TDP2 is a regulator of estrogen-responsive oncogene expression.

With its ligand estrogen, the estrogen receptor (ER) initiates a global transcriptional program, promoting cell growth. This process involves topoisomerase 2 (TOP2), a key protein in resolving topological issues during transcription by cleaving a DNA duplex, passing another duplex through the break, and repairing the break. Recent studies revealed the involvement of various DNA repair proteins in the repair of TOP2-induced breaks, suggesting potential alternative repair pathways in cases where TOP2 is halted after cleavage. However, the contribution of these proteins in ER-induced transcriptional regulation remains unclear. We investigated the role of tyrosyl-DNA phosphodiesterase 2 (TDP2), an enzyme for the removal of halted TOP2 from the DNA ends, in the estrogen-induced transcriptome using both targeted and global transcription analyses. MYC activation by estrogen, a TOP2-dependent and transient event, became prolonged in the absence of TDP2 in both TDP2-deficient cells and mice. Bulk and single-cell RNA-seq analyses defined MYC and CCND1 as oncogenes whose estrogen response is tightly regulated by TDP2. These results suggest that TDP2 may inherently participate in the repair of estrogen-induced breaks at specific genomic loci, exerting precise control over oncogenic gene expression.

Reciprocal regulation of forkhead box C1 and L1 cell adhesion molecule contributes to triple-negative breast cancer progression.

PURPOSE: The potential of targeting forkhead box C1 (FOXC1) as a therapeutic approach for triple-negative breast cancer (TNBC) is promising. However, a comprehensive understanding of FOXC1 regulation, particularly upstream factors, remains elusive. Expression of the L1 cell adhesion molecule (L1CAM), a transmembrane glycoprotein associated with brain metastasis, was observed to be positively associated with FOXC1 transcripts. Thus, this study aims to investigate their relationship in TNBC progression. METHODS: Publicly available FOXC1 and L1CAM transcriptomic data were obtained, and their corresponding proteins were analyzed in four TNBC cell lines. In BT549 cells, FOXC1 and L1CAM were individually silenced, while L1CAM was overexpressed in BT549-shFOXC1, MDA-MB-231, and HCC1937 cells. CCK-8, transwell, and wound healing assays were performed in these cell lines, and immunohistochemical staining was conducted in tumor samples. RESULTS: A positive correlation between L1CAM and FOXC1 transcripts was observed in publicly available datasets. In BT549 cells, knockdown of FOXC1 led to reduced L1CAM expression at both the transcriptional and protein levels, and conversely, silencing of L1CAM decreased FOXC1 protein levels, but interestingly, FOXC1 transcripts remained largely unaffected. Overexpressing L1CAM resulted in increased FOXC1 protein expression without significant changes in FOXC1 mRNA levels. This trend was also observed in BT549-shFOXC1, MDA-MB-231-L1CAM, and HCC1937-L1CAM cells. Notably, alterations in FOXC1 or L1CAM levels corresponded to changes in cell proliferation, migration, and invasion capacities. Furthermore, a positive correlation between L1CAM and FOXC1 protein expression was detected in human TNBC tumors. CONCLUSION: FOXC1 and L1CAM exhibit co-regulation at the protein level, with FOXC1 regulating at the transcriptional level and L1CAM regulating at the post-transcriptional level, and together they positively influence cell proliferation, migration, and invasion in TNBC.

Overcoming Resistance in Prostate Cancer Therapy Using a DZ-Simvastatin Conjugate.

Prostate cancer (PC), particularly its metastatic castration-resistant form (mCRPC), is a leading cause of cancer-related deaths among men in the Western world. Traditional systemic treatments, including hormonal therapy and chemotherapy, offer limited effectiveness due to tumors' inherent resistance to these therapies. Moreover, they often come with significant side effects. We have developed a delivery method using a tumor-cell-specific heptamethine carbocyanine dye (DZ) designed to transport therapeutic agents directly to tumor cells. This research evaluated simvastatin (SIM) as the antitumor payload because of its demonstrated chemopreventive effects on human cancers and its well-documented safety profile. We designed and synthesized a DZ-SIM conjugate for tumor cell targeting. PC cell lines and xenograft tumor models were used to assess tumor-cell targeting specificity and killing activity and to investigate the corresponding mechanisms. DZ-SIM treatment effectively killed PC cells regardless of their androgen receptor status or inherent therapeutic resistance. The conjugate targeted and suppressed xenograft tumor formation without harming normal cells of the host. In cancer cells, DZ-SIM was enriched in subcellular organelles, including mitochondria, where the conjugate formed adducts with multiple proteins and caused the loss of transmembrane potential, promoting cell death. The DZ-SIM specifically targets PC cells and their mitochondria, resulting in a loss of mitochondrial function and cell death. With a unique subcellular targeting strategy, the conjugate holds the potential to outperform existing chemotherapeutic drugs. It presents a novel strategy to circumvent therapeutic resistance, offering a more potent treatment for mCRPC.

Single protein encapsulated SN38 for tumor-targeting treatment.

BACKGROUND: The alkaloid camptothecin analog SN38 is a potent antineoplastic agent, but cannot be used directly for clinical application due to its poor water solubility. Currently, the prodrug approach on SN38 has resulted in 3 FDA-approved cancer therapeutics, irinotecan, ONIVYDE, and Trodelvy. However, only 2-8% of irinotecan can be transformed enzymatically in vivo into the active metabolite SN38, which severely limits the drug's efficacy. While numerous drug delivery systems have been attempted to achieve effective SN38 delivery, none have produced drug products with antitumor efficacy better than irinotecan in clinical trials. Therefore, novel approaches are urgently needed for effectively delivering SN38 to cancer cells with better efficacy and lower toxicity. METHODS: Based on the unique properties of human serum albumin (HSA), we have developed a novel single protein encapsulation (SPE) technology to formulate cancer therapeutics for improving their pharmacokinetics (PK) and antitumor efficacy and reducing their side effects. Previous application of SPE technology to doxorubicin (DOX) formulation has led to a promising drug candidate SPEDOX-6 (FDA IND #, 152154), which will undergo a human phase I clinical trial. Using the same SPE platform on SN38, we have now produced two SPESN38 complexes, SPESN38-5 and SPESN38-8. We conducted their pharmacological evaluations with respect to maximum tolerated dose, PK, and in vivo efficacy against colorectal cancer (CRC) and soft tissue sarcoma (STS) in mouse models. RESULTS: The lyophilized SPESN38 complexes can dissolve in aqueous media to form clear and stable solutions. Maximum tolerated dose (MTD) of SPESN38-5 is 250 mg/kg by oral route (PO) and 55 mg/kg by intravenous route (IV) in CD-1 mice. SPESN38-8 has the MTD of 45 mg/kg by IV in the same mouse model. PK of SPESN38-5 by PO at 250 mg/kg gave mouse plasma AUC0-∞ of 0.05 and 4.5 nmol × h/mL for SN38 and SN38 glucuronidate (SN38G), respectively, with a surprisingly high molar ratio of SN38G:SN38 = 90:1. However, PK of SPESN38-5 by IV at 55 mg/kg yielded much higher mouse plasma AUC0-∞ of 19 and 28 nmol × h/mL for SN38 and SN38G, producing a much lower molar ratio of SN38G:SN38 = 1.5:1. Antitumor efficacy of SPESN38-5 and irinotecan (control) was evaluated against HCT-116 CRC xenograft tumors. The data indicates that SPESN38-5 by IV at 55 mg/kg is more effective in suppressing HCT-116 tumor growth with lower systemic toxicity compared to irinotecan at 50 mg/kg. Additionally, SPESN38-8 and DOX (control) by IV were evaluated in the SK-LMS-1 STS mouse model. The results show that SPESN38-8 at 33 mg/kg is highly effective for inhibiting SK-LMS-1 tumor growth with low toxicity, in contrast to DOX's insensitivity to SK-LMS-1 with high toxicity. CONCLUSION: SPESN38 complexes provide a water soluble SN38 formulation. SPESN38-5 and SPESN38-8 demonstrate better PK values, lower toxicity, and superior antitumor efficacy in mouse models, compared with irinotecan and DOX.

RGCC-mediated PLK1 activity drives breast cancer lung metastasis by phosphorylating AMPKα2 to activate oxidative phosphorylation and fatty acid oxidation.

BACKGROUND: More than 90% of the mortality of triple-negative breast cancer (TNBC) patients is attributed to cancer metastasis with organotropism. The lung is a frequent site of TNBC metastasis. However, the precise molecular mechanism for lung-specific metastasis of TNBC is not well understood. METHODS: RNA sequencing was performed to identify patterns of gene expression associated with lung metastatic behavior using 4T1-LM3, MBA-MB-231-LM3, and their parental cells (4T1-P, MBA-MB-231-P). Expressions of RGCC, called regulator of cell cycle or response gene to complement 32 protein, were detected in TNBC cells and tissues by qRT-PCR, western blotting, and immunohistochemistry. Kinase activity assay was performed to evaluate PLK1 kinase activity. The amount of phosphorylated AMP-activated protein kinase α2 (AMPKα2) was detected by immunoblotting. RGCC-mediated metabolism was determined by UHPLC system. Oxidative phosphorylation was evaluated by JC-1 staining and oxygen consumption rate (OCR) assay. Fatty acid oxidation assay was conducted to measure the status of RGCC-mediated fatty acid oxidation. NADPH and ROS levels were detected by well-established assays. The chemical sensitivity of cells was evaluated by CCK8 assay. RESULTS: RGCC is aberrantly upregulated in pulmonary metastatic cells. High level of RGCC is significantly related with lung metastasis in comparison with other organ metastases. RGCC can effectively promote kinase activity of PLK1, and the activated PLK1 phosphorylates AMPKα2 to facilitate TNBC lung metastasis. Mechanistically, the RGCC/PLK1/AMPKα2 signal axis increases oxidative phosphorylation of mitochondria to generate more energy, and promotes fatty acid oxidation to produce abundant NADPH. These metabolic changes contribute to sustaining redox homeostasis and preventing excessive accumulation of potentially detrimental ROS in metastatic tumor cells, thereby supporting TNBC cell survival and colonization during metastases. Importantly, targeting RGCC in combination with paclitaxel/carboplatin effectively suppresses pulmonary TNBC lung metastasis in a mouse model. CONCLUSIONS: RGCC overexpression is significantly associated with lung-specific metastasis of TNBC. RGCC activates AMPKα2 and downstream signaling through RGCC-driven PLK1 activity to facilitate TNBC lung metastasis. The study provides implications for RGCC-driven OXPHOS and fatty acid oxidation as important therapeutic targets for TNBC treatment.

Androgen receptor in breast cancer and its clinical implication.

Breast cancer is a heterogeneous group of diseases characterized by diverse subtypes. Currently, the classification of breast cancer is based on the status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). In addition to these receptors, the presence of the androgen receptor (AR) in breast cancer cells adds a layer of complexity to our understanding of the disease. The role of AR in breast cancer is intricate, as it can alter diverse signaling pathways in the presence of different hormone receptors (HRs). This complex interplay between signaling pathways affects patient outcomes and prognosis, and the presence of AR has a significant effect. While AR positivity is common in breast cancer, the efficacy of utilizing AR blockade as a monotherapy has been limited, demonstrating only modest results. To address this challenge, substantial efforts have been directed toward comprehending the intricacies of AR's role and pathways in breast cancer development in the hope of understanding its utility as a biomarker or drug target. Multiple ongoing clinical trials are currently investigating combination treatments involving AR inhibitors and other agents to disrupt oncogenic signaling pathways and their crosstalk. Particularly in the context of triple-negative breast cancer (TNBC), where targeted therapeutic options are lacking, extensive research efforts have been dedicated to exploring the potential of AR-related interventions. This review aims to provide an overview of the various breast cancer subtypes with AR signaling mechanisms, and ongoing clinical trials that hold the potential to reshape future clinical approaches.

Antibody-Drug Conjugates in Breast Cancer: Current Status and Future Directions.

Antibody drug conjugates (ADCs) are novel medications that combine monoclonal antibodies with cytotoxic payloads, enabling the selective delivery of potent drugs to cancer cells expressing specific surface antigens. This targeted strategy seeks to optimize treatment effectiveness while reducing the risk of systemic toxicity, distinguishing ADCs from conventional chemotherapy. The rapid growth in ADC research has led to numerous developments and approvals for cancer treatment, with significant impacts on the management of breast cancer. ADCs like T-DXd for HER2-low disease and sacituzumab govitecan for triple negative breast cancer (TNBC) have provided valuable options for challenging subtypes of breast cancer. However, essential questions still need to be addressed, including the optimal order of ADCs amidst the growing number of newly developed ones and strategies to overcome resistance mechanisms. Preclinical studies have shed light on potential resistance mechanisms, emphasizing the potential benefit of combinational approaches with other agents such as immune checkpoint inhibitors (ICIs) and targeted tyrosine kinase inhibitors (TKIs) to enhance treatment effectiveness. Additionally, personalized approaches based on molecular profiling hold promise in tailoring ADC treatments to individual tumors, identifying unique molecular markers for each patient to optimize treatment efficacy while minimizing side effects.

Human Breast Organoid Models for Lactation Research.

The human mammary gland is the major organ involved in lactation. In the mammary gland, alveoli secrete milk and myoepithelial cells contract to propel the milk through branched structures called ducts and eventually to the nipple. It is through this process of lactation that infants receive milk, which is essential for proper infant growth and development. The lactation process is comprised of sophisticated interactive networks at the cellular level that are not well understood. Whereas the majority of published mammary gland lactation studies have relied on mouse mammary glands, recent advancements in techniques to study mammary glands enable in vitro reproduction of lactation using human-representative frameworks. Currently, the 3D breast organoid is the state-of-the-art model in human mammary gland research, utilizing induced pluripotent stem cells (iPSCs) or processed patient-derived breast tissues embedded in a special matrix that are then able to grow into complex structures that recapitulate aspects of native human breast tissue. Gaining comprehensive biological insight into the process of lactation through these breast tissue-mimetic 3D models is essential for further studies on lactation-associated human mammary gland diseases, human milk composition, and potential solutions to challenges in maternal milk accessibility. In this short review, the benefits and potential utility of 3D breast organoids in understanding the underlying science of lactation and advancing further human mammary gland studies are discussed.

Protein-encapsulated doxorubicin reduces cardiotoxicity in hiPSC-cardiomyocytes and cardiac spheroids while maintaining anticancer efficacy.

The chemotherapeutic doxorubicin (DOX) detrimentally impacts the heart during cancer treatment. This necessitates development of non-cardiotoxic delivery systems that retain DOX anticancer efficacy. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), cardiac fibroblasts (hiPSC-CFs), multi-lineage cardiac spheroids (hiPSC-CSs), patient-specific hiPSCs, and multiple human cancer cell lines to compare the anticancer efficacy and reduced cardiotoxicity of single protein encapsulated DOX (SPEDOX-6), to standard unformulated (UF) DOX. Cell viability assays and immunostaining in human cancer cells, hiPSC-ECs, and hiPSC-CFs revealed robust uptake of SPEDOX-6 and efficacy in killing these proliferative cell types. In contrast, hiPSC-CMs and hiPSC-CSs exhibited substantially lower cytotoxicity during SPEDOX-6 treatment compared with UF DOX. SPEDOX-6-treated hiPSC-CMs and hiPSC-CSs maintained their functionality, as indicated by sarcomere contractility assessment, calcium imaging, multielectrode arrays, and RNA sequencing. This study demonstrates the potential of SPEDOX-6 to alleviate cardiotoxic side effects associated with UF DOX, while maintaining its anticancer potency.

Single Protein Encapsulated SN38 for Tumor-Targeting Treatment.

Background: The alkaloid camptothecin analog SN38 is a potent antineoplastic agent, but cannot be used directly for clinical application due to its poor water solubility. Currently, the prodrug approach on SN38 has resulted in 3 FDA-approved cancer therapeutics, irinotecan, ONIVYDE, and Trodelvy. However, only 2-8% of irinotecan can be transformed enzymatically in vivo into the active metabolite SN38, which severely limits the drug's efficacy. While numerous drug delivery systems have been attempted to achieve effective SN38 delivery, none have produced drug products with antitumor efficacy better than irinotecan in clinical trials. Therefore, novel approaches are urgently needed for effectively delivering SN38 to cancer cells with better efficacy and lower toxicity. Methods: Based on the unique properties of human serum albumin (HSA), we have developed a novel single protein encapsulation (SPE) technology to formulate cancer therapeutics for improving their pharmacokinetics (PK) and antitumor efficacy and reducing their side effects. Previous application of SPE technology to doxorubicin (DOX) formulation has led to a promising drug candidate SPEDOX-6 (FDA IND #, 152154), which will undergo a human phase I clinical trial. Using the same SPE platform on SN38, we have now produced two SPESN38 complexes, SPESN38-5 and SPESN38-8. We conducted their pharmacological evaluations with respect to maximum tolerated dose, PK, and in vivo efficacy against colorectal cancer (CRC) and soft tissue sarcoma (STS) in mouse models. Results: The lyophilized SPESN38 complexes can dissolve in aqueous media to form clear and stable solutions. Maximum tolerated dose (MTD) of SPESN38-5 is 250 mg/kg by oral route (PO) and 55 mg/kg by intravenous route (IV) in CD-1 mice. SPESN38-8 has the MTD of 45 mg/kg by IV in the same mouse model. PK of SPESN38-5 by PO at 250 mg/kg gave mouse plasma AUC0-∞ of 0.0548 and 4.5007 (nmol × h/mL) for SN38 and SN38 glucuronidate (SN38G), respectively, with a surprisingly high molar ratio of SN38G:SN38 = 82:1. However, PK of SPESN38-5 by IV at 55 mg/kg yielded much higher mouse plasma AUC0-∞ of 18.80 and 27.78 nmol × h/mL for SN38 and SN38G, producing a much lower molar ratio of SN38G:SN38 = 1.48:1. Antitumor efficacy of SPESN38-5 and irinotecan (control) was evaluated against HCT-116 CRC xenograft tumors. The data indicates that SPESN38-5 by IV at 55 mg/kg is more effective in suppressing HCT-116 tumor growth with lower systemic toxicity compared to irinotecan at 50 mg/kg. Additionally, SPESN38-8 and DOX (control) by IV were evaluated in the SK-LMS-1 STS mouse model. The results show that SPESN38-8 at 33 mg/kg is highly effective for inhibiting SK-LMS-1 tumor growth with low toxicity, in contrast to DOX's insensitivity to SK-LMS-1 with high toxicity. Conclusion: SPESN38 complexes provide a water soluble SN38 formulation. SPESN38-5 and SPESN38-8 demonstrate better PK values, lower toxicity, and superior antitumor efficacy in mouse models, compared with irinotecan and DOX.

CDK4/6 Inhibitor Resistance in Hormone Receptor-Positive Metastatic Breast Cancer: Translational Research, Clinical Trials, and Future Directions.

The emergence of CDK4/6 inhibitors, such as palbociclib, ribociclib, and abemaciclib, has revolutionized the treatment landscape for hormone receptor-positive breast cancer. These agents have demonstrated significant clinical benefits in terms of both progression-free survival and overall survival. However, resistance to CDK4/6 inhibitors remains a challenge, limiting their long-term efficacy. Understanding the complex mechanisms driving resistance is crucial for the development of novel therapeutic strategies and the improvement of patient outcomes. Translational research efforts, such as preclinical models and biomarker studies, offer valuable insight into resistance mechanisms and may guide the identification of novel combination therapies. This review paper aims to outline the reported mechanisms underlying CDK4/6 inhibitor resistance, drawing insights from both clinical data and translational research in order to help direct the future of treatment for hormone receptor-positive metastatic breast cancer.

The molecular consequences of androgen activity in the human breast.

Estrogen and progesterone have been extensively studied in the mammary gland, but the molecular effects of androgen remain largely unexplored. Transgender men are recorded as female at birth but identify as male and may undergo gender-affirming androgen therapy to align their physical characteristics and gender identity. Here we perform single-cell-resolution transcriptome, chromatin, and spatial profiling of breast tissues from transgender men following androgen therapy. We find canonical androgen receptor gene targets are upregulated in cells expressing the androgen receptor and that paracrine signaling likely drives sex-relevant androgenic effects in other cell types. We also observe involution of the epithelium and a spatial reconfiguration of immune, fibroblast, and vascular cells, and identify a gene regulatory network associated with androgen-induced fat loss. This work elucidates the molecular consequences of androgen activity in the human breast at single-cell resolution.

Breast cancer liver metastasis: Pathogenesis and clinical implications.

Breast cancer is the most common malignant disease in female patients worldwide and can spread to almost every place in the human body, most frequently metastasizing to lymph nodes, bones, lungs, liver and brain. The liver is a common metastatic location for solid cancers as a whole, and it is also the third most common metastatic site for breast cancer. Breast cancer liver metastasis (BCLM) is a complex process. Although the hepatic microenvironment and liver sinusoidal structure are crucial factors for the initial arrest of breast cancer and progression within the liver, the biological basis of BCLM remains to be elucidated. Importantly, further understanding of the interaction between breast cancer cells and hepatic microenvironment in the liver metastasis of breast cancer will suggest ways for the development of effective therapy and prevention strategies for BCLM. In this review, we provide an overview of the recent advances in the understanding of the molecular mechanisms of the hepatic microenvironment in BCLM formation and discuss current systemic therapies for treating patients with BCLM as well as potential therapeutic development based on the liver microenvironment-associated signaling proteins governing BCLM.

miR766-3p and miR124-3p Dictate Drug Resistance and Clinical Outcome in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive disease with poor prognosis, which is mainly due to drug resistance. The biology determining the response to chemo-radiotherapy in HNSCC is poorly understood. Using clinical samples, we found that miR124-3p and miR766-3p are overexpressed in chemo-radiotherapy-resistant (non-responder) HNSCC, as compared to responder tumors. Our study shows that inhibition of miR124-3p and miR766-3p enhances the sensitivity of HNSCC cell lines, CAL27 and FaDu, to 5-fluorouracil and cisplatin (FP) chemotherapy and radiotherapy. In contrast, overexpression of miR766-3p and miR124-3p confers a resistance phenotype in HNSCC cells. The upregulation of miR124-3p and miR766-3p is associated with increased HNSCC cell invasion and migration. In a xenograft mouse model, inhibition of miR124-3p and miR766-3p enhanced the efficacy of chemo-radiotherapy with reduced growth of resistant HNSCC. For the first time, we identified that miR124-3p and miR766-3p attenuate expression of CREBRF and NR3C2, respectively, in HNSCC, which promotes aggressive tumor behavior by inducing the signaling axes CREB3/ATG5 and ß-catenin/c-Myc. Since miR124-3p and miR766-3p affect complementary pathways, combined inhibition of these two miRNAs shows an additive effect on sensitizing cancer cells to chemo-radiotherapy. In conclusion, our study demonstrated a novel miR124-3p- and miR766-3p-based biological mechanism governing treatment-resistant HNSCC, which can be targeted to improve clinical outcomes in HNSCC.

Human fibroblasts facilitate the generation of iPSCs-derived mammary-like organoids.

BACKGROUND: Breast cancer is the most common malignancy in women worldwide, and its treatment largely depends on mastectomy. Patients after mastectomy suffer from crippled body image, self-esteem, and quality of life. Post-mastectomy breast reconstruction can improve patients' psychosocial health. Although silicone and fat have been widely used for breast reconstruction, they have remarkable limitations. Our study aimed to establish an improved method for breast reconstruction from human-induced pluripotent stem cells (iPSCs). METHODS: We used a two-step procedure to induce mammary-like organoids (MLOs) from iPSCs and applied transcriptome sequencing to analyze the gene expression profiles during the development process from embryoid bodies (mEBs) to MLOs. Moreover, we evaluated the in vitro effect of fibroblasts cell line HFF (human foreskin fibroblasts) on the size and morphology of MLOs and explored the in vivo effect of HFF on regeneration rate of MLOs. RESULTS: MLOs had a similar gene expression profile and morphogenesis as the normal mammary glands. Furthermore, the addition of HFF increases the branching ratio and organoid diameters and facilitates the formation of multiple cell layers duct-like structures in MLOs in vitro. Finally, orthotopical transplantation of the MLOs to cleared mammary gland fad pad of NSG mice showed that HFF increases the formation of mammary gland-like structures. CONCLUSIONS: Fibroblasts facilitate iPSC-derived MLOs to generate mammary gland-like structures in both in vitro and in vivo conditions. Our findings lay a foundation for breast reconstruction by using iPSCs.

TOP1 inhibition induces bifurcated JNK/MYC signaling that dictates cancer cell sensitivity.

Rationale: Triple-negative breast cancer (TNBC) does not respond to anti-estrogen and anti-HER2 therapies and is commonly treated by chemotherapy. TNBC has a high recurrence rate, particularly within the first 3 years. Thus, there is an urgent clinical need to develop more effective therapies for TNBC. Topoisomerase I (TOP1) inhibitors cause DNA damage, making these drugs desirable for TNBC treatment since DNA repair machinery is defective in this subtype of breast cancer. Among the main molecular subtypes of breast cancer, the TNBC cell lines exhibited the highest TOP1 inhibition sensitivity. However, clinically used TOP1 inhibitors, such as topotecan and irinotecan, have shown limited clinical applications and the reasons remain unclear. Understanding the mechanism of differential responses to TOP1 blockade and identifying the predictive markers for cancer cell sensitivity will help further TOP1-targeted therapy for TNBC treatment and improve the clinical use of TOP1 inhibitors. Methods: Viability assays were used to evaluate breast cancer cell sensitivity to topotecan and other TOP1 inhibitors as well as TOP2 inhibitors. An in vitro-derived topotecan-resistant TNBC cell model and TNBC xenograft models were employed to confirm cancer cell response to TOP1 blockade. RNA-seq was used to identify potential predictive markers for TNBC cell response to TOP1 blockade. Western blotting and qRT-PCR were performed to measure the protein levels and RNA expression. ATAC-seq and luciferase reporter assays were used to examine MYC transcriptional regulations. The effects of MYC and JNK in cancer cell response to TOP1 inhibition were validated via loss-of-function and gain-of-function experiments. Results: We observed two distinct and diverging cancer cell responses - sensitive versus resistant to TOP1 inhibition, which was confirmed by TNBC xenograft mouse models treated by topotecan. TNBC cells exhibited bifurcated temporal patterns of ATR pathway activation upon TOP1 inhibitor treatment. The sensitive TNBC cells showed an "up then down" dynamic pattern of ATR/Chk1 signaling, while the resistant TNBC cells exhibited a "persistently up" profile. On the contrary, opposite temporal patterns of induced expression of MYC, a key regulator and effector of DNA damage, were found in TNBC cells treated by TOP1 inhibitors. Mechanistically, we showed that TOP1-induced JNK signaling upregulated MYC expression. Furthermore, pharmacological inhibition of ATR reversed TNBC cell resistance to topotecan, whereas MYC knockdown and JNK inhibition reduced cancer cell sensitivity. Conclusions: Dynamic temporal profiles of induced ATR/Chk1 and JNK activation as well as MYC expression, may predict cancer cell response to TOP1 inhibitors. JNK activation-mediated constitutive elevation of MYC expression may represent a novel mechanism governing cancer cell sensitivity to TOP1-targeting therapy. Our results may provide implications for identifying TNBC patients who might benefit from the treatment with TOP1 inhibitors.

A chemokine regulatory loop induces cholesterol synthesis in lung-colonizing triple-negative breast cancer cells to fuel metastatic growth.

Triple-negative breast cancer (TNBC) has a high propensity for organ-specific metastasis. However, the underlying mechanisms are not well understood. Here we show that the primary TNBC tumor-derived C-X-C motif chemokines 1/2/8 (CXCL1/2/8) stimulate lung-resident fibroblasts to produce the C-C motif chemokines 2/7 (CCL2/7), which, in turn, activate cholesterol synthesis in lung-colonizing TNBC cells and induce angiogenesis at lung metastatic sites. Inhibiting cholesterol synthesis in lung-colonizing breast tumor cells by pulmonary administration of simvastatin-carrying HER3-targeting nanoparticles reduces angiogenesis and growth of lung metastases in a syngeneic TNBC mouse model. Our findings reveal a novel, chemokine-regulated mechanism for the cholesterol synthesis pathway and a critical role of metastatic site-specific cholesterol synthesis in the pulmonary tropism of TNBC metastasis. The study has implications for the unresolved epidemiological observation that use of cholesterol-lowering drugs has no effect on breast cancer incidence but can unexpectedly reduce breast cancer mortality, suggesting interventions of cholesterol synthesis in lung metastases as an effective treatment to improve survival in individuals with TNBC.

Current Status of Breast Organoid Models.

Breast cancer (BC) is the most frequently diagnosed malignancy among women globally. Although mouse models have been critical in advancing the knowledge of BC tumorigenesis and progression, human breast models comprising the breast tissue microenvironment are needed to help elucidate the underlying mechanisms of BC risk factors. As such, it is essential to identify an ex vivo human breast tissue mimetic model that can accurately pinpoint the effects of these factors in BC development. While two-dimensional models have been invaluable, they are not suitable for studying patient-specific tumor biology and drug response. Recent developments in three-dimensional (3D) models have led to the prominence of organized structures grown in a 3D environment called "organoids." Breast organoids can accurately recapitulate the in vivo breast microenvironment and have been used to examine factors that affect signaling transduction, gene expression, and tissue remodeling. In this review, the applications, components, and protocols for development of breast organoids are discussed. We summarize studies that describe the utility of breast organoids, including in the study of normal mammary gland development and tumorigenesis. Finally, we provide an overview of protocols for development of breast organoids, and the advantages and disadvantages of different techniques in studies are described. The included studies have shown that breast organoids will continue to serve as a crucial platform for understanding of progression of BC tumors and the testing of novel therapeutics.