International Journal of
Molecular Sciences
Review
Hormone, Targeted, and Combinational Therapies for Breast
Cancers: From Humans to Dogs
Chiao-Hsu Ke 1,2 , Chao-Nan Lin 1,2,3
Citation: Ke, C.-H.; Lin, C.-N.; Lin, C.-S.
Hormone, Targeted, and
Combinational Therapies for Breast
Cancers: From Humans to Dogs. Int. J.
Mol. Sci. 2024, 25, 732.
https://doi.org/10.3390/ijms25020732
Academic Editor:
Rocío García-Becerra
Received: 18 November 2023
Revised: 2 January 2024
Accepted: 5 January 2024
Published: 5 January 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2024, 25, 732. https://doi.org/10.3390/ijms25020732
and Chen-Si Lin 4, *
1 Sustainable Swine Research Center, National Pingtung University of Science and Technology,
Pingtung 91201, Taiwan; f08629002@ntu.edu.tw (C.-H.K.); cnlin6@mail.npust.edu.tw (C.-N.L.)
2 Animal Disease Diagnostic Center, College of Veterinary Medicine, National Pingtung University of Science
and Technology, Pingtung 91201, Taiwan
3 Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science
and Technology, Pingtung 91201, Taiwan
4 Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University,
Taipei 10617, Taiwan
* Correspondence: cslin100@ntu.edu.tw; Tel.: +886-233-661-286
Abstract: Breast cancer (BC) is the most frequent cancer in women. In female dogs, canine mammary
gland tumor (CMT) is also the leading neoplasm. Comparative oncology indicates similar tumor
behaviors between human BCs (HBCs) and CMTs. Therefore, this review summarizes the current
research in hormone and targeted therapies and describes the future prospects for HBCs and CMTs.
For hormone receptor-expressing BCs, the first medical intervention is hormone therapy. Monoclonal
antibodies against Her2 are proposed for the treatment of Her2+ BCs. However, the major obstacle
in hormone therapy or monoclonal antibodies is drug resistance. Therefore, increasing alternatives
have been developed to overcome these difficulties. We systemically reviewed publications that
reported inhibitors targeting certain molecules in BC cells. The various treatment choices for humans
decrease mortality in females with BC. However, the development of hormone or targeted therapies in
veterinary medicine is still limited. Even though some clinical trials have been proposed, severe side
effects and insufficient case numbers might restrict further explorations. This difficulty highlights the
urgent need to develop updated hormone/targeted therapy or novel immunotherapies. Therefore,
exploring new therapies to provide more precise use in dogs with CMTs will be the focus of future
research. Furthermore, due to the similarities shared by humans and dogs, well-planned prospective
clinical trials on the use of combinational or novel immunotherapies in dogs with CMTs to obtain
solid results for both humans and dogs can be reasonably anticipated in the future.
Keywords: hormone therapy; targeted therapy; comparative oncology; canine mammary gland tumor
1. Introduction
In 2022, female breast cancer (BC) was the most commonly diagnosed cancer in
the United States, with an estimated 287,850 new cases (31%). BC is the second most
common cause of cancer-related deaths among females, corresponding to an estimated
61,360 deaths [1]. In breast tissues, the hormones estrogen and progesterone are the primary
regulators for proliferation and differentiation [2]. They exert cellular functions and activate
specific nuclear receptors, namely, estrogen receptors (ERs) and progesterone receptors
(PRs) [3]. Once activated, nuclear receptors directly regulate the transcription of genes
that promote cell proliferation and development [4]. BCs are heterogeneous and can be
divided into several subtypes according to their molecular and histological features [5].
At the molecular level, five subtypes have been identified: luminal A (ER/PR+, Her2−),
luminal B (ER/PR+, Her2+), human epidermal receptor 2 (Her2)-enriched (ER/PR−,
Her2+), triple-negative (ER/PR−, Her2−), and normal-like (ER/PR+, Her2−, Ki67−) [6].
These classifications provide important clues for treatment decision making [7]. At present,
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2024, 25, 732 2 of 26

the hormone receptors (HRs) ER and/or PR are expressed in about 70% of all BC patients.
Therefore, hormonotherapy is a major therapeutic strategy [8]. The most frequent medicines
are estrogen blockers and aromatase inhibitors (AIs), which prevent hormone production
from the ovaries [9]. Furthermore, in the case of BC expressing Her2, several antibodies
targeting tumors have been reported. These antibodies and/or analogs bind to different
sites of Her2 and disturb the proliferative signaling pathways in cancer cells [10] (Figure 1).
In recent years, comparative oncology has been widely proposed. Canine cancers share
several biological and pathological similarities with their human counterparts [11]. In addi-
tion to the spontaneous tumor initiation, the clinical similarities between human and canine
mammary tumors (CMTs) include hormonal etiology, the molecular characteristics of steroid
receptors, and the courses of the diseases [12]. In canines, as in humans, hormones (estrogen
and progesterone) moderate the growth and development of mammary glands and participate
in carcinogenesis [13]. Approximately 50% of CMTs are malignant [14–16], but recent studies
have shown an increasing trend in malignant tumors [17]. The prevalence varies by geo-
graphic location, and in countries where ovariectomy is not routinely performed, it is more
prevalent. In these countries, the prevalence of CMTs in female dogs is three-times higher than
that in women [18]. Although clinical staging and histological grading might help standardize
treatment protocols, no precision therapies are available in dogs with CMTs. Therefore, over
40% of dogs with CMTs die within one year of diagnosis [19]. This difficulty highlights the
urgent need to develop updated therapeutic protocols and targeted therapies.
Though BC is still the most frequently diagnosed type of tumor, BC mortality rates
have gradually decreased in recent years. This decline indicates the progress of advances
in anti-cancer therapies [20]. Many studies have found highly similar tumor behaviors in
HBCs and CMTs. This review summarizes the current hormone, target, and combinational
Int. J. Mol. Sci. 2024, 25, x FOR PEER therapies
REVIEW in HBCs. The comparative oncology and published clinical and 3basic of 28 data on
established anti-tumor therapies in CMTs are also described here.

Figure 1. The estrogen receptor, progesterone receptor, and Her2 pathways in breast cancer cells.
Figure 1. The estrogen receptor, progesterone receptor, and Her2 pathways in breast cancer cells.
Two steroid hormones, estrogen and progesterone, directly penetrate through the cell membrane
Two steroid
and then bindhormones, estrogen and
to the corresponding progesterone,
receptor monomer, directly penetrate
which is throughand
then dimerized thetransported
cell membrane and
then bind to the corresponding receptor monomer, which is then dimerized and transported into
into the nucleus to activate signaling. The extracellular estrogens bind to the membrane receptors
and activate the PI3K/AKT signaling. Her2 promotes cell survival and proliferation by activating
the nucleus to activate signaling. The extracellular estrogens bind to the membrane receptors and
MAPK and PI3K/AKT pathways. Activation of RAS kinase triggers the activation of the MAPK sig-
activate the PI3K/AKT signaling. Her2 promotes cell survival and proliferation by activating MAPK
naling cascade, which includes the phosphorylation of RAF, MEK, and MAPK. Upon phosphoryla-
tion by MEK, MAPK translocates into the nucleus, where it regulates the transcription of genes in-
volved in cell proliferation. Furthermore, progesterone-induced activation of c-Src also provides a
hormone-induced MAPK phosphorylation signaling, thus modulating tumorigenesis. c-Src, proto-
oncogene tyrosine-protein kinase Src; ER, estrogen receptor; Her2, human epidermal growth factor re-
ceptor2; mER, membrane estrogen receptor; mTOR, mammalian target of rapamycin; MAPK, mitogen-
activated protein kinase; MEK, MAPK kinase; PI3K, phosphatidylinositol-3-kinase; PR, progesterone re-
Int. J. Mol. Sci. 2024, 25, 732 3 of 26

and PI3K/AKT pathways. Activation of RAS kinase triggers the activation of the MAPK signaling
cascade, which includes the phosphorylation of RAF, MEK, and MAPK. Upon phosphorylation by
MEK, MAPK translocates into the nucleus, where it regulates the transcription of genes involved in
cell proliferation. Furthermore, progesterone-induced activation of c-Src also provides a hormone-
induced MAPK phosphorylation signaling, thus modulating tumorigenesis. c-Src, proto-oncogene
tyrosine-protein kinase Src; ER, estrogen receptor; Her2, human epidermal growth factor receptor2;
mER, membrane estrogen receptor; mTOR, mammalian target of rapamycin; MAPK, mitogen-
activated protein kinase; MEK, MAPK kinase; PI3K, phosphatidylinositol-3-kinase; PR, progesterone
receptor; RAF, Rapidly Accelerated Fibrosarcoma; RAS, Rat sarcoma protein; SOS, Son of sevenless.

2. Hormone Therapies
2.1. Selective Estrogen Receptor Modulators
Numerous hormone-related drugs were approved from 1977 to recent years (Figure 2).
Among these agents, tamoxifen has solid evidence that supports its usefulness as a hor-
mone therapy in treating early breast cancers [21] and metastatic BC in postmenopausal
females [22]. The relatively low toxicity of tamoxifen has led to multiple clinical trials. The
first single-arm phase 2 trial of tamoxifen was initiated in 1969, in which 22% of recruited
patients responded to treatment [23]. Tamoxifen was then compared with other medical
interventions in several clinical trials. Although no significant differences in survival were
found compared with other agents or medicines, patients with tamoxifen treatment re-
ported less severe side effects [24]. Several clinical trials produced similar findings. One
clinical trial evaluated patients with receptor-positive BC, who were randomized to five
years of tamoxifen or placebo after surgery. In females treated with tamoxifen, the disease-
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 of 28
free survival (DFS) rate was significantly higher than that of the placebo group [25]. In
a clinical trial, 1285 patients with BC underwent tumorectomy with axillary lymph node
clearance and were treated with tamoxifen for two years. That study demonstrated that
clearance
a two-year and were treated
course with tamoxifen
of tamoxifen for two
therapy was years.
highly That study
associated demonstrated
with a reductionthat
in arecur-
two-year course of tamoxifen therapy was highly associated with a reduction in
rence and an improvement in overall survival time [26]. Randomized trials of tamoxifen recur-
rence and an improvement in overall survival time [26]. Randomized trials of tamoxifen
versus other selective estrogen receptor modulators (SERMs) were conducted in hopes of
versus other selective estrogen receptor modulators (SERMs) were conducted in hopes of
providing greater clinical efficacies than those of tamoxifen. Unfortunately, these agents,
providing greater clinical efficacies than those of tamoxifen. Unfortunately, these agents,
idocifene and toremifene, turned out to be equally or less effective than tamoxifen in treat-
idocifene and toremifene, turned out to be equally or less effective than tamoxifen in treat-
ing advanced BC [27,28]. However, persistent therapy has been debated, especially if the
ing advanced BC [27,28]. However, persistent therapy has been debated, especially if the
duration exceeds five years. Therefore, tamoxifen remains the leading SERM, and interest
duration exceeds five years. Therefore, tamoxifen remains the leading SERM, and interest in
in developing analogs with better anti-tumor activities and reduced toxicity continues [29].
developing analogs with better anti-tumor activities and reduced toxicity continues [29].

Figure
Figure 2.
2.FDA-approved
FDA-approvedhormone
hormonetherapies forfor
therapies breast cancers.
breast cancers.

2.2. Aromatase
2.2. AromataseInhibitors
Inhibitors
In postmenopausal
In postmenopausalfemales,
females,estrogen
estrogenisis poorly
poorly produced
producedby by ovarian
ovariantissues
tissues and
and is
is pre-
dominantly synthesized
predominantly from
synthesized nongranular
from sources
nongranular by the
sources by aromatase
the aromataseenzyme. Aromatase
enzyme. Aroma-is dis-
tributed
tase in several in
is distributed tissues, including
several tissues, subcutaneous fat, liver, muscle,
including subcutaneous andmuscle,
fat, liver, breast cancer cells [30].
and breast
cancer cellsinhibition
Therefore, [30]. Therefore, inhibition
of aromatase hasofbeen
aromatase
proposedhas asbeen proposedstrategy
a treatment as a treatment strat-
for breast cancer.
egy forThebreast
firstcancer.
two generations of aromatase inhibitors (AIs) were effective but simulta-
The first
neously twosevere
caused generations of aromatase
side effects inhibitors
because (AIs)
they also were effective
inhibited steroid buthormones,
simultane-such
ously caused severe side effects because they also inhibited steroid
as cortisol and aldosterone. Due to the increased specificity of third-generation hormones, suchAIs,
as the
cortisol and aldosterone. Due to the increased specificity of third-generation AIs, the se-
lective inhibition of aromatase did not interfere with the other steroid biosynthesis. These
novel AIs, anastrozole, letrozole, and exemestane, have comparable efficacy and have sig-
nificantly better activities than tamoxifen [31]. These qualities led to the introduction of
the first trial that randomized postmenopausal females to tamoxifen or anastrozole. The
Int. J. Mol. Sci. 2024, 25, 732 4 of 26

selective inhibition of aromatase did not interfere with the other steroid biosynthesis. These
novel AIs, anastrozole, letrozole, and exemestane, have comparable efficacy and have
significantly better activities than tamoxifen [31]. These qualities led to the introduction
of the first trial that randomized postmenopausal females to tamoxifen or anastrozole.
The results showed that anastrozole provided a superior DFS [32] and overall survival
time (OST) to those of tamoxifen [33]. In a head-to-head clinical trial, the well-tolerated
efficacy of letrozole was compared with that of tamoxifen. This phase 3, double-blind trial
recruited 8010 patients and compared their 5-year survival rates. The results showed that
the 5-year survival rate was higher in the letrozole group than in the tamoxifen group.
Furthermore, some side effects, including thromboembolism, endometrial cancer, and
vaginal bleeding, were more common in the tamoxifen group [34]. Therefore, AIs are
well-tolerated drugs with manageable side effects in general. The common side effects
include musculoskeletal symptoms, osteopenia, osteoporosis, and a heightened risk of
fracture [35]. However, the fracture risk appears to increase when patients are undergoing
active treatment. The differences in fracture risk between cases with and without AIs were
not significantly different after the treatment was completed [36]. Furthermore, because
of the lack of partial estrogenic effects, AIs might not enhance the risks of endometrial
cancers and thromboembolisms, as tamoxifen does [35,36]. Based on the clinical relevance
of anastrozole and letrozole, the FDA approved the use of these two AIs for the initial
therapy of hormone-sensitive breast cancer. A clinical trial that compared the efficacy and
safety of letrozole and anastrozole was reported. That study included 4136 postmenopausal
patients and found that letrozole and anastrozole had similar efficacies [37].
Although AI therapies have met with success in BC in past decades, patients with ta-
moxifen therapy usually experienced recurrence because of drug resistance and developed
side effects. The focus then shifted to combinational therapy, and AIs have been regarded
as sequential therapy after tamoxifen treatment [38,39]. The clinical results suggest that
postmenopausal patients benefit from a 2-year course of tamoxifen followed by a 3-year
course of anastrozole. A decrease in risk for recurrence was noted in patients receiving
tamoxifen and anastrozole compared with those receiving tamoxifen only [38]. This result
suggested that the sequential use of AIs and tamoxifen provided clinical benefits in BC
patients (Table 1).

Table 1. Previous clinical trials reporting hormone therapies against breast cancers. BC, breast cancer;
HR+, hormone receptor positive; PFS, progress-free survival; AEs, adverse effects.

References or
Drugs Indication Efficacy
Accession Number
Late or recurrent carcinoma of the breast 22% of patients responded to treatment [23]
Tamoxifen Hormone receptor-positive BC Increased PFS [25]
Early breast cancer Increased PFS and decreased death [26]
Patients with invasive operable breast cancer who Anastrozole is an effective and well-tolerated with
Anastrozole [32,33]
had completed primary therapy fewer AEs than tamoxifen.
Letrozole reduced the risk of recurrent diseases as
Letrozole HR+ BC (phase 3 trial) NCT00004205
compared with tamoxifen.

2.3. Resistance to Hormone Therapies

Several molecular mechanisms have been proposed for hormone therapy resistance.
Pathways that lead to estrogen-independent sustained activation of estrogen receptors render
patients resistant to these hormone drugs: the acquired mutation of ESR1, upregulation of miR-
NAs, and autophagy. The wild-type ESR1 is bound by an estrogen ligand to enable coactivator
recruitment, whereas mutated ESR1 (ESR1-MUT) is constitutively active and unaffected by the
AI depletion of estrogen [40,41]. Therefore, in the absence of a ligand, ESR1-MUT has increased
stability in the active conformation, increased binding to coactivators, and decreased prote-
olytic degradation [42,43]. The upregulation of miRNAs can be associated with anti-hormone
therapies through the activation of alternate pathways or the inhibition of ER expression.
Int. J. Mol. Sci. 2024, 25, 732 5 of 26

MiR-155 stimulated the activation of STAT3 signaling pathways, which is associated with
cell survival, thus leading to resistance to tamoxifen [44,45]. MiR-221 and miR-222 regulated
several oncogenic pathways, such as Wnt, and their high expression was found to be correlated
with tamoxifen resistance [46,47]. In breast cancer cells, autophagy allows cells to survive under
endoplasmic reticulum stress, which increases the drug resistance following anti-hormone
therapies [48]. For example, the mammalian target of rapamycin (mTOR) kinase is the major
component of two mTOR complexes, mTORC1 and mTORC2. mTORC1 is a major down-
stream target of the PI3K/AKT pathway and a negative regulator of autophagy. Therefore, the
downregulation of mTORC1 by PI3K/AKT signaling inhibition results in increased autophagy,
which facilitates the resistance to tamoxifen in breast cancer cells [49]. Abnormal interactions
between the cyclin D-CDK 4/6-retinoblasoma (Rb) pathways, aberrant modification by histone
deacetylases (HDACs), and dysregulation of the cellular microenvironment and immune
responses also cause resistance to hormone therapy in breast cancer [50]. Thus, these studies
on therapy resistance trigger the development of new drugs/agents targeting these pathways.
Int. J. Mol. Sci. 2024, 25, x FOR PEER Although
REVIEW the newly developed drugs fail to reverse drug resistance, they are expected 6 of 28 to
reinstate sensitivity to endocrine therapy through the inhibition of other pathways or targets.

3. Targeted Therapies
3. Targeted Therapies
In recent years, targeted therapies, including Her-2-targeted and small molecular
In recent years, targeted therapies, including Her-2-targeted and small molecular
agents, have gradually been approved by the FDA in the treatment of BC (Figure 3).
agents, have gradually been approved by the FDA in the treatment of BC (Figure 3). A
A summary of the included studies is shown in Table 2.
summary of the included studies is shown in Table 2.

Figure 3. Timeline of FDA-approved targeted therapies for anti-breast cancer agents.

Figure 3. Timeline of FDA-approved targeted therapies for anti-breast cancer agents.
Table 2. Previous clinical trials using small molecular inhibitors in treating breast cancers.
Table 2. Previous clinical trials using small molecular inhibitors in treating breast cancers.
Reference or
Medical Intervention Phase Indication Efficacy Reference or
Accession
Medical Intervention Phase Indication Efficacy Accession
Number
Number
Buparlisib plus fulvestrant: well-toler-
Buparlisib plus fulvestrant: well-tolerated.
ated.increased alanine
Serious AEs: NCT01610284
3 HR+/Her2− BC NCT01610284
Buparlisib 3 HR+/Her2− BC Serious AEs: increased alanine
aminotransferase ami-
and aspartate (BELLE-2)
aminotransferase (BELLE-2)
notransferase and aspartate aminotrans-
Buparlisib plus fulvestrant: increased PFS
Seriousferase
AEs: increased alanine
NCT01633060
3 HR+/Her2− BC Buparlisib aminotransferase, increased
plus fulvestrant: aspartate
increased (BELLE-3)
aminotransferase, hyperglycemia,
PI3K inhibitors
Buparlisib PFS
hypertension
Serious AEs: increased alanine ami- NCT01633060
[51]
3 Her2−
2/3 HR+/Her2− BCBC No difference in PFS (compared to paclitaxel)
notransferase, increased aspartate ami- (BELLE-4)
(BELLE-3)
2 HR+/Her2− BC No difference in PFS (compared to fulvestrant) NCT01437566
Pictilisib notransferase, hyperglycemia,
No significant hyperten-
benefit from adding pictilisib NCT01740336
PI3K inhibitors 2 HR+/Her2− BC
sion
to paclitaxel (PEGGY)
1 HR+/Her2− BC No difference in Well-tolerated
PFS (compared to NCT01219699
[51]
Alpelisib 2/3 1b Her2− BC
ER+/HT-resistant BC Well-tolerated NCT01219699
3 HR+/Her2− BC paclitaxel) prolonged PFS
Alpelisib-fulvestrant (BELLE-4)
NCT02437318
No difference in PFS (compared to ful-
2 HR+/Her2− BC NCT01437566
vestrant)
Pictilisib
No significant benefit from adding pic- NCT01740336
2 HR+/Her2− BC
tilisib to paclitaxel (PEGGY)
1 HR+/Her2− BC Well-tolerated NCT01219699
Alpelisib 1b ER+/HT-resistant BC Well-tolerated NCT01219699
Int. J. Mol. Sci. 2024, 25, 732
Medical Intervention
AKT inhibitors
mTOR inhibitors
CDK4/6 inhibitors
HDAC inhibitors
PARP inhibitors
6 of 26
Table 2. Cont.
Reference or
Phase Indication Efficacy Accession
Number
NCT01992952
2 ER+/Her2− BC Increased PFS
Capivasertib (FAKTION)
2 Metastatic TNBC Increased PFS and OST [52]
Everolimus significantly increased
2 ER+ BC NCT00107016
letrozole efficacy.
Everolimus Everolimus plus tamoxifen increased clinical
2 HT-resistant BC [53]
benefit rate, PFS, and OST.
3 HR+ BC Everolimus plus an AI increased PFS. NCT00863655
3 HR+/Her2− BC Palbociclib plus letrozole increased PFS. NCT01740427
Palbociclib 3 HR+/Her2− BC Palbociclib plus fulvestrant increased PFS. NCT01942135
Ribociclib plus letrozole increased PFS but
3 HR+/Her2− BC NCT01958021
Ribociclib a higher rate of myelosuppression was found.
Ribociclib plus fulvestrant increased PST
3 HR+/Her2− BC NCT02422615
and OST
[54]
2 HR+/Her2− BC Well-tolerated
Abemeciclib (MONARCH 1)
Improved PFS, ORR, and a tolerable safety NCT02107703
3 HR+/Her2− BC
profile were found. (MONARCH 2)
Tucidinostat plus exemestane improved PFS;
Tucidinostat 3 HR+ BC however, grade 3–4 hematological AEs were NCT02482753
more common.
Well-tolerated and increased PFS were found,
Olaparib 3 Her2− BC but there was no statistically significant NCT02000622
improvement in OST.
Advanced BC with Talazoparib increased PFS; however, no
Talazoparib 3 NCT01945775
BRCA1/2 mutation significant increased OST was found.
3.1. Her2-Targted Therapies
3.1.1. Monoclonal Antibodies
Human epidermal growth factor receptor-2 (Her2) is overexpressed in 15% to 20% of
early-stage BCs [55], which makes it an ideal target for medical intervention. A humanized
monoclonal antibody against Her2, trastuzumab (Herceptin), was first approved by the FDA
for Her2+ BC patients. Trastuzumab binds to the extracellular domain of Her2, suppresses
cell cycle arrest, and mediates antibody-dependent cell-mediated cytotoxicity [56]. Because
of its specificity to Her2, trastuzumab provides remarkable success in treating Her2+ BC
patients. Clinical studies have shown that a combination of trastuzumab and chemotherapy
produces more favorable outcomes than chemotherapy alone [57,58]. Therefore, trastuzumab
was recommended as the treatment choice for patients with Her2+ BC, which revolutionized
the outcomes for patients suffering from this disease. However, although trastuzumab provides
improved outcomes, some patients develop drug resistance and disease relapse [59].
Resistant mechanisms to trastuzumab encompass various factors, including steric
effects, alternative elevations of different tyrosine kinase receptors, and intracellular al-
terations. Steric effects refer to structural mutations in the Her2 protein, resulting in the
inhibition of trastuzumab binding. This leads to the overexpression of other tyrosine kinase
receptors, thereby evading the Her2 signaling pathway blockage [60]. In other words,
during this mutation, a truncated isoform with constitutive kinase activity emerges, by-
passing trastuzumab’s neutralizing effects [61]. Notably, trastuzumab fails to prevent Her3
dimerization, potentially leading to trastuzumab-mediated inhibitory signaling pathways
being compromised by the overexpression of Her3 [62]. Suppression of the Her2 signaling
pathway by trastuzumab prompts cancer cells to compensate through increased reliance
on Her3 signaling [63]. Last, understudied mechanisms contributing to trastuzumab
resistance involve intracellular alterations. Research indicates that patients with PTEN
loss exhibit significantly lower overall responses to trastuzumab compared to those with
wild-type PTEN [64]. Clinical findings suggest that trastuzumab-treated patients lack-
Int. J. Mol. Sci. 2024, 25, 732 7 of 26

ing PIK3CA-activating mutations experience improved PFS compared to their mutated

counterparts [65]. In vitro studies further demonstrate increased trastuzumab resistance in
Her2+ breast cancer cells with PIK3CA-activating mutations [66]. These results highlight
the role of the constitutive activation of the PI3K/AKT pathway, stemming from PTEN de-
ficiency or PIK3CA-activating mutations, in contributing to trastuzumab resistance. While
the detailed mechanisms necessitate further elucidation, the emergence of trastuzumab
resistance and disease relapse in several breast cancer patients underscores the urgency for
the development of novel medical interventions.
Previous studies suggested that antibodies targeting multiple domains in Her2 could
potentially elicit synergistic anti-neoplastic effects [67]. Therefore, the development of
the second-generation humanized monoclonal antibody, pertuzumab, was proposed. Per-
tuzumab binds to Her1, Her3, and Her4, effectively inhibiting downstream tumor signaling
pathways [68]. Recognizing the synergistic effects demonstrated by trastuzumab and
pertuzumab, the idea of combining these two monoclonal antibodies with chemotherapy
gained traction, leading to the initiation of clinical trials. In a clinical trial, patients with
Her2+ BC were randomized into two groups: one receiving pertuzumab, trastuzumab, and
docetaxel (pertuzumab combination), and the other receiving trastuzumab and docetaxel
(placebo combination). The results from this trial revealed a significant improvement in
median overall survival, extending to 56.5 months with the addition of pertuzumab, high-
lighting the promising efficacy of this combined therapeutic approach [69]. Another phase
2 clinical study involving women with Her2+ BC corroborated these findings, showing that
patients administered pertuzumab, trastuzumab, and docetaxel exhibited a significantly
enhanced treatment response rate compared to those receiving trastuzumab and docetaxel
alone [70]. These clinical trials collectively underscore the enhanced survival and treatment
responses achieved through pertuzumab and trastuzumab combinational therapies.
In light of these discoveries, the field of developmental therapeutic antibodies has wit-
nessed a surge in momentum, propelling the clinical advancement of novel HER2-targeted
monoclonal antibodies. Margetuximab is a chimeric anti-Her2 monoclonal antibody en-
gineered with an Fc domain modification to enhance binding to FcγRIIIa and reduce
affinity for its inhibitory counterpart, FcγRIIB [71]. Antibody-dependent cytotoxicity as-
says have demonstrated the heightened activity of margetuximab against Her2+ cancer
cells compared to trastuzumab surrogates in donors possessing a low-affinity variant of
FcγRIIIa [71]. A phase 1 clinical trial recruited patients with metastatic BC. Notably, 11 out
of 28 BC patients exhibited tumor reduction, all of whom had previously received at least
one Her2-targeted therapy [72]. The clinical efficacy of margetuximab is currently under
scrutiny in a phase 3 trial, comparing margetuximab combined with chemotherapy against
trastuzumab plus chemotherapy. This trial involves patients with Her2+ metastatic BC who
have experienced progression following Her2-targeted therapy. Preliminary results from
536 patients with Her2+ metastatic BC revealed that margetuximab led to an improvement
in PFS compared to trastuzumab (median of 6 months vs. 5 months) [73].

3.1.2. Trastuzumab Biosimilars

Clinical trials have demonstrated the ideal efficacy and safety of trastuzumab; however,
over 90% of patients in several low-income countries had no access to trastuzumab owing
to economic restraints [74]. A lower-cost alternative, trastuzumab biosimilars, has the poten-
tial to increase accessibility in the treatment of Her2+ BC. Biosimilars are biological agents
that are created from living cells, contain versions of the reference products’ substances, and
have similar pharmacokinetic and pharmacodynamic properties to the original products [10].
Although they may have minor differences from their parental components, biosimilars usu-
ally share similar biological characteristics, efficacy, and safety [75]. Several trastuzumab
biosimilars have undergone phase 3 trials in patients with early and metastatic BC [76–78].
A multicenter phase 3 randomized clinical trial assessed the efficacy of trastuzumab and a
trastuzumab biosimilar (HD201) in patients with Her2+ BC. HD201 demonstrated equiva-
lence to the referent trastuzumab in terms of efficacy in the response rate and a similar safety
Int. J. Mol. Sci. 2024, 25, 732 8 of 26

profile [76]. Similar results were proposed in a clinical trial recruiting patients with Her2+
metastatic BC. A proposed trastuzumab biosimilar resulted in an equivalent overall response
rate compared with trastuzumab [77]. A phase 3 randomized clinical trial included women
with Her2+ early BC and evaluated the efficacy and safety between trastuzumab and a
trastuzumab biosimilar (ABP980). The results showed that the trastuzumab biosimilar
(ABP980) shared high similar efficacy and safety with trastuzumab [78]. Based on that
success, five trastuzumab biosimilars have been approved by the US FDA, with additional
candidates still underdeveloped. Trastuzumab-dkst (MYL1401O) was the first to receive
FDA approval in 2017, followed by tastuzumab-pkrb (CT-P6) in 2018 and trastuzumab-dttb
(SB3), trastuzumab-qyyp (PF-05280015), and trastuzumab-anns (ABP980) in 2019 [10]. After
FDA approval, one study evaluated the effectiveness of MYL1401O in patients with early
Her2+ BC. That study demonstrated similar response rates in patients receiving MYL1401O
plus chemotherapy or trastuzumab plus chemotherapy [79]. Taken together, the clinical
relevance of trastuzumab biosimilars has been revealed, showing its value as an excellent
cost-effective alternative to trastuzumab.
The progress of biosimilars presents significant potential for improving access to biological
therapies and curbing healthcare expenses. However, tackling these challenges is pivotal for
promoting a more well-rounded exploration of biosimilars. Thus, a comprehensive strategy
is essential to surmount economic constraints in the biosimilars and antibody landscapes.
Collaborative efforts involving regulators, industry stakeholders, and healthcare professionals
might pave the way for a more sustainable exploration of biosimilars.

3.1.3. Tyrosine Kinase Inhibitor

Lapatinib is a reversible inhibitor of EGFR (Her1) and Her2 [80,81]. Therefore,
trastuzumab and lapatinib have complementary mechanisms of action and synergistic
anti-tumor activity in Her2+ BC. A phase 3 clinical trial (NeoALTTO) recruited patients
with Her2+ tumors greater than 2 cm in diameter. Significantly higher response rates were
found in patients receiving trastuzumab plus lapatinib than in those with trastuzumab
alone [82]. An extended study reported, however, that overall survival and PFS were
similar between these groups [83]. In a phase 2 EORTC trial, Her2+ BC patients benefited
from trastuzumab and lapatinib therapy with an increased response rate. However, PFS
did not increase compared with the control arm [84]. Although lapatinib plus trastuzumab
increased pathological complete response and improved some outcomes for Her2+ BC
patients, these therapies seem to have mild impacts on extended overall survival. These
findings might result from trastuzumab resistance, but lapatinib is reported to overcome
trastuzumab resistance in patients [80,81]. Therefore, the clinical utility of lapatinib in
combination with other drugs for trastuzumab-resistant BC has been proposed [85]. A clin-
ical trial demonstrated that women with Her2+-advanced BC benefited from lapatinib
plus capecitabine, which significantly increased PFS. The median PFS was 8.4 months
in the combination therapy group, as compared with 4.4 months in the control arm [86].
The efficacy and safety of lapatinib plus letrozole were well tolerated. Postmenopausal
women with HR+/HER2+ BC were randomized to treatment with lapatinib plus letrozole
versus letrozole plus placebo. The addition of lapatinib to letrozole was well tolerated and
resulted in a significantly increased PFS and overall response rate [87,88]. Taken together,
lapatinib in combination with capecitabine was superior to capecitabine alone, and the
lapatinib–letrozole combination was more efficacious than letrozole alone [10]. Thus, these
combinations have been approved by the FDA for BC treatment.
In contrast to lapatinib, neratinib is an irreversible pan-HER TKI that targets Her1,
Her2, and Her4 [89]. A phase 1/2 clinical study assessed the dose-escalation, safety, efficacy,
and pharmacokinetics in patients with Her2+ BC. Though a high response rate was found
(73%) in patients receiving neratinib plus paclitaxel, a high toxicity rate was also identified,
suggesting the need for a dose reduction [90]. A multicenter, randomized, phase 3 trial
(ExteNET) was then conducted to assess the benefits and risks of neratinib. Patients who
had Her2+ early-stage BC and had experienced trastuzumab therapy were recruited and
Int. J. Mol. Sci. 2024, 25, 732
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 109of
of 28
26

randomly assigned to receive neratinib or placebo. This trial demonstrated that neratinib
that neratinib for 12 months significantly improved 2-year invasive PFS when given to
for 12 months significantly improved 2-year invasive PFS when given to women with
women with Her2-positive
Her2-positive breast cancer breast cancer after therapy
after trastuzumab trastuzumab
[91]. therapy [91].
In
In clinical practice, lapatinib and neratinib are the only
clinical practice, lapatinib and neratinib are the only TKIs
TKIs approved
approved byby the
the FDA
FDA
for
for Her2+ BC patients. Several TKI candidates, such as pyrotinib (approved by the Chinse
Her2+ BC patients. Several TKI candidates, such as pyrotinib (approved by the Chinse
regulatory authority) and
regulatory authority) and tucatinib,
tucatinib, are
are still
still under
under investigation.
investigation. Whether
Whether treatment
treatment with
with
aa single
single multi-kinase
multi-kinase inhibitor
inhibitor or combined treatment with other anti-tumor drugs,
or combined treatment with other anti-tumor drugs, TKIs TKIs
have
have aa high
high potential in the
potential in the treatment
treatment ofof trastuzumab-resistant
trastuzumab-resistant BC BC patients.
patients.

3.2. PI3K/AKT/mTOR Pathway Target Inhibitors

3.2.1. PI3K Inhibitors
dysregulation in the phosphoinositide
Genetic dysregulation 3-kinase (PI3K)
phosphoinositide 3-kinase (PI3K) pathway, known as
mutations of the PI3K catalytic alpha subunit (PIK3CA), is usually observed in BC. The
first-generation PI3K
first-generation PI3K inhibitors
inhibitors (PI3Ki)
(PI3Ki) are
are known
known asas pan-inhibitors
pan-inhibitors of
of PI3K, as they
PI3K, as they target
target
all four
all four isoforms of class
isoforms of class II PI3Ks
PI3Ks (α,
(α, β, γ, and
β, γ, δ) [92,93].
and δ) [92,93]. Because
Because these
these inhibitors
inhibitors provide
provide
widely
widely inhibitory
inhibitory activities,
activities, they
they lead
lead to
to aa high
high prevalence
prevalence of
of side
side effects,
effects, which
which limit
limit their
their
therapeutic purposes and cause treatment difficulties [94–96]. The receptor
therapeutic purposes and cause treatment difficulties [94–96]. The receptor mechanisms mechanisms
involved in
involved in the
the progression
progression of of BC
BC are
are summarized
summarized in in Figure
Figure 4.
4.

Figure
Figure 4.
4. Crosstalk
Crosstalk between
between signaling
signaling and
and pharmacological
pharmacological mechanisms
mechanisms of of hormone
hormone therapies
therapies and
and
targeted agents. AIs, aromatase inhibitors; CDK, cyclin-dependent kinases; EGFR,
targeted agents. AIs, aromatase inhibitors; CDK, cyclin-dependent kinases; EGFR, epidermal epidermal
growth
growth factor receptor; ER, estrogen receptor; Her2, human epidermal growth factor receptor2;
factor receptor; ER, estrogen receptor; Her2, human epidermal growth factor receptor2; Her3, human
Her3, human epidermal growth factor receptor3; mER, membrane estrogen receptor; mTOR, mam-
epidermal growth factor receptor3; mER, membrane estrogen receptor; mTOR, mammalian target
malian target of ra-pamycin; MEK, mitogen-activated protein kinase; PI3K, phosphatidylino-sitol-
of ra-pamycin;
3-kinase; MEK, mitogen-activated
RAF, Rapidly protein kinase;
Accelerated Fibrosarcoma; RAS,PI3K, phosphatidylino-sitol-3-kinase;
Rat sar-coma RAF,
protein; Rb, retinoblastoma;
Rapidly selective
SERMs, Accelerated Fibrosarcoma;
estrogen receptor RAS, Rat sar-coma
modulators; protein;
SOS, Son Rb, retinoblastoma;
of sevenless; SERMs,
TKI, tyrosine kinaseselective
inhibi-
estrogen receptor modulators; SOS, Son of sevenless; TKI, tyrosine kinase inhibitor.
tor.
Int. J. Mol. Sci. 2024, 25, 732 10 of 26

In BC, PIK3CA-activating mutations occur in about 40% of cases, and the pan-PI3Ki
most commonly used are buparlisib and pictilisib. A phase 3 clinical trial investigated the
efficacy and safety of buparlisib plus fulvestrant in HR+/Her2− postmenopausal patients,
whose BC was progressive during or after treatment with AIs. These patients were then
randomized to receive buparlisib plus fulvestrant or placebo. However, the trial demonstrated
relatively mild effects on PFS, and many patients receiving buparlisib treatment withdrew
from the trial due to the severe side effects. These adverse effects included higher transaminase
levels, hyperglycemia, rash, and mood disorders that influenced the potential benefits of
therapy [97]. The abilities of buparlisib plus fulvestrant were then tested in another trial. The
results showed that combinational therapy was correlated with improved PFS, especially
in those with PIK3CA mutations [98]. In the phase 2/3 clinical trial, the combination of
buparlisib plus paclitaxel in BC patients as first-line treatment was evaluated. Unfortunately,
combinational therapy did not improve the PFS of patients, as compared with paclitaxel alone.
Furthermore, buparlisib treatment led to a higher frequency of serious side effects, including
diarrhea, rash, hyperglycemia, and nausea [51]. The discrepancy between these trials suggested
that the effects and the tolerability of buparlisib still need to be elucidated.
Pictilisib, an orally delivered drug, is a pan-PI3Ki, which shares similarly high efficacy
and side effects with those of buparlisib. A randomized, double-blind, phase 2 clinical trial in-
vestigated the efficacy and safety of pictilisib plus fulvestrant in HR+/Her2− postmenopausal
patients but found no difference in median PFS in the combination treatments. However,
patients with pictilisib suffered from drug toxicity, rash, diarrhea, increased transaminase level,
hyperglycemia, pneumonitis, broncho-pneumonitis, and fatigue. These severe side effects
might limit the treatment effectiveness of pictilisib [99]. Another phase 2 randomized placebo-
controlled clinical trial failed to meet the primary endpoint. This study evaluated the benefit of
pictilisib plus paclitaxel versus paclitaxel alone in HR+/Her2− BC patients. No significant
differences in PFS and overall responses were found, but high toxicity was induced by
the pictilisib intervention. Thus, the severe side effects caused by pictilisib resulted in
discontinuation of the treatment [100].
Due to the low specificity of pan-PI3Ki, which usually causes considerable adverse
effects, PI3K isoform-specific inhibitors were gradually proposed. Alpelisib and taselisib
have stronger inhibitory effects and lower incidence of adverse events than pan-PI3Ki.
Alpelisib was the first oral inhibitor approved by the US FDA, and it can specifically target
the p110α isoform of wild-type PI3Kα [101,102]. A phase 1 trial was primarily carried
out. Patients with BC and PKI3CA mutations were recruited and demonstrated high
sensitivity to a single use of alpelisib. Frequent side effects included hyperglycemia, nau-
sea, rash, diarrhea, fatigue, and mucositis, but a well-tolerated effect was observed. The
median PFS was increased by 5.5 months among all the PIK3-mutated BC patients [103].
A phase 1b clinical study showed an increased median PFS in patients with PKI3CA al-
terations [104]. These findings triggered the motivation to conduct clinical trials on a
larger scale. A phase 3 randomized trial was conducted to assess the safety and efficacy
of alpelisib plus fulvestrant versus fulvestrant alone in postmenopausal women with
lumina BC. In the PKI3CA-mutated group, the median PFS of combination therapy sig-
nificantly increased compared with that of fulvestrant alone [95,105]. However, alpelisib
demonstrated a high incidence of side effects, such as hyperglycemia, diarrhea, nausea, and
rash. Due to these issues, 25% of patients in this trial shifted to fulvestrant therapy [106].
Current research and innovative strategies in the field of PI3K inhibitors are actively
working towards mitigating adverse effects and enhancing the safety profile of PI3K
inhibitors. However, their clinical application has faced challenges due to side effects that
may affect patient tolerance and treatment outcomes [107]. One well-studied strategy is
isoform-specific inhibition, where studies investigate inhibitors designed to selectively
target PI3K isoforms [108]. By homing in on specific isoforms, it is anticipated that the
adverse effects associated with non-selective inhibition can be relieved. This approach
aims to achieve a more precise therapeutic effect while reducing off-target effects. The
exploration of isoform-specific inhibition holds significant promise for improving the safety
Int. J. Mol. Sci. 2024, 25, 732 11 of 26

profile of PI3K inhibitors, thereby advancing their clinical efficacy in cancer therapy. For
example, taselisib is a selective PI3Ki; it can selectively inhibit p100α, β, and γ isoforms,
thereby exhibiting better selectivity for the mutant PI3Kα isoform [109]. In a phase 1 study,
taselisib treatment was associated with markable tumor-suppressing effects in patients
with PIK3CA-mutated metastatic BC [109]. Based on these encouraging findings, a phase 3
trial was conducted and showed outstanding effects for the combination of taselisib and
fulvestrant against PIK3CA-mutated BC patients. This trial recruited postmenopausal
women and demonstrated that median PFS was significantly increased in PIK3CA-mutant
patients with combinational therapy. Conversely, with PIK3CA-wild-type tumors, no
significant difference was found between patients receiving taselisib plus fulvestrant and
fulvestrant alone. However, further studies suggested that a high incidence of adverse
effects caused by taselisib, including diarrhea and hyperglycemia, resulted in treatment
discontinuation [110,111].
Taken together, PI3K gene mutations are related to tumor proliferation and metastasis,
and PKI3CA mutations usually occur in BC patients. The pan-inhibition of PI3K often led
to serious side effects instead of satisfactory therapeutic effects. Studies were then shifted
to PI3K isoform-specific inhibitors that can specify the target; however, ways to minimize
the adverse effects caused by selective PI3Ki will require further investigations.

3.2.2. AKT Inhibitors

AKT is a vital characteristic of the PI3K intracellular pathway; it regulates cell prolifera-
tion, survival, and metabolism [112]. Three AKT isoforms, namely, AKT1, AKT2, and AKT3,
have been reported. In cancer cells, AKT1 is involved in cell proliferation and growth,
thereby facilitating tumor development and suppressing apoptosis. AKT2 participates in
cytoskeleton dynamics and, therefore, favors tumor invasion and metastasis. Although
the activation of AKT3 in cancer cells remains controversial, it has been speculated to be
involved in cell proliferation [113,114].
Hyperactivation of the PI3K/AKT/mTOR pathway is an oncogenic driver in BC
and has a correlation with hormone therapy resistance. AKT is altered in about 7% of
BC [115], so it represents a potential therapeutic target in patients with BC [116]. The
main clinical trial was proposed by FAKTION and investigated the efficacy and safety
of an AKT inhibitor, capivasertib. This phase 2 clinical study enrolled postmenopausal
women with HR+/Her2− advanced breast cancers who had relapsed or progressed after
previous AI treatment. Patients receiving capivasertib plus fulvestrant had improved PFS
(10.3 months), as compared with those receiving fulvestrant alone (4.8 months) [117]. Trials
that investigated AKT inhibitors with chemotherapy were also reported. A randomized
placebo-controlled phase 2 trial (PAKT) evaluated the efficacy of capivasertib plus paclitaxel.
The population was stratified by PIK3CA/AKT1/PTEN mutational status by next-generation
sequencing. In patients with PIK3CA/AKT1/PTEN mutation, PFS was significantly extended
with capivasertib plus paclitaxel (9.3 months) compared with controls (3.7 months) [52].
Furthermore, in an updated report, these patients showed a favorable trend in OST for
capivasertib plus paclitaxel [118].
Therefore, AKT inhibitors represent a promising avenue for cancer therapy, particu-
larly in breast cancer where the PI3K/AKT/mTOR pathway is frequently dysregulated.
Clinical trials evaluating AKT inhibitors have shown encouraging results, with capivasertib
being a notable example, especially in patients with specific molecular alterations. While
AKT inhibitors show promising efficacies in clinics, their development faces certain chal-
lenges. One challenge is the potential for off-target effects, as AKT is involved in various
cellular processes beyond cancer. Achieving selectivity for cancer cells while minimizing
adverse effects on normal cells remains a critical consideration. Second, the complexity of
the PI3K/AKT/mTOR signaling pathways involves multiple components and crosstalk
with other signaling pathways. Identifying the optimal combination therapies and un-
derstanding the context-specific effects of AKT inhibition are areas that require further
exploration. In the future, advancements in drug design and technology may lead to the
Int. J. Mol. Sci. 2024, 25, 732 12 of 26

development of more selective and potent AKT inhibitors, minimizing off-target effects
and improving overall therapeutic profiles.

3.2.3. mTOR Inhibitors

mTOR is a downstream target of the PI3K/AKT and adenosine monophosphate-
activated protein kinase (AMPK) signaling pathways. As a member of these pathways,
mTOR is a regulator of cell proliferation and metabolism. The activation of mutations is
usually found in the mTOR pathway, including PIK3CA, AKT1, and mTOR mutations.
Therefore, drugs/agents targeting mTOR have been developed for cancer treatment. The
mTOR inhibitor, everolimus, is a targeted drug for HR+/Her2− metastatic BC patients.
In a randomized phase 2 clinical study, patients with ER+ BC were enrolled. The effi-
cacy of everolimus plus letrozole was compared against letrozole alone. The results showed
that clinical responses were better in patients receiving everolimus, which increased the
letrozole efficacy in the neoadjuvant setting [119]. Furthermore, that study assessed the
Ki67 expression in tumors at baseline and after 15 days of medical intervention. In tumors
of the everolimus and placebo arms, the Ki67 expression significantly decreased in the
everolimus group. This result indicated a greater anti-neoplastic response for everolimus-
treated patients. Another phase 2 study evaluated efficacies between everolimus plus
tamoxifen and tamoxifen alone. This clinical trial recruited patients with HR+/Her2−
advanced BC who had failed AI treatments. The clinical benefit rate of everolimus
plus tamoxifen was 61%, as compared with 42% with tamoxifen alone. Improved PFS
was found in the combination arm (8.6 months), as compared with the tamoxifen arm
(4.5 months) [53]. The phase 3 trial (BOLERO-2) enrolled patients with HR+ advanced BC,
who were randomized to receive everolimus combined with exemestane versus exemestane
alone. The median PFS was significantly improved in the everolimus plus exemestane arm
(6.9 months) compared with the control arm (2.8 months) [120]. An extended report out-
lined that, at a median follow-up of 18 months, improved PFS was found in patients with
the combination therapy, as compared with those who received exemestane alone [121].

3.3. CDK4/6 Inhibitors

Cyclin-dependent kinase (CDK) belongs to the serine/threonine protein kinase family
and is involved in cell cycle regulation. CDK4 and CDK6 are correlated to D-type cyclins
and promote the initiation of progression from the G1 phase to the S phase. Thus, CDK4/6
inhibitors can effectively inhibit the progression of cancer cells from the G1 phase to the
S phase. In recent years, CDK inhibitors have been proposed for the management of
hormone receptor-positive breast cancers. Three CDK4/6 inhibitors, palbociclib, ribociclib,
and abemacicilib, are approved by the US FDA for breast cancers in combination with
hormone therapies [122–124]. Double suppression of CDK4/6 and ER signals, known as
palbociclib with an AI, can control the cell cycle and block the proliferation of breast cancer
cells, so it is the first-line choice in postmenopausal females [125].
Palbociclib was the first CDK4/6 inhibitor to be approved by the FDA for breast cancer
in combination with AIs in 2015. In phase 2 and 3 clinical trials, postmenopausal patients
were given palbociclib plus letrozole or placebo plus letrozole. The results showed that PFS
was significantly higher in the palbociclib plus letrozole groups [126,127]. After these trials,
a study that assessed the efficacy and safety of palbociclib plus fulvestrant was conducted
to evaluate women with advanced BC. Patients with these drugs improved their PFS, with a
58% reduction in progression [128]. Palbociclib is generally well tolerated, with certain side
effects. Fatigue, nausea, leukopenia, and neutropenia are commonly reported [122–124].
Nevertheless, the overall tolerability was considered to be manageable, which led to a
positive quality of life in BC patients [129,130].
In 2017, ribociclib became the second CDK4/6 inhibitor to receive FDA approval
for HR+/HER2- advanced or metastatic BC. A clinical trial compared patients receiving
ribociclib plus letrozole or letrozole alone. The results were similar to those for palbociclib,
in which patients with ribociclib in combination with letrozole had increased PFS [131].
Int. J. Mol. Sci. 2024, 25, 732 13 of 26

The efficacy and tolerability of the combination were reported in an extended study [132],
which also noted that improved PFS and OST were found. In addition to AIs, ribociclib is
also being investigated in combination with fulvestrant, and this combinational therapy
significantly increased the PFS in BC patients [133]. The results showed that patients
benefited from ribociclib plus fulvestrant, showing an increased OST and a decreased
relative risk of death [134]. A phase 3 clinical trial evaluated the efficacy and safety of
ribociclib as first-line therapy for females with breast cancers. Compared with the placebo
group, the groups with ribociclib plus tamoxifen or AIs had significantly higher PFS [135]
and OST [136].
Abemaciclib is noted to be the most potent CDK4/6 inhibitor, as it can cross the blood–
brain barrier due to its unique structure. This ability may be beneficial for patients with
brain metastases, and this medicine has also been approved by the FDA for women with
advanced BC. Abemaciclib showed promising clinical activities after 12 months in patients,
with an overall response rate of 19.7% and a 6-month increase in PFS [54,122]. Furthermore,
patients significantly benefited from the combination of abemaciclib and fulvestrant, with
higher PFS [137] and OST [137] compared with those with fulvestrant alone.
For BC treatment, CDK4/6 inhibitors are usually combined with fulvestrant or AIs and
have shown well-tolerated efficacy, with manageable side effects. These findings suggested
that CDK4/6 inhibitors combined with fulvestrant are a potentially promising choice for
the first-line treatment of patients with BC.

3.4. HDAC Inhibitors

Histone deacetylase (HDAC) is an enzyme that regulates gene expression and modifies
the chromosome structure. Epigenetic alteration modified by HDAC results in aberrant
gene expression, leading to disease progression and resistance to hormone therapy in
BC patients [138,139]. These modifications/alterations can be reversibly modulated by
HDAC inhibitors [140]. Tucidinostat, also known as chidamide, is a selective HDAC
inhibitor that suppresses Class I and Class IIb HDACs. A randomized, phase 3 clinical trial
enrolled postmenopausal women with HR+/Her2− BC who had relapsed or progressed
after at least one hormone therapy. Tucidinostat plus exemestane significantly improved
PFS compared with placebo plus exemestane in patients. Though hematological adverse
events were more common in the tucidinostat plus exemestane group, these findings
provide a new treatment option for patients with relapsed diseases [141].

3.5. PARP Inhibitors

DNA damage and deficiencies of repair are vital features of cancer pathology. Normal
cells will maintain their genome integrity through DNA damage response (DDR) path-
ways, which protect them from DNA damage [142]. At least 450 proteins or molecules
are involved in DDR pathways, including poly (ADP-ribose) polymerase 1 (PARP1) and
PARP2 [143]. Furthermore, PARPs moderate transcription, apoptosis, and immune func-
tion [144]. These multiple mechanisms could result in increased PARP inhibitor efficacy.
PARP inhibitors bind to PARP, inhibiting PARylation, and also trap inactivated PARP
in DNA, thereby blocking replication forks, which, in turn, leads to collapse and the gener-
ation of double-strand breaks in cancer cells [143,145,146]. Two PARP inhibitors, Olaparib
and talazoparib, have been approved by the US FDA and European Medicines Agency
(EMA) as monotherapy for BC patients. Several PARP inhibitors (niraparib, rucaparib,
and veliparib) are also being investigated; for example, veliparib is currently in phase 3
clinical trials for the treatment of HER2- metastatic BC and has showed promising out-
comes [147]. A randomized, multicenter, phase 3 clinical trial (OlympiAD) compared the
efficacy and safety of olaparib versus single-agent standard therapy of the physician’s
choice (TPC: capecitabine, eribulin, or vinorelbine) in patients with Her2- metastatic BC.
Median PFS was significantly increased with olaparib (7.0 months) compared with the
control arm (4.2 months) [148]. However, in an extended analysis, no significant differ-
ences were detected in median overall survival with olaparib (19.3 months) versus controls
Int. J. Mol. Sci. 2024, 25, 732 14 of 26

(17.1 months) [149]. EMBRACA was a randomized, multicenter, phase 3 clinical trial
that assessed the efficacy and safety of talazoparib versus single-agent standard TPC. In
that study, median PFS was significantly longer in patients with talazoparib treatment
(8.6 months) than in those with TPC therapy (5.6 months) [150]. In the final overall survival
analysis, however, no significant difference was found in patients receiving talazoparib
(19.3 months) or TPC (19.5 months) [151]. Although no significant difference in overall
survival was found, olaparib and talazoparib monotherapies have demonstrated significant
PFS benefits compared with chemotherapy. Common side effects are manageable through
supportive treatment and dose interruptions/reductions. Therefore, the advent of PARP
inhibitors has potential benefits for BC treatment.

4. Therapies for Triple-Negative Breast Cancer (TNBC)

4.1. Immune Checkpoint Inhibitors: Programmed Death-Ligand 1 (PD-L1)
Immune checkpoint inhibition using the antibodies against PD-L1 has shed light on
TNBC. Several anti-PD-L1 antibodies, for example, atezolizumab, avelumab, and durval-
umab, were investigated and reported to significantly increase OST in TNBC patients.
A phase 3 clinical trial evaluated the effects of atezolizumab in combination with nab-
paclitaxel. Among the patients with PD-L1-positive tumors, these combinational therapies
significantly prolonged the OST (25 months) compared with placebo plus nab-paclitaxel
(15.5 months) [152]. A clinical trial recruited 33 patients with metastatic TNBC and ex-
amined the safety, tolerability, and clinical activity of atezolizumab. All patients showed
at least one treatment-related adverse event, and 73% of patients experienced grade 3
adverse events. Nevertheless, no death was observed, and the median PFS and OST were
5.5 months and 14.7 months, respectively [153].
A phase 1b clinical trial using avelumab reported a higher overall response rate in patients
with PD-L1-positive tumors (22.2% versus 2.6%) [154]. Several trials are also conducted with
durvalumab for patients with metastatic TNBC. A phase 1b trial reported the therapeutic effects
of durvalumab and a peptide vaccine in patients with stage 2 or 3 TNBC. The promising results
of PD-L1 therapies in TNBC progressed the utility in TNBC. Furthermore, PD-L1 antibodies
alone or in combination with different anti-tumor agents are currently under investigation.

4.2. MicroRNA-Based Gene Therapy and Biomimetic Carriers

The ongoing and recent discovery of the regulatory effects of miRNA remains in
a preclinical state, in which miRNA can downregulate PD-L1 expression in TNBC cells.
Furthermore, the use of miRNA as a therapy for TNBC not only targets the tumor cells but
also microenvironmental components. Numerous studies showed that several miRNAs
exhibited anti-tumor properties in TNBC cells. For example, miR-195-5p and miR-497-5p
can inhibit tumoral PD-L1 expression in MDA-MB-231 cells. MiR-424-50 can inhibit PD-L1
transcript expression in MDA-MB-231 cells and stimulate interferon production in the
tumor microenvironment [155]. Taken together, these preliminary studies provide further
direction for the utility of miRNA in TNBC patients. The use of miRNAs as therapeutics is
a promising new potential modality for the treatment of TNBC.
Carriers that deliver therapeutic agents to target TNBCs were also reported. These
techniques may provide the patients with personalized, specific, and safe tumor-associated
delivery in the future. Molinaro et al. reported that leukosomes, a kind of biomimetic
nanovesicle, can effectively deliver a chemotherapeutic agent to tumors in the syngeneic
TNBC murine model [156]. The leukosomes can recruit immune cells within the tumors,
thereby decreasing the tumor volume. Kang et al. demonstrated that neutrophil membrane-
coated nanoparticles can capture circulating tumor cells and accumulate in metastatic
tumors in mice bearing TNBC cells [157].
Some limitations in microRNA-based therapy for breast cancers should be considered.
First, the variability in certain microRNAs across different patients might influence the
efficacy of the TNBC patients [158]. Individual variation in microRNAs will alter the bio-
genesis of the microRNA, which makes significant impacts on the transcription, maturation,
Int. J. Mol. Sci. 2024, 25, 732 15 of 26

and target specificity [159]. Therefore, microRNA-based therapy responses vary from
patient to patient, and these dissimilarities result from genetic variability [160,161]. Second,
the biological functions of microRNA in different types of tumors should be taken into
consideration. For example, in breast cancer cells, miR-142-3p stimulated cell apoptosis
and cell cycle arrest [162], whereas miR-142-3p increased the cell viability of gastric cancer
cells [163]. In summary, although the potential of microRNA-based therapy is fascinating,
many more obstacles need to be addressed. Further investigation of microRNA target-
ing genes and their functions is still needed to elucidate and, thereby, provide precision
medicine for patients with TNBC.

4.3. Single-Cell Sequencing and Personalized Medicine

Single-cell sequencing reveals the transcript profiles of single cells, giving the possi-
bility to differentiate among cell populations that are not distinguishable by cell surface
markers and morphology. This approach has revolutionized our ability to study the im-
mune system and allowed us to break through the bottleneck of oncology studies. In
a murine model, two TNBC cell lines (4T1 and EMT6) were observed [164]. Zhou et al.
identified the upregulation of the ETV6 gene in TNBC using single-cell RNA-seq [165].
These studies highlight that single-cell sequencing could help us to dissect the intratumoral
heterogeneity and identify the critical genes of TNBC. This novel methodology represents
a clinically relevant tool to map specific cellular populations spatially and temporally
through disease progression and along treatment.

5. Comparative Oncology
5.1. Epidemiology and Molecular Characteristics
Historical data suggest that almost half of CMTs are malignant [14–16]. Unfortunately,
an increasing tendency toward malignant tumors has been observed over the years [17],
especially in countries/regions where ovariectomy is not routinely performed. In fe-
male dogs and women, mammary cancer is the most frequently diagnosed malignancy
and the leading cause of cancer-related death in women [12,166]. Furthermore, CMTs
and HBCs share several similar characteristics in various aspects, such as hormonal re-
ceptors, metastatic patterns, and the roles of carcinogens, that impact the onset of these
diseases [167]. Many pathological, molecular, and phenotypic similarities also exist be-
tween HBC and CMT [168,169]. Comparative genetic expression profiles revealed that cell
cycle activation, WNT-β-Catenine signaling, PI3K/AKT, and ERK signaling were highly
consistent in HBC and CMT. Furthermore, loss-of-function mutations in the tumor suppres-
sors CDKN2A, PTEN, CDH1, and TP53 were also observed in CMTs [170]. Because of these
features, CMTs are a good spontaneous model for the study of HBC [171,172] (Figure 5).
Surgery is the treatment of choice in both HBC and CMT. Adjuvant therapies, espe-
cially hormone and targeted therapies, are only provided in HBC. One of the underlying
reasons is that the question of whether dogs with CMTs might benefit from adjuvant
therapies remains unanswered [173,174]. These results suggest that the clinical relevance
between treatment choices and these hormone receptors remains poorly reported and still
needs to be elucidated. Therefore, as previously noted for HBC, we summarized current
investigations on hormone and targeted therapies for CMTs.
 Int.J.J.Mol.
Int. Mol.Sci.
Sci.2024,
2024,25,
25,732
x FOR PEER REVIEW 17 of 28
16 of 26

Figure5.5.Comparative
Figure Comparativeoncology
oncologyinin humans
humans and
and dogs.
dogs. Humans
Humans and
and dogs
dogs share
share similar
similar living
living envi-
environ-
ronments. Both these two species are exposed to similar toxic substances, immunogens, and
ments. Both these two species are exposed to similar toxic substances, immunogens, and pathogens.patho-
gens. Because of these external stimuli, human breast cancers and canine mammary gland tumors
Because of these external stimuli, human breast cancers and canine mammary gland tumors de-
develop from similar genetic/epigenetic alterations and metabolic changes. Furthermore, dogs are
velop from similar genetic/epigenetic alterations and metabolic changes. Furthermore, dogs are
immunocompetent individuals, and the tumors share high similarity in tumor microenvironments
immunocompetent individuals, and the tumors share high similarity in tumor microenvironments
with human counterparts.
with human counterparts.
5.2.Hormone
5.2. Hormoneand
andTargeted
TargetedTherapy
TherapyininCanine
CanineMammary
MammaryGland
GlandTumors
Tumors
5.2.1.Hormone
5.2.1. HormoneTherapy
Therapy
Indogs,
In dogs,as asin
inhumans,
humans,hormones
hormones(estrogen
(estrogenand andprogesterone)
progesterone)manipulate
manipulatethe thegrowth
growth
anddevelopment
and developmentof ofmammary
mammaryglands glandsand andparticipate
participatein incarcinogenesis
carcinogenesis [13]. [13]. The
The expres-
expres-
sion of
sion ofERERandandPR PRisiscorrelated
correlated withwith better
better clinical
clinical outcomes
outcomes in in female
female dogs
dogs [175,176].
[175,176].
Decreasedexpression
Decreased expressionof ofER
ERandandPR PRhashasbeen
beendocumented
documentedin incanine
caninemalignant
malignantmammary
mammary
tumors[177,178].
tumors [177,178]. These findings
findings reinforce
reinforce thatthatERERandandPR PRare areideal
idealtherapeutic
therapeutictargets
targetsin
inCMTs.
CMTs.However,
However,the theexpression
expressionof of PR/ER
PR/ERremains
remains undetermined
undetermined in canine canine CMTs.
CMTs. ER ER
and/or PR are expressed
and/or expressed in in about
about 66%
66% of of all
all malignant
malignant MGT MGT cases
cases [178],
[178],which
whichisishighly
highly
consistentwith
consistent withhumans.
humans. Conversely,
Conversely, one one publication
publication reported
reported thatthat nearly
nearly 70%70% of ofMGT
MGT
samples
sampleswerewerenegatively
negativelystained
stainedforforERERor orPRPR[179].
[179].InInhuman
humanmedicine,
medicine,ER+ ER+breast
breastcancer
cancer
isisthe
themost
mostcommon
commonsubgroup
subgroup(above(above70%)70%)[180].
[180].
Tamoxifen
Tamoxifenisiswidely
widelyutilized
utilized inin
thethe
treatment
treatment of human
of human breast cancer
breast (HBC).
cancer However,
(HBC). How-
in dogs,
ever, in severe
dogs, adverse effects (vulvar
severe adverse edema, vaginal
effects (vulvar edema, purulent discharge,
vaginal purulent and pyome-
discharge, and
tra) are repeatedly
pyometra) reported,
are repeatedly and they
reported, andoutweigh the possible
they outweigh benefits
the possible of thisofhormone
benefits this hor-
therapy [181,182].
mone therapy Other anti-estrogen
[181,182]. Other anti-estrogentherapies, such assuch
therapies, indole-3-carbinol (an estrogen
as indole-3-carbinol (an es-
receptor antagonist), have been studied for the treatment of CMTs.
trogen receptor antagonist), have been studied for the treatment of CMTs. These studies These studies were
in vitro [183,184] or murine studies [185], and further clinical trials
were in vitro [183,184] or murine studies [185], and further clinical trials assessing theassessing the efficacy
and safety
efficacy aresafety
and needed.
are Furthermore, despite the
needed. Furthermore, effectiveness
despite of AIs as of
the effectiveness hormone therapy,
AIs as hormone
no clinicalno
therapy, trials on the
clinical use
trials onofthe
this
usestrategy in dogs in
of this strategy with
dogsCMTswithhave
CMTs been
havereported. Ther-
been reported.
apeutic targeting
Therapeutic of PRs
targeting has has
of PRs beenbeen
wellwellstudied
studied in in
HBC,
HBC, butbutanti-progestin
anti-progestinhas hasrecently
recently
Int. J. Mol. Sci. 2024, 25, 732 17 of 26

been employed in veterinary oncology. Mifepristone and onapristone can decrease the
viability of CMT cell lines [186]. Aglepristone, a PR antagonist, effectively diminished
the expression of PR and the proliferation of CMTs. Furthermore, dogs that benefited
from aglepristone had increased PFS and overall survival time [187]. In addition to that
trial, several underdeveloped hormone therapies remain in veterinary medicine. However,
severe side effects and limited clinical studies in dogs might restrict further explorations.

5.2.2. Her2-Targeted Therapies

The targeted therapy with anti-Her2 agents in HBC is well established. In veterinary
medicine, to clarify the application of trastuzumab that can be applicable in dogs, the
sequences and epitopes of the Her family have been analyzed. The amino acid homology
values of Her1 and Her2 are, respectively, 91% and 92% between canines and humans.
Cetuximab (antibody against Her1) epitopes only differ by four amino acids in dogs, and the
trastuzumab (antibody against Her2) binding sites were identical in humans and dogs in an in
silico study. Furthermore, cetuximab or trastuzumab significantly inhibited CMT cell growth
and triggered cell cycle arrest [188]. However, no clinical trials have been published on the
use of Her2 inhibitors or “caninized” monoclonal antibodies in CMTs. One of the possible
reasons is that the clinical benefits of Her2 amplifications and their association with Her2
overexpression are not straightforward in CMTs [189,190]. Her2 status has been investigated
in numerous studies to emphasize the similarity between humans and dogs, but validated
methods for canine-specific Her2 clinical relevance are still lacking [191]. These unique findings
indicate that the incidence and the clinical significance of Her2 in MCTs need to be further
elucidated. Therefore, the role of Her2 expression in CMT needs to be scrutinized to determine
its values for diagnosis, therapeutics, and prognosis in dogs.
The use of hormone and Her2-targeted therapies in canines is still far from becoming
routine. Despite the similarities between humans and dogs, CMTs show certain discrep-
ancies that have perplexed researchers (Table 3). Furthermore, no consistent classification
based on HRs exists, and whether dogs with MCTs might benefit from hormone/Her2-
targeted therapies still needs to be elucidated in the future.

Table 3. Comparative table of currently used anti-BC or anti-CMT therapies in humans and dogs.

Human Dogs
Hormone therapy Tamoxifen FDA-approved Serious AEs in dogs
AIs FDA-approved -
Aglepristone - Increased PFS and OST in dogs
Mifepristone - CMT cell lines
Onapristone - CMT cell lines
Targeted therapy Her-2 mAbs FDA-approved -
Trastuzumab biosimilars FDA-approved -
Tyrosine kinase inhibitors FDA-approved -
PI3K/AKT/mTOR inhibitors FDA-approved -
HDAC inhibitors FDA-approved -
PARP inhibitors FDA-approved -

6. Conclusions and Future Prospectives

In conclusion, our exploration of clinical trials in hormone receptor-positive breast
cancer (HR+ BC) revealed a transformative era marked by the integration of conventional
hormone therapy with targeted medicines. The field of hormone therapies for breast cancer
has witnessed significant advancements, particularly with the success of SERMs like tamox-
ifen and Ais, such as anastrozole, letrozole, and exemestane. However, several challenges
should be addressed, mainly in the form of resistance mechanisms. Combinational therapy,
such as the use of AIs following tamoxifen, has shown promising efficacies, offering clinical
benefits and reducing recurrence risk. The success of inhibitors targeting specific pathways
and molecules in clinical trials has introduced novel strategies for treating HR+ BCs. The
Int. J. Mol. Sci. 2024, 25, 732 18 of 26

diverse range of targeted therapies reflects a growing understanding of the molecular

complexities of breast cancer. Combining these therapies, exploring novel agents, and
addressing challenges related to resistance and side effects are critical for advancing breast
cancer treatment and improving patient outcomes. However, in the context of canine
mammary tumors (CMTs), hormone therapy for dogs has often resulted in severe adverse
effects, overshadowing the potential therapeutic benefits. The utilization of Her2 or other
targeted therapies in dogs is still in the developmental stage and has not become routine.
While dogs are considered excellent models for human oncology, there is substantial
room for improvement in developing novel therapies in veterinary medicine. Clinical
trials in dogs with CMTs are limited, with few demonstrating effective anti-tumor efficacy
and manageable side effects. Notably, novel immunotherapies in dogs with CMTs have
been reported gradually, but the low case numbers hinder the derivation of conclusive and
extrapolative results. To address these gaps, future research directions are recommended.
First, conducting well-planned, large-scale, prospective randomized clinical trials in dogs
with CMTs might obtain robust and conclusive results. Through these trials, a more
comprehensive understanding of the efficacy and safety of hormone therapy, targeted
therapies, and novel immunotherapies can be proposed. Second, dogs with spontaneous
CMTs are ideal natural models for studying HR+ BC due to their similar physiological
behaviors shared with humans. Immunocompetent dogs can mimic the complete immune
system against cancer cells, reflecting the current state of cancer research and offering
insights into trends in immune system activation. Last, comparative studies should be
performed. Dogs with CMTs can provide more relevant and translatable insights into
patients with HR+ BC compared to rodent models. Therefore, dogs are valuable bridges
between preclinical research and clinical trials in humans. Through these concerted efforts,
future research in veterinary medicine can significantly contribute to the development of
effective and safe therapies for both dogs with CMTs and humans with BC. This integrated
approach holds promise for advancing our understanding of cancer treatment, especially
in the context of hormone and targeted therapies, and shaping the landscape of clinical
oncology for both species.

Author Contributions: Conceptualization, C.-H.K. and C.-S.L.; investigation, C.-H.K.; resources,

C.-N.L.; writing—original draft preparation, C.-H.K.; writing—review and editing, C.-N.L. and
C.-S.L.; supervision, C.-S.L.; project administration, C.-H.K.; funding acquisition, C.-S.L. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflicts of interest.

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