1 inhibitor, was evaluated in combination with endocrine therapy and palbociclib in HR-positive patients who had failed prior CDK4/6 inhibitor treatment. The results so far have suggested a possible intervention for CDK4/6 inhibitor-resistant patients (114).
Histone deacetylase (HDAC) can increase the activation of CDK4/6 and inhibit cell cycle arrest by suppressing p21 expression in CDK4/6 inhibitor-resistant tumors (101). In terms of this, HDAC inhibitors can inhibit BC proliferation (115), induce apoptosis (116), and suppress DNA repair (117), producing an anti-cancer effect. In the ACE trial (ClinicalTrials.gov Identifier: NCT02482753), the use of chidamide, an HDAC inhibitor, with exemestane significantly improved progression-free survival in CDK4/6 inhibitors-resistant patients (118).
While the anti-apoptotic protein B-cell lymphoma (BCL)-2 is highly responsive to estrogen (119), targeting BCL-2 may be a potential option for CDK4/6 inhibitor resistance. The ongoing VERONICA trial (ClinicalTrials.gov Identifier: NCT03584009) has indicated that BCL-2 inhibition, although not statically significant, may be expected to be a treatment option for CDK4/6 inhibitor-resistant patients (120).
Last but not least, CDK4/6 inhibitors increase the expression of PD-L1 raising the possibility of using CDK4/6 inhibitors as a priming approach to attract T cells into immune-cold, T cell-excluded tumors because pre-existing tumor-infiltrating CD8+ T cells are more likely to respond to anti-PD-1 (121). Studies using a MMTV-rtTA/tetO-HER2 transgenic mouse model have shown that CDK4/6 inhibitors may enhance the susceptibility of tumors to PD-1/PD-L1 blockade, through the promotion of the CD8+ T cell-mediated clearance of cancer cells (94). Further investigation has suggested that the addition of PD-L1 inhibitors to CDK4/6 inhibitors suppresses cell-cycle gene-mediated proliferation and increases MHC expression in BC cells. Such combination immunotherapy also increases the activation of T-cells, macrophages, and dendritic cells, leading to higher efficiency of antigen presentation (121). Several ongoing clinical trials using CDK4/6 inhibitors in combination with PD-L1 inhibitors, aromatase inhibitors, or selective estrogen-receptor degraders in hormone-positive BC patients (ClinicalTrials.gov Identifier: NCT02778685; NCT02778685; NCT03294694) may provide us with a better understanding of this immune-priming effects in the future. However, since cytotoxic T cells depend on CDKs [6], the long-term effects of CDK4/6 inhibitors on T cell proliferation and activation should be carefully monitored.
Traditional Chinese herbs. Because of the different mechanisms in BC resistance to CDK4/6 inhibitors, finding a new drug may be a more realistic approach to deal with this clinically significant malignancy. Traditional Chinese medicine (TCM) has been widely studied for cancer treatment in recent years. These include crude extracts or active compounds derived from plants (122).
For example, extracts of rhizome bollbostemmatis could induce DNA fragmentation in a three-dimensional BC culture (123). Germacrone extracted from Rhizma curcuma can induce apoptosis of doxorubicin-resistant MCF-7 cells (124). In addition, active compounds, such as ganoderic acid, emodin azide methyl-anthraquinone derivative, and quercetin can suppress BC cell proliferation and induce apoptosis (125, 126). Because many TCMs can modulate the host’s immunity, they may complement immunotherapy.
In addition, several reports have suggested that complementary use of TCMs during cytotoxic chemotherapy may reduce adverse effects, prolong patients’ survival, and prevent recurrence (127). However, despite no evidence regarding TCM treatment in CDK4/6 inhibitor-resistant BC, this has put forward a future potential therapeutic approach.
Other alternative drugs. Triazoles and their derivatives have also been used extensively as anti-cancer, anti-viral, anti-microbial, anti-inflammatory, and anti-oxidant agents (128). 1,2,3-triazole derivatives have displayed high cytotoxicity against doxorubicin-resistant MCF-7 cells (129). Because of the usefulness of triazoles and the flavonoid compound isolated from TCM, several triazole-bridged flavonoid dimers [such as Ac22(Az8)2 and Ac15(Az8)2] have recently been synthesized as a highly selective breast cancer resistance protein (BCRP) inhibitors (130, 131), revealing that triazole-based compounds might also be promising candidates for future BC treatment.
Conclusion
CDK4/6 inhibitors are currently the effectual standard therapy in combination with endocrine therapy in HR-positive BC. Despite that, issues regarding resistance to CDK4/6 inhibitors in a clinical setting is an inevitable issue. Current knowledge of the molecular mechanisms of CDK4/6 inhibitor resistance is far from complete, as it is based mainly on single-agent studies or cell line models. As such a resistance problem may arise from different intrinsic or acquired mechanisms, future studies on the current topic are therefore recommended to elucidate the mechanism. At the same time, newer therapeutic options or alternative drugs may require continuous development to combat resistance.
Footnotes
Authors’ Contributions
CMC and HYPL made substantial contributions to the concept, literature research and article writing. HYPL was involved in the production of figures and revising the article. All Authors gave their final approval of the version to be published.
Conflicts of Interest
The Authors declare that there are no conflicts of interest related to the manuscript.
Funding
This work was financially supported by Buddhist Tzu Chi Medical Foundation (Grant number: TCMMP110-01-03(112)). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Received September 15, 2023.
- Revision received October 17, 2023.
- Accepted October 18, 2023.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
