Research progress in tumor angiogenesis and drug resistance in breast cancer

VEGF and VEGFR

The discovery of VEGF has greatly advanced understanding of tumor angiogenesis and enabled its potential targeting in cancer therapy. The binding of VEGF-A to VEGFR2 is crucial for angiogenesis. Notably, VEGF-A binding leads to the tyrosine phosphorylation of VEGFR2, which in turn further activates downstream signaling pathways including the MAPK and PI3K pathways. Furthermore, pathways such as PLCγ¹⁰, ERK1/2, and PI3K¹¹ stimulate endothelial cell division. The activation of endothelial nitric oxide synthase (eNOS)¹² increases vascular permeability¹³ (Figure 1). A recent study has revealed that VEGF inhibition effectively inhibits tumor angiogenesis, promotes vascular normalization, and inhibits tumor growth¹⁴. Nevertheless, the normalization of blood vessels by targeting VEGF anti-tumor angiogenesis therapy is short-lived. Therefore, investigating the appropriate dose and time window of drugs to promote and maintain tumor vascular normalization may become a primary focus of future research on anti-tumor angiogenesis¹⁵.

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Figure 1

The hypoxia-inducible factor HIF promotes the transcription of angiogenesis-related factors. The VEGF/VEGFR signaling axis promotes angiogenesis via MAPK, PI3K, PLCγ, ERK1/2, and eNOS. The PDGF/PDGFR signaling axis promotes angiogenesis via MAPK, PI3K-AKT, and STAT. The EGF/EGFR signaling axis promotes angiogenesis via MAPK, PI3K, PLCγ, and STAT. Other signaling axes (PDGF/HGF/IGF) promote angiogenesis through MAPK, PI3K, STAT, and RAGE.

PDGF and PDGF receptor (PDGFR)

PDGF is a growth factor that is secreted by platelets and their stromal cells and is involved in the regulation of angiogenesis. This growth factor comprises the following 4 subunits: PDGF-A, PDGF-B, PDGF-C, and PDGF-D¹⁶. PDGFR is divided primarily into PDGFR-α and PDGF-β. Studies have focused on the binding between PDGF-B and PDGFR-β, and subsequent promotion of angiogenesis. PDGFR-β is significantly expressed in breast cancer cells^17,18. Phosphorylated PDGFR regulates cell proliferation and migration via PI3K-AKT¹⁹ and other signaling pathways, thereby participating in tumor angiogenesis (Figure 1). Wang et al. have reported that decreased levels of PDGF-B promote vascular normalization of breast cancer cells, increase cytotoxic drug delivery, and inhibit tumor growth¹⁷. In a mouse model of triple-negative breast cancer with lung metastasis formation, PDGFR-β blockade has been found to decrease cancer cell growth and migration, and consequently prevent lung metastasis²⁰. Nevertheless, the specific mechanism of the PDGF/PDGFRs axis in the angiogenesis of breast cancer cells remains unclear. Further basic experiments are warranted to elucidate the relevant mechanisms.

Epidermal growth factor (EGF) and EGF receptor (EGFR)

EGF is a mediator with crucial roles in the proliferation, survival, differentiation, and migration of vascular endothelial cells, through EGFR binding. EGFR is activated by phosphorylation and is involved in regulation of tumor angiogenesis via its downstream signaling pathways (MAPK, PI3K/AKT/PKB, STAT, and PLCγ/PKC)²¹ (Figure 1). HER2, a subtype of EGFR, is a frequent target in breast cancer treatment. EGFR-mediated downregulation of the JAK-1/STAT-3 signaling pathway has been found to inhibit angiogenesis, significantly decrease the volume of breast cancer tissues, and inhibit tumor growth²¹. EGFR and VEGFR frequently share downstream signaling pathways. Furthermore, the expression of EGFR leads to an increase in VEGFR levels and consequently plays a crucial role in promoting angiogenesis²². Therefore, targeting EGFR to downregulate VEGFR expression is advantageous in cancer treatment. Nevertheless, many patients who undergo targeted EGFR therapy exhibit progression after 1 year²³. Therefore, mechanisms underlying resistance and strategies to reverse resistance via targeted EGFR therapy must be further explored.

Other factors

Beyond the VEGF/VEGFR, EGF/EGFR, and PDGF/PDGFR axes, many other growth factors play indispensable roles in angiogenesis, such as fibroblast growth factor (FGF)/FGFR4, hepatocyte growth factor (HGF)/c-Met, insulin-like growth factor (IGF)/IGFR, and transforming growth factor (TGF-β) (Figure 1). Angiogenesis-related FGFR4 is significantly upregulated in breast cancer, and promotes vascular endothelial cell proliferation and breast cancer angiogenesis via the PI3K/AKT signaling pathway²⁴. HGF activates c-Met through phosphorylation of the tyrosine residues Y1234 and Y1235. Adaptor proteins bind several substrates, and downstream MAPK and STAT signaling pathways are consequently activated, thereby promoting angiogenesis in breast cancer²⁵. Muoio et al. have reported that elevated IGF expression in patients with obesity and diabetes promotes breast cancer angiogenesis via the S7A1/RAGE downstream signaling pathway²⁶ (Figure 1). Notably, TGF-β increases angiogenesis in breast cancer by modulating endothelial–mesenchymal transition, potentially through TGF-β-induced expression of Snail and Slug²⁷. Various growth factors play important roles in the angiogenesis of breast cancer. Therefore, anti-angiogenic therapies against these vascular factors can be beneficial. Further studies are warranted to validate the possibility of targeting single or multiple growth factors to increase the anti-angiogenic efficacy of breast cancer treatment.

HIF-induced angiogenesis

Under hypoxia, the activity of FIN-1 and PHDs, acting as oxidase-related groups, is inhibited, thus leading to decreased HIF-1α hydroxylation and proteasome degradation. Consequently, the cytoplasmic level of HIF-1α increases, and HIF-1α forms a heterodimer with HIF-1β. This heterodimer translocates to the nucleus, where HIF-1 binds the activating protein CREB (P300) and the hypoxia reactive element (HRE), and subsequently initiates a series of hypoxia-induced cascades, including angiogenesis, through increased transcription of target genes, such as VEGF²⁸. Recent studies have elucidated the role of HIF-1 in breast cancer angiogenesis through the SNHG1/miR-199a-3p/TFAM axis²⁹. Beyond HIF-1, the hypoxia-induced HIF-2-dependent pathway promotes angiogenesis in breast cancer by upregulating levels of the lncRNA RAB1B-AS231 through HIF-2. This event in turn increases the transcription of angiogenesis-related factor VEGFA. Knockdown of HIF-2 has been shown to eliminate the increase in RAB1B-AS231 expression, thereby decreasing angiogenesis in breast cancer³⁰ (Figure 2C). Given the current limitations of targeted VEGF treatment regimens, targeting HIF, a key regulator of angiogenesis in breast cancer, may emerge as a promising new therapeutic approach.

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Figure 2

A. Hypoxia inhibits the activity of FIH-1 and PHD, thereby promoting the expression of HIF-1. Subsequently, HIF-1 stimulates the expression of VEGF, which in turn facilitates angiogenesis. B. HIF-1 induces endothelial to mesenchymal transition by promoting the activity of the transcription factors Twist, Snail, and ZEB2, thereby stimulating angiogenesis. However, its specific mechanism is unclear. C. Hypoxia promotes HIF-2 expression through a lncRNA that also promotes angiogenesis.

Epithelial–endothelial transition (EET)

Epithelial–mesenchymal transition (EMT) is a prevalent phenomenon. Research has revealed that the same key factors are involved in both EET and EMT. Consequently, EET is generally considered a phenotype of EMT³¹. Under hypoxia, breast cancer cells show a decrease in tight junction proteins (such as E-cadherin and occludin) and an increase in vimentin and VE-cadherin. Tumor epithelial cell transition to endothelial cell phenotypes subsequently promotes the proliferation of tumor vascular endothelial cells and angiogenesis³² (Figure 2A) Numerous EET-associated signaling pathways are directly or indirectly regulated by hypoxia. Transcription factors such as Twist, Snail, and ZEB2, which are directly regulated by hypoxia, play major roles in this process and share a frequent promoter: the HRE. Under hypoxia, binding of HIF to HRE promotes the transcription of Twist, Snail, and ZEB2^33–35 (Figure 2B). Twist proteins downregulate the expression of E-cadherin in breast cancer, thereby promoting EET. This process may be involved in EET progression via activation of Wnt/β-catenin signaling by the JPX/miR-33a-5p/Twist1 axis³⁶. However, this signaling pathway was discovered and validated by Pan et al. in the EET in lung cancer, and further research is warranted to confirm its presence in breast cancer³⁷. The Snail gene has been found to interact with RNF20 (E120 ubiquitin-protein ligase with monoubiquitinated H3BK2) and G9a (methyltransferase of H3K9me2) in breast cancer, and to subsequently inhibit the expression of E-cadherin. The Snail gene also promotes tumor EET and induces tumor angiogenesis^38,39. Consequently, hypoxia plays a crucial role in tumor angiogenesis, and reversing the tumor hypoxic microenvironment may aid in the treatment of anti-angiogenic and tumor vascular normalization.

Immediate effects

Hypoxia directly induces the transcription of drug resistance-associated genes, namely MDR1, MRP1, and BRCP, which are members of the same ABC transporter family. The expression of ABC transporters increases under hypoxic conditions, thus facilitating drug efflux, decreasing effective drug concentrations, and fostering drug resistance in breast cancer. Expression of the multidrug resistance gene (MDR1) frequently contributes to drug resistance in various cancers. Che et al. have demonstrated that HIF-1α directly binds the MDR1 promoter and promotes MDR1 expression, as confirmed with ChIP assays⁴¹. In breast cancer cells, AGR2 expression decreases the degradation of HIF-1α and increases MDR1 transcription, thus decreasing epirubicin uptake and promoting doxorubicin (DOX) resistance in breast cancer cells⁴². MDR1 is regulated through hypoxia-induced Notch1 signaling and contributes to the development of tumor resistance⁴³. Under hypoxic conditions, increased HIF-1α activity promotes the BRCP expression⁴¹. DOX has been found to induce the production of reactive oxygen species, thereby synergistically upregulating the expression of MDR1 and BRCP in conjunction with the high activity of HIF-1α under hypoxia. This process limits the uptake of DOX and promotes the emergence of drug resistance⁴⁴.

Indirect effects

The hypoxia-induced acidic microenvironment of tumors can affect drug efficacy⁴⁵, and promote the phenotypic expression of breast cancer stem cells (CSCs) and the acquisition of MDR in breast cancer. In a hypoxic environment, HIF-1α is essential for promoting the expression of the target gene VEGF, which in turn stimulates angiogenesis, exacerbates hypoxia, and supports the development of drug resistance in breast cancer. Under hypoxia, enhanced anaerobic metabolism leads to the accumulation of acidic substances, such as lactic acid, and an increase in the microenvironment pH. The pH value of the TME is typically 10 to 30 times higher than that of normal tissues⁴⁵. The efficacy of DOX against breast cancer cells is limited by the low pH TME induced by hypoxia simulation. Simultaneously, the degree of pH decrease precisely corresponds to the extent of tumor growth delay⁴⁶. Breast CSCs exhibit unlimited proliferation and diverse differentiation potential, and consequently promote tumor immune evasion. Cancer stem cells usually overexpress ABC transporters, which contribute to dysregulation of the signal transduction network and play essential roles in tumor MDR⁴⁷. HIF is involved in the expression of CSC in breast cancer. Through FACS, Brooks et al. have observed that HIF is highly active under hypoxic conditions and promotes the transcription of the target gene ITGA6, which, in conjunction with other integrins, is enriched in breast stem cells and promotes the acquisition of MDR phenotypes in breast cancer⁴⁸. HIF participates in the expression of glutathione S-transferase omega 1 (GSTO1), thereby promoting endoplasmic reticulum release of Ca²⁺; inducing the recruitment of CSC through the PYK2 → SRC → STAT3 signal transduction induced by cisplatin; and contributing to the occurrence of drug resistance in breast cancer⁴⁹ (Figure 3A). HIF-1α activity increases under hypoxia. HIF-1α, which promotes transcription under hypoxia, directly promotes the expression of cancer stem cell phenotypes in liver cancer. However, whether HIF-1α directly promotes the expression of breast cancer stem cell phenotypes has not been validated (Figure 3B). The RAS/RAF/MEK/ERK kinase cascade enhances the translation of HIF-1α by phosphorylating eukaryotic translation initiation factor 4E (eIF-4E). Furthermore, this kinase cascade enhances the transcriptional activity of HIF-1α by binding the TAD sequence of HIF-1α through the transcriptional co-activators p300/CBP (Figure 3C). In breast cancer cells, loss of the tumor suppressor gene PTEN is frequently observed. The loss of PTEN promotes activation of the PI3K/AKT pathway, and its downstream mTOR protein activates eIF-4E and consequently promotes the translation of HIF-1α protein^50,51 (Figure 3D). HIF-1α forms a heterodimer with HIF-1β in the cytoplasm, translocates into the nucleus, and binds specific HREs on DNA, where it acts as a transcription factor for the downstream target gene VEGF (Figure 3E). Subsequently, the proliferation of endothelial cells increases, angiogenesis in breast cancer is improved, and hypoxia is further exacerbated. Thus, the development of drug resistance in breast cancer occurs through various direct and indirect mechanisms.

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Figure 3

A. Cisplatin-induced HIF-1α facilitates the release of calcium ions from the endoplasmic reticulum. The released calcium ions in turn promote the expression of glutathione S-transferase omega 1 (GSTO1). GSTO1 induces the recruitment of cancer stem cells (CSCs) through the PYK2 → SRC → STAT3 signal transduction pathway. STAT3 activity promotes the BCSC phenotype by upregulating the expression of the pluripotency factor KLF4. B. Hypoxia-induced HIF-1α directly promotes the cancer stem cell phenotype in liver cancer. However, whether HIF-1α directly promotes the breast cancer stem cell phenotype remains to be validated. C. The RAS/RAF/MEK/ERK kinase cascade enhances the translation of HIF-1α by phosphorylating the eukaryotic translation initiation factor 4E (eIF-4E). Furthermore, this cascade pathway facilitates the interaction between transcriptional co-activators p300/CBP and HIF-1α, thereby augmenting the binding capacity of HIF-1α to HRE. D. Loss of PTEN promotes activation of the PI3K/AKT/mTOR pathway, thereby activating eIF-4E, which in turn promotes HIF-1α translation. E. HIF-1α forms a heterodimer with HIF-1β in the cytoplasm, translocates into the nucleus, and acts as a transcription factor binding specific HREs on DNA, thus promoting transcription of the downstream target gene VEGF. F. VEGF binding to VEGFR on the surfaces of T cells promotes TOX expression and leads to T cell exhaustion. G. VEGF binds VEGFR and promotes the expression of FASL ligands in epithelial cells, thereby facilitating T cell apoptosis. H. VEGF/VEGFR signaling promotes the transcription of miRNAs (miR-142-5p, miR-183-5p, and miR-222-3p) and facilitates their extracellular transport via exosomes, thereby promoting the polarization of M2 macrophages.

Drug concentration

The drugs used to treat tumors typically must reach effective concentrations within tumors to exert anti-tumor effects. Drug concentrations are affected by the abnormal and immature tumor vasculature system, as well as by the activation of drug efflux transporters under the hypoxic tumor environment⁵².

MDR1, also known as permeability glycoprotein (P-gp), is a transmembrane transport protein that recognizes and controls drug efflux from tumor tissues and eventually promotes drug resistance in breast cancer by altering drug distribution in tumors⁵². HIF-1α is highly expressed after tumor angiogenesis, and subsequently binds P-gp, and upregulates P-gp gene expression. This upregulation then promotes drug efflux. Consequently, achieving effective drug concentrations in the tumor becomes difficult and ultimately leads to drug resistance in breast cancer. HIF-1α inhibition decreases P-gp levels in tumors and partially reverses MDR caused by MDR1 (P-gp)⁴¹. A recent study has described the use of novel nanomolecular materials to directly downregulate P-gp gene expression and enhance the sensitivity of breast cancer cells to chemotherapy⁵².

Drug efficacy

Antineoplastic drugs typically require certain conditions to exert optimal therapeutic effects. For example, compared to cells with an inactive cell cycle, chemotherapy drugs are usually more effective against active tumor cells. Tumor cells typically undergo active DNA replication, and chemotherapy drugs act on the cell cycle⁵³. Abnormal tumor vascular systems often affect pH levels and create a hypoxic environment, which in turn affects the efficacy of antineoplastic drugs.

Tumor angiogenesis often results in a low-pH and hypoxic TME, which eventually limits drug efficacy. The low-pH TME has been reported to promote the resistance of breast cancer cells to DOX⁴⁶. In addition, the hypoxic microenvironment caused by impaired oxygen delivery due to abnormal vascular systems affects drug efficacy. In breast cancer treatment, chemotherapeutic drugs, such as docetaxel, are among the most frequently used therapeutic agents. Typically, these drugs exert therapeutic effects by interfering with DNA synthesis, inducing apoptosis, and inhibiting cell division in breast cancer cells. Although chemotherapeutic drugs are more effective against actively proliferating cancer cells than proliferating inactive cancer cells, decreased aerobic oxidation in cancer cells under hypoxia decreases cell activity and proliferation, and consequently limits the effectiveness of chemotherapeutic drugs⁵³. However, no specific measures are currently available to prevent or treat drug resistance caused by angiogenesis in breast cancer. Therefore, investigating the mechanisms of action of various drugs and devising strategies to prevent drug resistance will be imperative to achieve more precise and effective targeting of breast cancer cells.

Tumor-associated internal resistance

The oncogenic signaling of the MAPK pathway associated with breast cancer is linked to the c-myc pathway through the VEGF promoter, which promotes VEGF transcription; subsequently, secreted proteins inhibit the recruitment and infiltration of specific T-cells in tumors⁵⁷. In addition, the loss of PTEN, a tumor suppressor gene, plays a major role in the invasion and progression of breast cancer. PTEN loss and VEGF overexpression are closely correlated in breast cancer. PTEN loss promotes VEGF expression by inducing HIF-1α and activating the PI3K-AKT pathway^58,59. PTEN loss in breast cancer is associated with resistance to chemotherapy⁵⁸ and immune checkpoint-associated immunotherapy, which may be associated with the activation of the PI3K-AKT pathway, thereby affecting T-cell infiltration in tumors⁶⁰. Both miR526b and miR655 regulate PTEN expression and upregulate VEGF expression, and hence promote angiogenesis in breast cancer. Altered PTEN pathway expression has been found to enhance the efficacy of anti-PD-1 and anti-CTLA-4 antibodies in mouse models⁶⁰ and to improve the chemoresistance of breast cancer cells to DOX⁶¹. Moreover, miR526b and miR655 may serve as targets for improving drug resistance in breast cancer via the PTEN/PI3K pathway. The WNT pathway, which is often involved in tumorigenesis signaling, is crucial for tumor angiogenesis. This pathway, under stabilization by β-catenin, is closely associated with immune suppression in tumors⁶². Moreover, this pathway has been associated with increased β-catenin levels, and decreased DC chemokine CCL103 and mature CD4 DC levels in tumors. The Wnt/β-catenin pathway, targeting suppresses stemness and angiogenesis, improves MDR in breast cancer⁶². Compared with mice with Wnt/β-catenin pathway activation, mice lacking β-catenin exhibit markedly enhanced immunotherapeutic efficacy⁵⁴. Conventionally, breast cancer has not been considered an immunogenic tumor. However, several recent studies have shown that breast cancer cells express immune inhibitory ligands, such as PD-1, which suppress CD8 T-cell activity and lead to immune resistance in breast cancer⁶³. In addition, tumor-associated intrinsic pathways such as MAPK/PI3K, WNT/β-catenin, and PD-1 are closely associated with tumor angiogenesis, and interactions among these pathways affect DC maturation, and T-cell recruitment and activation, and subsequently promote drug resistance in breast cancer. Targeting the key factor VEGFR2 promotes tumor vascular normalization, thus increasing immune cell infiltration and activation. Furthermore, low-dose anti-angiogenic therapy promotes osteopontin (OPN) secretion by CD8⁺ T-cells. Subsequently, OPN induces tumor cells to produce TGF-β, which in turn upregulates PD-1 expression on immune cells and ultimately increases the sensitivity of breast cancer to PD-1 immunotherapy. The combination of anti-angiogenic therapy and anti-PD-1 immunotherapy has been found to effectively overcome drug resistance in breast cancer⁶⁴.

Tumor-associated external resistance

Beyond abnormal vascularization due to tumor angiogenesis, hypoxia, and low pH induce an immunosuppressive TME⁶⁵. VEGF highly expressed on the endothelium of peripheral tumor blood vessels binds VEGFR1, and subsequently inhibits DC maturation and antigen presentation, and impedes T-cell activation and infiltration⁶⁶. High VEGF expression promotes the accumulation of peripheral myeloid-derived suppressor cells (MDSCs), which are closely associated with cancer angiogenesis and immunosuppression⁶⁷. MDSCs regulate breast CSCs via the CXCL2-CXCR2 pathway, thereby inducing resistance to docetaxel⁶⁸. Furthermore, MDSC targeting and inhibition via the SDF1α/CXCR4 axis enhance the anti-tumor activity of chimeric antigen receptor (CAR)-T-cell immunotherapy in breast cancer⁶⁹. High VEGF expression during angiogenesis in breast cancer decreases CD8 T-cell levels via the HMG-bOX (TOX) pathway, thereby increasing tumor-cell tolerance to immunotherapy⁷⁰ (Figure 3F). The combination of VEGF, interleukin-10, and prostaglandin E2 promotes the expression of the death ligand FasL in endothelial cells. FasL is an ectopic expression product in mouse solid tumors and is undetectable in normal vascular systems. FasL promotes the tumor-specific exclusion of cytotoxic T-cells, facilitates the formation of an immunosuppressive TME, and leads to the development of drug resistance⁷¹ (Figure 3G). In addition, angiopoietin 2 and placental growth factor, which are associated with tumor angiogenesis, induce macrophage polarization toward the M2 phenotype, thereby leading to the development of an immunosuppressive TME⁷². Moreover, vascular endothelial cells in the breast cancer microenvironment release several miRNAs (miR-142-5p, miR-183-5p, and miR-222-3p) that are transported through extracellular vesicles, and subsequently regulate macrophage remodeling and promote macrophage polarization toward the M2 phenotype⁷³ (Figure 3H). Anti-angiogenesis treatment to promote tumor vessel normalization has been found to reverse the tumor immunosuppressive microenvironment and improve cancer treatment efficacy⁷⁴. Thus, the combination of anti-angiogenesis therapy and immunotherapy may enhance the efficacy of immunotherapy against breast cancer. However, several challenges exist in the application of such combination treatment strategies. Future studies should focus on the appropriate dosages of anti-angiogenic drugs and their potential toxicity after combined treatment, to overcome persisting challenges.