Enhanced Therapeutic Effect of Optimized Melittin-dKLA,a Peptide Agent Targeting M2-like Tumor-Associated Macrophages in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is characterized by a high possibility of metastasis. M2-like tumor-associated macrophages (TAMs) are the main components of the tumor microenvironment (TME) and play a key role in TNBC metastasis. Therefore, TAMs may be a potential target for reducing TNBC metastasis. Melittin-dKLA, a peptide composed of fused melittin and pro-apoptotic peptide d(KLAKLAK)2 (dKLA), showed a potent therapeutic effect against cancers by depleting TAMs. However, melittin has a strong adverse hemolytic effect. Hence, we attempted to improve the therapeutic potential of melittin-dKLA by reducing toxicity and increasing stability. Nine truncated melittin fragments were synthesized and examined. Of the nine peptides, the melittin-dKLA8-26 showed the best binding properties to M2 macrophages and discriminated M0/M1/M2. All fragments, except melittin, lost their hemolytic effects. To increase the stability of the peptide, melittin-dKLA8-26 fragment was conjugated with PEGylation at the amino terminus and was named PEG-melittin-dKLA8-26. This final drug candidate was assessed in vivo in a murine TNBC model and showed superior effects on tumor growth, survival rates, and lung metastasis compared with the previously used melittin-dKLA. Taken together, our study showed that the novel PEG-melittin-dKLA8-26 possesses potential as a new drug for treating TNBC and TNBC-mediated metastasis by targeting TAMs.     

Aurora Kinase A and Bcl-xL Inhibition Suppresses Metastasis in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a heterogeneous disease that accounts for 10–15% of all breast cancer cases. Within TNBC, the treatment of basal B is the most challenging due to its highly invasive potential, and thus treatments to suppress metastasis formation in this subgroup are urgently needed. However, the mechanisms underlying the metastatic ability of TNBC remain unclear. In the present study, we investigated the role of Aurora A and Bcl-xL in regulating basal B cell invasion. We found gene amplification and elevated protein expression in the basal B cells, which also showed increased invasiveness in vitro, compared to basal A cells. Chemical inhibition of Aurora A with alisertib and siRNA-mediated knockdown of BCL2L1 decreased the number of invading cells compared to non-treated cells in basal B cell lines. The analysis of the correlation between AURKA and BCL2L1 expression in TNBC and patient survival revealed significantly decreased relapse-free survival (n = 534, p = 0.012) and distant metastasis-free survival (n = 424, p = 0.017) in patients with primary tumors exhibiting a high combined expression of AURKA and BCL2L1. Together, our findings suggest that high levels of Aurora A and Bcl-xL promote metastasis, and inhibition of these proteins may suppress metastasis and improve patient survival in basal B TNBC.     

An Eleven-microRNA Signature Related to Tumor-Associated Macrophages Predicts Prognosis of Breast Cancer

The dysregulation of microRNAs (miRNAs) has been known to play important roles in tumor development and progression. However, the understanding of the involvement of miRNAs in regulating tumor-associated macrophages (TAMs) and how these TAM-related miRNAs (TRMs) modulate cancer progression is still in its infancy. This study aims to explore the prognostic value of TRMs in breast cancer via the construction of a novel TRM signature. Potential TRMs were identified from the literature, and their prognostic value was evaluated using 1063 cases in The Cancer Genome Atlas Breast Cancer database. The TRM signature was further validated in the external Gene Expression Omnibus {"type":"entrez-geo","attrs":{"text":"GSE22220","term_id":"22220"}}GSE22220 dataset. Gene sets enrichment analyses were performed to gain insight into the biological functions of this TRM signature. An eleven-TRM signature consisting of mir-21, mir-24-2, mir-125a, mir-221, mir-22, mir-501, mir-365b, mir-660, mir-146a, let-7b and mir-31 was constructed. This signature significantly differentiated the high-risk group from the low-risk in terms of overall survival (OS)/ distant-relapse free survival (DRFS) (p value < 0.001). The prognostic value of the signature was further enhanced by incorporating other independent prognostic factors in a nomogram-based prediction model, yielding the highest AUC of 0.79 (95% CI: 0.72–0.86) at 5-year OS. Enrichment analyses confirmed that the differentially expressed genes were mainly involved in immune-related pathways such as adaptive immune response, humoral immune response and Th1 and Th2 cell differentiation. This eleven-TRM signature has great potential as a prognostic factor for breast cancer patients besides unravelling the dysregulated immune pathways in high-risk breast cancer.     

Current Advancements of Plant-Derived Agents for Triple-Negative Breast Cancer Therapy through Deregulating Cancer Cell Functions and Reprogramming Tumor Microenvironment

Triple-negative breast cancer (TNBC) is defined based on the absence of estrogen, progesterone, and human epidermal growth factor receptor 2 receptors. Currently, chemotherapy is the major therapeutic approach for TNBC patients; however, poor prognosis after a standard chemotherapy regimen is still commonplace due to drug resistance. Abnormal tumor metabolism and infiltrated immune or stromal cells in the tumor microenvironment (TME) may orchestrate mammary tumor growth and metastasis or give rise to new subsets of cancer cells resistant to drug treatment. The immunosuppressive mechanisms established in the TME make cancer cell clones invulnerable to immune recognition and killing, and turn immune cells into tumor-supporting cells, hence allowing cancer growth and dissemination. Phytochemicals with the potential to change the tumor metabolism or reprogram the TME may provide opportunities to suppress cancer metastasis and/or overcome chemoresistance. Furthermore, phytochemical intervention that reprograms the TME away from favoring immunoevasion and instead towards immunosurveillance may prevent TNBC metastasis and help improve the efficacy of combination therapies as phyto-adjuvants to combat drug-resistant TNBC. In this review, we summarize current findings on selected bioactive plant-derived natural products in preclinical mouse models and/or clinical trials with focus on their immunomodulatory mechanisms in the TME and their roles in regulating tumor metabolism for TNBC prevention or therapy.     

EpCAM- and EGFR-Specific Antibody Drug Conjugates for Triple-Negative Breast Cancer Treatment

Triple-negative breast cancer (TNBC) is a group of heterogeneous and refractory breast cancers with the absence of estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor receptor 2 (HER2). Over the past decade, antibody drug conjugates (ADCs) have ushered in a new era of targeting therapy. Since the epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) are over expressed on triple-negative breast cancer, we developed novel ADCs by conjugating benzylguanine (BG)-modified monomethyl auristatin E (MMAE) to EpCAM- and EGFR-specific SNAP-tagged single chain antibody fragments (scFvs). Rapid and efficient conjugation was achieved by SNAP-tag technology. The binding and internalization properties of scFv-SNAP fusion proteins were confirmed by flow cytometry and fluorescence microscopy. The dose-dependent cytotoxicity was evaluated in cell lines expressing different levels of EGFR and EpCAM. Both ADCs showed specific cytotoxicity to EGFR or EpCAM positive cell lines via inducing apoptosis at a nanomolar concentration. Our study demonstrated that EGFR specific scFv-425-SNAP-BG-MMAE and EpCAM-specific scFv-EpCAM-SNAP-BG-MMAE could be promising ADCs for the treatment of TNBC.     

Cracking the Breast Cancer Glyco-Code through Glycan-Lectin Interactions: Targeting Immunosuppressive Macrophages

The immune microenvironment of breast cancer (BC) is composed by high macrophage infiltrates, correlated with the most aggressive subtypes. Tumour-associated macrophages (TAM) within the BC microenvironment are key regulators of immune suppression and BC progression. Nevertheless, several key questions regarding TAM polarisation by BC are still not fully understood. Recently, the modulation of the immune microenvironment has been described via the recognition of abnormal glycosylation patterns at BC cell surface. These patterns rise as a resource to identify potential targets on TAM in the BC context, leading to the development of novel immunotherapies. Herein, we will summarize recent studies describing advances in identifying altered glycan structures in BC cells. We will focus on BC-specific glycosylation patterns known to modulate the phenotype and function of macrophages recruited to the tumour site, such as structures with sialylated or N-acetylgalactosamine epitopes. Moreover, the lectins present at the surface of macrophages reported to bind to such antigens, inducing tumour-prone TAM phenotypes, will also be highlighted. Finally, we will discuss and give our view on the potential and current challenges of targeting these glycan-lectin interactions to reshape the immunosuppressive landscape of BC.     

EZH2–CCF–cGAS Axis Promotes Breast Cancer Metastasis

Cytoplasmic chromatin fragments (CCF) are recognized by the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS), which activates the cGAS–STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway and promotes the production of inflammatory factors and breast cancer metastasis. However, the mechanisms by which CCF are formed in tumor cells and CCF activation cGAS promotes breast cancer metastasis remain unclear. Here, we report that the enhancer of zeste homolog 2 (EZH2) can promote the formation of CCF and activate the cGAS–STING pathway to promote breast cancer metastasis. Further research found that the EZH2-mediated CCF formation depended on high mobility group A1 (HMGA1), while the stability of EZH2 required ubiquitin-specific peptidase 7 (USP7), indicating that the EZH2–HMGA1–USP7 complex regulated CCF formation. Moreover, EZH2 can activate cGAS through CCF, requiring USP7 to deubiquitinate cGAS and stabilize cGAS. In vivo experimental results showed that EZH2 could promote breast cancer metastasis through CCF. Our findings highlight a new target for breast cancer metastasis. Targeting the EZH2–CCF–cGAS axis may be a potential therapeutic strategy for inhibiting breast cancer metastasis.     

CTI-2 Inhibits Metastasis and Epithelial-Mesenchymal Transition of Breast Cancer Cells by Modulating MAPK Signaling Pathway

Although some breast cancer patients die due to tumor metastasis rather than from the primary tumor, the molecular mechanism of metastasis remains unclear. Therefore, it is necessary to inhibit breast cancer metastasis during cancer treatment. In this case, after designing and synthesizing CTI-2, we found that CTI-2 treatment significantly reduced breast cancer cell metastasis in vivo and in vitro. Notably, with the treatment of CTI-2 in breast cancer cells, the expression level of E-cadherin increased, while the expression level of N-cadherin and vimentin decreased. In addition, after CTI-2 treatment, those outflow levels for p-ERK, p-p38, and p-JNK diminished, while no significant changes in the expression levels of ERK, JNK, or p38 were observed. Our conclusion suggested that CTI-2 inhibits the epithelial-mesenchymal transition (EMT) of breast carcinoma cells by inhibiting the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, thereby inhibiting the metastasis of breast tumor cells. Therefore, we believe that CTI-2 is another candidate for breast tumor medication.     

The In Vivo Selection Method in Breast Cancer Metastasis

Metastasis is a complex event in cancer progression and causes most deaths from cancer. Repeated transplantation of metastatic cancer cells derived from transplanted murine organs can be used to select the population of highly metastatic cancer cells; this method is called as in vivo selection. The in vivo selection method and highly metastatic cancer cell lines have contributed to reveal the molecular mechanisms of cancer metastasis. Here, we present an overview of the methodology for the in vivo selection method. Recent comparative analysis of the transplantation methods for metastasis have revealed the divergence of metastasis gene signatures. Even cancer cells that metastasize to the same organ show various metastatic cascades and gene expression patterns by changing the transplantation method for the in vivo selection. These findings suggest that the selection of metastasis models for the study of metastasis gene signatures has the potential to influence research results. The study of novel gene signatures that are identified from novel highly metastatic cell lines and patient-derived xenografts (PDXs) will be helpful for understanding the novel mechanisms of metastasis.     

SIRT1/FOXO Signaling Pathway in Breast Cancer Progression and Metastasis

Breast cancer is the second most common cancer in women. The roles of the SIRT and FoxO proteins in tumor progression are known, but their roles in metastasis have not yet been clearly elucidated. In our study, we investigated the roles of SIRT and FoxO proteins their downstream pathways, proteins p21 and p53, in tumor progression and metastasis. We evaluated these proteins in vitro using metastatic 4TLM and 67NR cell lines, as well as their expression levels in tumor-bearing mice. In addition, the regulatory role of SIRT and FoxO proteins in different transduction cascades was examined by IPA core analysis, and clinicopathological evidence was investigated in the TCGA database. In primary tumors, the expression levels of SIRT1, p21, p53, E2F1 and FoxO proteins were higher in 67NR groups. In metastatic tissues, the expression levels of SIRT1, E2F1 and FoxO proteins were found to be enhanced, whereas the levels of p53 and p21 expression were noted to be reduced. IPA analysis also provided empirical evidence of the mechanistic involvement of SIRT and FoxO proteins in tumor progression and metastasis. In conclusion, SIRT1 was found to co-operate with FoxO proteins and to play a critical role in metastasis. Additional research is required to determine why overexpression of SIRT1 in metastatic tissues has oncogenic effects.     

The Role of MicroRNAs in Breast Cancer Migration, Invasion and Metastasis

MicroRNAs (miRNAs) are a major class of small, noncoding RNA molecules that regulate gene expression by targeting mRNAs to trigger either translational repression or mRNA degradation. They have recently been more widely investigated due to their potential role as targets for cancer therapy. Many miRNAs have been implicated in several human cancers, including breast cancer. miRNAs are known to regulate cell cycle and development, and thus may serve as useful targets for exploration in anticancer therapeutics. The link between altered miRNA signatures and breast cancer development and metastasis can be observed either through the loss of tumor suppressor miRNAs, such as let-7s, miR-30a/31/34a/125s/200s/203/205/206/342 or the overexpression of oncogenic miRNAs, such as miR-10b/21/135a/155/221/222/224/373/520c in breast cancer cells. Some of these miRNAs have also been validated in tumor specimens of breast cancer patients, underscoring their potential roles in diagnostics, as well as targets for novel therapeutics for breast cancer. In this review article, we will provide an overview and update of our current understanding of the mode of action of several of these well characterized miRNAs in breast cancer models. Therefore, better understanding of the gene networks orchestrated by these miRNAs may help exploit the full potential of miRNAs in regards to cancer diagnosis, treatment, and therapeutics.     

Spatial and Quantitative Analysis of Tumor-Associated Macrophages: Intratumoral CD163-/PD-L1+ TAMs as a Marker of Favorable Clinical Outcomes in Triple-Negative Breast Cancer

Tumor-associated macrophages (TAMs) and abnormalities in cancer cells affect cancer progression and response to therapy. TAMs are a major component of the tumor microenvironment (TME) in breast cancer, with their invasion affecting clinical outcomes. Programmed death-ligand 1 (PD-L1), a target of immune checkpoint inhibitors, acts as a suppressive signal for the surrounding immune system; however, its expression and effect on TAMs and the clinical outcome in breast cancer are unknown. In this study, we used high-throughput multiple immunohistochemistry to spatially and quantitatively analyze TAMs. We subjected 81 breast cancer specimens to immunostaining for CD68, CD163, PD-1, PD-L1, CD20, and pan-CK. In both stromal and intratumoral areas, the triple-negative subtype had significantly more CD68/CD163, CD68/PD-L1, and CD163/PD-L1 double-positive cells than the estrogen receptor (ER)/progesterone receptor (PR) subtype. Interestingly, a higher number of CD68+/PD-L1+/CK-/CD163- TAMs in the intratumoral area was correlated with a favorable recurrence rate (p = 0.048). These findings indicated that the specific subpopulation and localization of TAMs in the TME affect clinical outcomes in breast cancer.     

Development of a Patient-Derived Xenograft (PDX) of Breast Cancer Bone Metastasis in a Zebrafish Model

Bone metastasis is a complex process that needs to be better understood in order to help clinicians prevent and treat it. Xenografts using patient-derived material (PDX) rather than cancer cell lines are a novel approach that guarantees more clinically realistic results. A primary culture of bone metastasis derived from a 67-year-old patient with breast cancer was cultured and then injected into zebrafish (ZF) embryos to study its metastatic potential. In vivo behavior and results of gene expression analyses of the primary culture were compared with those of cancer cell lines with different metastatic potential (MCF7 and MDA-MB-231). The MCF7 cell line, which has the same hormonal receptor status as the bone metastasis primary culture, did not survive in the in vivo model. Conversely, MDA-MB-231 disseminated and colonized different parts of the ZF, including caudal hematopoietic tissues (CHT), revealing a migratory phenotype. Primary culture cells disseminated and in later stages extravasated from the vessels, engrafting into ZF tissues and reaching the CHT. Primary cell behavior reflected the clinical course of the patient’s medical history. Our results underline the potential for using PDX models in bone metastasis research and outline new methods for the clinical application of this in vivo model.     

TIAM2 Contributes to Osimertinib Resistance,Cell Motility,and Tumor-Associated Macrophage M2-like Polarization in Lung Adenocarcinoma

Background: Osimertinib-based therapy effectively improves the prognosis of lung adenocarcinoma (LUAD) patients with epidermal growth factor receptor mutations. However, patients will have cancer progression after approximately one year due to the occurrence of drug resistance. Extensive evidence has revealed that lipid metabolism and tumor-associated macrophage (TAM) are associated with drug resistance, which deserves further exploration. Methods: An osimertinib resistance index (ORi) was built to investigate the link between lipid metabolism and osimertinib resistance. The ORi was constructed and validated using TCGA and GEO data, and the relationship between ORi and immune infiltration was discussed. Weighted gene co-expression network analysis based on the M2/M1 macrophage ratio determined the hub gene TIAM2 and the biological function of TIAM2 in LUAD was verified in vitro. Results: ORi based on nine lipid metabolism-related genes was successfully constructed, which could accurately reflect the resistance of LUAD patients to osimertinib, predict the prognosis, and correlate with M2-like TAM. Additionally, TIAM2 was found to increase osimertinib tolerance, enhance cell motility, and promote M2-like TAM polarization in LUAD. Conclusions: The lipid metabolism gene is strongly connected with osimertinib resistance. TIAM2 contributes to osimertinib resistance, enhances cell motility, and induces M2-like TAM polarization in LUAD.     

The Interaction of the Senescent and Adjacent Breast Cancer Cells Promotes the Metastasis of Heterogeneous Breast Cancer Cells through Notch Signaling

Chemotherapy is one of the most common strategies for tumor treatment but often associated with post-therapy tumor recurrence. While chemotherapeutic drugs are known to induce tumor cell senescence, the roles and mechanisms of senescence in tumor recurrence remain unclear. In this study, we used doxorubicin to induce senescence in breast cancer cells, followed by culture of breast cancer cells with conditional media of senescent breast cancer cells (indirect co-culture) or directly with senescent breast cancer cells (direct co-culture). We showed that breast cancer cells underwent the epithelial–mesenchymal transition (EMT) to a greater extent and had stronger migration and invasion ability in the direct co-culture compared with that in the indirect co-culture model. Moreover, in the direct co-culture model, non-senescent breast cancer cells facilitated senescent breast cancer cells to escape and re-enter into the cell cycle. Meanwhile, senescent breast cancer cells regained tumor cell characteristics and underwent EMT after direct co-culture. We found that the Notch signaling was activated in both senescent and non-senescent breast cancer cells in the direct co-culture group. Notably, the EMT process of senescent and adjacent breast cancer cells was blocked upon inhibition of Notch signaling with N-[(3,5-difluorophenyl)acetyl]-l-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) in the direct co-cultures. In addition, DAPT inhibited the lung metastasis of the co-cultured breast cancer cells in vivo. Collectively, data arising from this study suggest that both senescent and adjacent non-senescent breast cancer cells developed EMT through activating Notch signaling under conditions of intratumoral heterogeneity caused by chemotherapy, which infer the possibility that Notch inhibitors used in combination with chemotherapeutic agents may become an effective treatment strategy.     

Biphasic α2β1 Integrin Expression in Breast Cancer Metastasis to Bone

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2β1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2β1 in the context of bone metastasis. In this study, we aimed to understand the role of α2β1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2β1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2β1 expression and bone-metastatic potential. Inhibiting α2β1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.