Microenvironment-induced CREPT expression by cancer-derived small extracellular vesicles primes field cancerization.

Rationale: Cancer local recurrence increases the mortality of patients, and might be caused by field cancerization, a pre-malignant alteration of normal epithelial cells. It has been suggested that cancer-derived small extracellular vesicles (CDEs) may contribute to field cancerization, but the underlying mechanisms remain poorly understood. In this study, we aim to identify the key regulatory factors within recipient cells under the instigation of CDEs. Methods: In vitro experiments were performed to demonstrate that CDEs promote the expression of CREPT in normal epithelial cells. TMT-based quantitative mass spectrometry was employed to investigate the proteomic differences between normal cells and tumor cells. Loss-of-function approaches by CRISPR-Cas9 system were used to assess the role of CREPT in CDEs-induced field cancerization. RNA-seq was performed to explore the genes regulated by CREPT during field cancerization. Results: CDEs promote field cancerization by inducing the expression of CREPT in non-malignant epithelial cells through activating the ERK signaling pathway. Intriguingly, CDEs failed to induce field cancerization when CREPT was deleted, highlighting the importance of CREPT. Transcriptomic analyses revealed that CDEs elicited inflammatory responses, primarily through activation of the TNF signaling pathway. CREPT, in turn, regulates the transduction of downstream signals of TNF by modulating the expression of TNFR2 and PI3K, thereby promoting inflammation-to-cancer transition. Conclusion: CREPT not only serves as a biomarker for field cancerization, but also emerges as a target for preventing the cancer local recurrence.

Tafazzin mediates tamoxifen resistance by regulating cellular phospholipid composition in ER-positive breast cancer.

Tamoxifen is the frontline therapeutic agent for the estrogen receptor-positive (ER + ) subtype of breast cancer patients, which accounts for 70-80% of total breast cancer incidents. However, clinical resistance to tamoxifen has become increasingly common, highlighting the need to identify the underlying cellular mechanisms. In our study, we employed a genome-scale CRISPR-Cas9 loss-of-function screen and validation experiments to discover that Tafazzin (TAZ), a mitochondrial transacylase, is crucial for maintaining the cellular sensitivity of ER+ breast cancer cells to tamoxifen and other chemotherapies. Mechanistically, we found that cardiolipin, whose synthesis and maturation rely on TAZ, is required to maintain cellular sensitivity to tamoxifen. Loss of metabolic enzymatic activity of TAZ causes ERα downregulation and therapy resistance. Interestingly, we observed that TAZ deficiency also led to the upregulation of lysophosphatidylcholine (LPC), which in turn suppressed ERα expression and nuclear localization, thereby contributing to tamoxifen resistance. LPC is further metabolized to lysophosphatidic acid (LPA), a bioactive molecule that supports cell survival. Thus, our findings suggest that the depletion of TAZ promotes tamoxifen resistance through an LPC-LPA phospholipid synthesis axis, and targeting this lipid metabolic pathway could restore cell susceptibility to tamoxifen treatment.

Osteoclast Cancer Cell Metabolic Cross-talk Confers PARP Inhibitor Resistance in Bone Metastatic Breast Cancer.

The majority of patients with late-stage breast cancer develop distal bone metastases. The bone microenvironment can affect response to therapy, and uncovering the underlying mechanisms could help identify improved strategies for treating bone metastatic breast cancer. Here, we observed that osteoclasts reduced the sensitivity of breast cancer cells to DNA damaging agents, including cisplatin and the PARP inhibitor (PARPi) olaparib. Metabolic profiling identified elevated glutamine production by osteoclasts. Glutamine supplementation enhanced the survival of breast cancer cells treated with DNA damaging agents, while blocking glutamine uptake increased sensitivity and suppressed bone metastasis. GPX4, the critical enzyme responsible for glutathione oxidation, was upregulated in cancer cells following PARPi treatment through stress-induced ATF4-dependent transcriptional programming. Increased glutamine uptake and GPX4 upregulation concertedly enhanced glutathione metabolism in cancer cells to help neutralize oxidative stress and generate PARPi resistance. Analysis of paired patient samples of primary breast tumors and bone metastases revealed significant induction of GPX4 in bone metastases. Combination therapy utilizing PARPi and zoledronate, which blocks osteoclast activity and thereby reduces the microenvironmental glutamine supply, generated a synergistic effect in reducing bone metastasis. These results identify a role for glutamine production by bone-resident cells in supporting metastatic cancer cells to overcome oxidative stress and develop resistance to DNA-damaging therapies. SIGNIFICANCE: Metabolic interaction between osteoclasts and tumor cells contributes to resistance to DNA-damaging agents, which can be blocked by combination treatment with PARP and osteoclast inhibitors to reduce bone metastatic burden.

Tumor-derived Jagged1 promotes cancer progression through immune evasion.

Immune checkpoint inhibitor (ICI) therapy is generating remarkable responses in individuals with cancer, but only a small portion of individuals with breast cancer respond well. Here we report that tumor-derived Jagged1 is a key regulator of the tumor immune microenvironment. Jagged1 promotes tumorigenesis in multiple spontaneous mammary tumor models. Through Jagged1-induced Notch activation, tumor cells increase expression and secretion of multiple cytokines to help recruit macrophages into the tumor microenvironment. Educated macrophages crosstalk with tumor-infiltrating T cells to inhibit T cell proliferation and tumoricidal activity. In individuals with triple-negative breast cancer, a high expression level of Jagged1 correlates with increased macrophage infiltration and decreased T cell activity. Co-administration of an ICI PD-1 antibody with a Notch inhibitor significantly inhibits tumor growth in breast cancer models. Our findings establish a distinct signaling cascade by which Jagged1 promotes adaptive immune evasion of tumor cells and provide several possible therapeutic targets.

Deubiquitinase USP20 promotes breast cancer metastasis by stabilizing SNAI2.

SNAI2/SLUG, a metastasis-promoting transcription factor, is a labile protein that is degraded through the ubiquitin proteasome degradation system. Here, we conducted comprehensive gain- and loss-of-function screens using a human DUB cDNA library of 65 genes and an siRNA library of 98 genes, and identified USP20 as a deubiquitinase (DUB) that regulates SNAI2 ubiquitination and stability. Further investigation of USP20 demonstrated its function in promoting migration, invasion, and metastasis of breast cancer. USP20 positively correlates with SNAI2 protein level in breast tumor samples, and higher USP20 expression is associated with poor prognosis in ER- breast cancer patients.

ASB13 inhibits breast cancer metastasis through promoting SNAI2 degradation and relieving its transcriptional repression of YAP.

Transcription factor SNAI2 plays key roles during development and has also been known to promote metastasis by inducing invasive phenotype and tumor-initiating activity of cancer cells. However, the post-translational regulation of SNAI2 is less well studied. We performed a dual-luciferase-based, genome-wide E3 ligase siRNA library screen and identified ASB13 as an E3 ubiquitin ligase that targets SNAI2 for ubiquitination and degradation. ASB13 knockout in breast cancer cells promoted cell migration and decreased F-actin polymerization, while overexpression of ASB13 suppressed lung metastasis. Furthermore, ASB13 knockout decreased YAP expression, and such regulation is dependent on an increased protein level of SNAI2, which in turn represses YAP transcription. YAP suppresses tumor progression in breast cancer, as YAP knockout increases tumorsphere formation, anchorage-independent colony formation, cell migration in vitro, and lung metastasis in vivo. Clinical data analysis reveals that ASB13 expression is positively correlated with improved overall survival in breast cancer patients. These findings establish ASB13 as a suppressor of breast cancer metastasis by promoting degradation of SNAI2 and relieving its transcriptional repression of YAP.

Bone vascular niche E-selectin induces mesenchymal-epithelial transition and Wnt activation in cancer cells to promote bone metastasis.

How disseminated tumour cells engage specific stromal components in distant organs for survival and outgrowth is a critical but poorly understood step of the metastatic cascade. Previous studies have demonstrated the importance of the epithelial-mesenchymal transition in promoting the cancer stem cell properties needed for metastasis initiation, whereas the reverse process of mesenchymal-epithelial transition is required for metastatic outgrowth. Here we report that this paradoxical requirement for the simultaneous induction of both mesenchymal-epithelial transition and cancer stem cell traits in disseminated tumour cells is provided by bone vascular niche E-selectin, whose direct binding to cancer cells promotes bone metastasis by inducing mesenchymal-epithelial transition and activating Wnt signalling. E-selectin binding activity mediated by the α1-3 fucosyltransferases Fut3/Fut6 and Glg1 are instrumental to the formation of bone metastasis. These findings provide unique insights into the functional role of E-selectin as a component of the vascular niche critical for metastatic colonization in bone.

Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

Triple-negative breast cancer (TNBC) patients have the worst prognosis and distant metastasis-free survival among all major subtypes of breast cancer. The poor clinical outlook is further exacerbated by a lack of effective targeted therapies for TNBC. Here we show that ectopic expression and therapeutic delivery of the secreted protein Tubulointerstitial nephritis antigen-like 1 (Tinagl1) suppresses TNBC progression and metastasis through direct binding to integrin α5ß1, αvß1, and epidermal growth factor receptor (EGFR), and subsequent simultaneous inhibition of focal adhesion kinase (FAK) and EGFR signaling pathways. Moreover, Tinagl1 protein level is associated with good prognosis and reversely correlates with FAK and EGFR activation status in TNBC. Our results suggest Tinagl1 as a candidate therapeutic agent for TNBC by dual inhibition of integrin/FAK and EGFR signaling pathways.

Therapeutic Antibody Targeting Tumor- and Osteoblastic Niche-Derived Jagged1 Sensitizes Bone Metastasis to Chemotherapy.

Bone metastasis is a major health threat to breast cancer patients. Tumor-derived Jagged1 represents a central node in mediating tumor-stromal interactions that promote osteolytic bone metastasis. Here, we report the development of a highly effective fully human monoclonal antibody against Jagged1 (clone 15D11). In addition to its inhibitory effect on bone metastasis of Jagged1-expressing tumor cells, 15D11 dramatically sensitizes bone metastasis to chemotherapy, which induces Jagged1 expression in osteoblasts to provide a survival niche for cancer cells. We further confirm the bone metastasis-promoting function of osteoblast-derived Jagged1 using osteoblast-specific Jagged1 transgenic mouse model. These findings establish 15D11 as a potential therapeutic agent for the prevention or treatment of bone metastasis.

Tumor-Stroma Interactions in Bone Metastasis: Molecular Mechanisms and Therapeutic Implications.

Metastasis and associated complications are the major cause of death for cancer patients. The incidence of bone metastasis is among the highest in cancers arising from breast, prostate, and lung. Common skeletal-related events caused by bone metastasis include aberrant bone remodeling (osteolytic, osteoblastic, and mixed), bone pain, fracture, spinal cord compression, and life-threatening hypercalcemia. It is now known that interactions between tumor cells and bone stroma lie at the core of major steps of bone-metastasis progression. Approved pharmaceutical drugs for the treatment of bone metastasis, including bisphosphonate and denosumab, were designed to target bone stromal cell components. In recent years, research in our laboratory and others has revealed intricate tumor-stroma interactions as the driving force behind osteolytic bone-metastasis development, providing a set of new candidates for future drug development. Moreover, recent studies also indicate existence of distinct bone niches in supporting hematopoietic stem cell renewal and differentiation. These niche components are likely utilized by metastatic cancer cells for seeding, progression, and therapy resistance of bone metastasis. Future studies in this direction may discover additional therapeutic targets for bone-metastasis treatment.

Cradle of evil: osteogenic niche for early bone metastasis.

Bone metastasis often emerges long after the initial dissemination of cancer cells. In this issue of Cancer Cell, Wang and colleagues demonstrate that disseminated breast cancer cells engage osteogenic niches in the bone through heterotypic adherins junctions. This interaction activates mTOR signaling in cancer cells and supports their expansion to micrometastases.

PKD1 phosphorylation-dependent degradation of SNAIL by SCF-FBXO11 regulates epithelial-mesenchymal transition and metastasis.

Metastatic dissemination is often initiated by the reactivation of an embryonic development program referred to as epithelial-mesenchymal transition (EMT). The transcription factor SNAIL promotes EMT and elicits associated pathological characteristics such as invasion, metastasis, and stemness. To better understand the posttranslational regulation of SNAIL, we performed a luciferase-based, genome-wide E3 ligase siRNA library screen and identified SCF-FBXO11 as an important E3 that targets SNAIL for ubiquitylation and degradation. Furthermore, we discovered that SNAIL degradation by FBXO11 is dependent on Ser-11 phosphorylation of SNAIL by protein kinase D1 (PKD1). FBXO11 blocks SNAIL-induced EMT, tumor initiation, and metastasis in multiple breast cancer models. These findings establish the PKD1-FBXO11-SNAIL axis as a mechanism of posttranslational regulation of EMT and cancer metastasis.

The Fbw7 tumor suppressor targets KLF5 for ubiquitin-mediated degradation and suppresses breast cell proliferation.

Fbw7 is a tumor suppressor frequently inactivated in cancers. The KLF5 transcription factor promotes breast cell proliferation and tumorigenesis through upregulating FGF-BP. The KLF5 protein degrades rapidly through the ubiquitin proteasome pathway. Here, we show that the Skp1-CUL1-Fbw7 E3 ubiquitin ligase complex (SCF(Fbw7)) targets KLF5 for ubiquitin-mediated degradation in a GSK3beta-mediated KLF5 phosphorylation-dependent manner. Mutation of the critical S303 residue in the KLF5 Cdc4 phospho-degrons motif ((303)SPPSS) abolishes the protein interaction, ubiquitination, and degradation by Fbw7. Inactivation of endogenous Fbw7 remarkably increases the endogenous KLF5 protein abundances. Endogenous Fbw7 suppresses the FGF-BP gene expression and breast cell proliferation through targeting KLF5 for degradation. These findings suggest that Fbw7 inhibits breast cell proliferation at least partially through targeting KLF5 for proteolysis. This new regulatory mechanism of KLF5 degradation may result in useful diagnostic and therapeutic targets for breast cancer and other cancers.

KLF5 promotes breast cell survival partially through fibroblast growth factor-binding protein 1-pERK-mediated dual specificity MKP-1 protein phosphorylation and stabilization.

Krüpple-like transcription factor 5 (KLF5) is a zinc-finger transcription factor promoting cell survival and tumorigenesis in multiple cancers. A high expression level of KLF5 has been shown to be associated with shorter breast cancer patient survival. However, the role of KLF5 and mechanism of KLF5 actions in breast cancer remain unclear. In this study, we found that KLF5 knockdown by small interfering RNA in two breast cell lines, MCF10A and BT20, induces apoptosis. Interestingly, a pro-survival phosphatase, dual specificity mitogen-activated protein kinase phosphatase 1 (MKP-1), is down-regulated by KLF5 ablation. Consistently, KLF5 overexpression increases the MKP-1 protein expression in Hs578T and MCF7. We further found that MKP-1 is essential and sufficient for KLF5 to promote breast cell survival. However, MKP-1 is not a KLF5 direct transcription target because the MKP-1 mRNA level is not regulated by KLF5. By cycloheximide chase assays, we found that KLF5 decreases MKP-1 protein degradation via activating the ERK signaling. Inhibition of pERK by the pharmacological inhibitor U0126 specifically blocks KLF5-induced MKP-1 phosphorylation and stabilization. Additionally, constitutive activation of ERK by constitutively activated MEK1 rescues the KLF5 depletion-induced MKP-1 down-regulation. Consistently, the phosphorylation-deficient MKP-1 mutant cannot be stabilized by KLF5. Finally, the activation of ERK by KLF5 is very likely through the KLF5 direct target gene FGF-BP in breast cells. These findings suggest that KLF5 is a pro-survival factor that promotes breast cell survival partially through pERK-mediated MKP-1 phosphorylation and stabilization. The KLF5-FGF-BP-pERK-MKP-1 signaling axis may provide new therapeutic targets for invasive breast cancer.