A CXCL1 paracrine network links cancer chemoresistance and metastasis.

Metastasis and chemoresistance in cancer are linked phenomena, but the molecular basis for this link is unknown. We uncovered a network of paracrine signals between carcinoma, myeloid, and endothelial cells that drives both processes in breast cancer. Cancer cells that overexpress CXCL1 and 2 by transcriptional hyperactivation or 4q21 amplification are primed for survival in metastatic sites. CXCL1/2 attract CD11b(+)Gr1(+) myeloid cells into the tumor, which produce chemokines including S100A8/9 that enhance cancer cell survival. Although chemotherapeutic agents kill cancer cells, these treatments trigger a parallel stromal reaction leading to TNF-α production by endothelial and other stromal cells. TNF-α via NF-kB heightens the CXCL1/2 expression in cancer cells, thus amplifying the CXCL1/2-S100A8/9 loop and causing chemoresistance. CXCR2 blockers break this cycle, augmenting the efficacy of chemotherapy against breast tumors and particularly against metastasis. This network of endothelial-carcinoma-myeloid signaling interactions provides a mechanism linking chemoresistance and metastasis, with opportunities for intervention.

Tumor self-seeding by circulating cancer cells.

Cancer cells that leave the primary tumor can seed metastases in distant organs, and it is thought that this is a unidirectional process. Here we show that circulating tumor cells (CTCs) can also colonize their tumors of origin, in a process that we call "tumor self-seeding." Self-seeding of breast cancer, colon cancer, and melanoma tumors in mice is preferentially mediated by aggressive CTCs, including those with bone, lung, or brain-metastatic tropism. We find that the tumor-derived cytokines IL-6 and IL-8 act as CTC attractants whereas MMP1/collagenase-1 and the actin cytoskeleton component fascin-1 are mediators of CTC infiltration into mammary tumors. We show that self-seeding can accelerate tumor growth, angiogenesis, and stromal recruitment through seed-derived factors including the chemokine CXCL1. Tumor self-seeding could explain the relationships between anaplasia, tumor size, vascularity and prognosis, and local recurrence seeded by disseminated cells following ostensibly complete tumor excision.

ECM1 secreted by HER2-overexpressing breast cancer cells promotes formation of a vascular niche accelerating cancer cell migration and invasion.

The tumor microenvironment is increasingly recognized as key player in cancer progression. Investigating heterotypic interactions between cancer cells and their microenvironment is important for understanding how specific cell types support cancer. Forming the vasculature, endothelial cells (ECs) are a prominent cell type in the microenvironment of both normal and neoplastic breast gland. Here, we sought out to analyze epithelial-endothelial cross talk in the breast using isogenic non-tumorigenic vs. tumorigenic breast epithelial cell lines and primary ECs. The cellular model used here consists of D492, a breast epithelial cell line with stem cell properties, and two isogenic D492-derived EMT cell lines, D492M and D492HER2. D492M was generated by endothelial-induced EMT and is non-tumorigenic while D492HER2 is tumorigenic, expressing the ErbB2/HER2 oncogene. To investigate cellular cross talk, we used both conditioned medium (CM) and 2D/3D co-culture systems. Secretome analysis of D492 cell lines was performed using mass spectrometry and candidate knockdown (KD), and overexpression (OE) was done using siRNA and CRISPRi/CRISPRa technology. D492HER2 directly enhances endothelial network formation and activates a molecular axis in ECs promoting D492HER2 migration and invasion, suggesting an endothelial feedback response. Secretome analysis identified extracellular matrix protein 1 (ECM1) as potential angiogenic inducer in D492HER2. Confirming its involvement, KD of ECM1 reduced the ability of D492HER2-CM to increase endothelial network formation and induce the endothelial feedback, while recombinant ECM1 (rECM1) increased both. Interestingly, NOTCH1 and NOTCH3 expression was upregulated in ECs upon treatment with D492HER2-CM or rECM1 but not by CM from D492HER2 with ECM1 KD. Blocking endothelial NOTCH signaling inhibited the increase in network formation and the ability of ECs to promote D492HER2 migration and invasion. In summary, our data demonstrate that cancer-secreted ECM1 induces a NOTCH-mediated endothelial feedback promoting cancer progression by enhancing migration and invasion. Targeting this interaction may provide a novel possibility to improve cancer treatment.

Microenvironment in metastasis: roadblocks and supportive niches.

In many cancers, malignant cells can spread from the primary tumor through blood circulation and initiate metastasis in secondary organs. Metastatic colonization may depend not only on inherent properties of cancer cells, but also on suitable microenvironments in distant sites. Increasing evidence suggests that the nature of the microenvironment may determine the fate of disseminated cancer cells, providing either hindrance or support for cancer cell propagation. This can result in strong selective pressure where the vast majority of cancer cells, invading a secondary organ, are either eliminated or maintained in a dormant state. The ability of cancer cells to fend off or circumvent anti-metastatic signals from the stroma and the capacity to manipulate the local microenvironment towards a supporting environment, a metastatic niche, may be essential for metastatic growth. The molecular interactions between cancer cells and the stroma are still enigmatic, but recent studies are beginning to reveal their nature. Here, we discuss the interactive relationship between metastatic cancer cells and host stroma, involving selection and adaptation of metastasis-initiating cells and host tissue remodeling. Understanding the dynamic and continuously evolving cross talk between metastatic cancer cells and the stroma may be crucial when developing cancer treatments.

The molecular composition of the metastatic niche.

In cancer, the microenvironment plays an important role of supporting the outgrowth of new tumors in distant organs i.e. the formation of metastasis. The interplay between cancer cells and the host stroma leads to generation of an active microenvironment termed a metastatic niche that effectively supports cancer progression and outgrowth of metastasis. The generation and development of the niche is intricately linked to cancer progression. Metastatic niches are highly dynamic interactions that can be forged by diverse mechanisms and continue to develop as the cancer progresses. The composition of the niche is increasingly being characterized and new niche components are being identified. The extracellular matrix (ECM), secreted enzymes, growth factors, cytokines and other molecules that carry information to cancer cells are essential parts of the metastatic niche. The sources of this molecular milieu are multiple cell types - local or recruited to the site of metastasis - and in some cases the cancer cells themselves. To understand metastatic progression it is essential to dissect the niche composition and identify the sources of niche components. With future analyses of the metastatic niche, significant opportunities can arise to identify novel targets for cancer therapy. Targeting the metastatic niche may be essential to treat and inhibit the progression of metastasis.

Endogenous human microRNAs that suppress breast cancer metastasis.

A search for general regulators of cancer metastasis has yielded a set of microRNAs for which expression is specifically lost as human breast cancer cells develop metastatic potential. Here we show that restoring the expression of these microRNAs in malignant cells suppresses lung and bone metastasis by human cancer cells in vivo. Of these microRNAs, miR-126 restoration reduces overall tumour growth and proliferation, whereas miR-335 inhibits metastatic cell invasion. miR-335 regulates a set of genes whose collective expression in a large cohort of human tumours is associated with risk of distal metastasis. miR-335 suppresses metastasis and migration through targeting of the progenitor cell transcription factor SOX4 and extracellular matrix component tenascin C. Expression of miR-126 and miR-335 is lost in the majority of primary breast tumours from patients who relapse, and the loss of expression of either microRNA is associated with poor distal metastasis-free survival. miR-335 and miR-126 are thus identified as metastasis suppressor microRNAs in human breast cancer.

NADK-mediated de novo NADP(H) synthesis is a metabolic adaptation essential for breast cancer metastasis.

Metabolic reprogramming and metabolic plasticity allow cancer cells to fine-tune their metabolism and adapt to the ever-changing environments of the metastatic cascade, for which lipid metabolism and oxidative stress are of particular importance. NADPH is a central co-factor for both lipid and redox homeostasis, suggesting that cancer cells may require larger pools of NADPH to efficiently metastasize. NADPH is recycled through reduction of NADP+ by several enzymatic systems in cells; however, de novo NADP+ is synthesized only through one known enzymatic reaction, catalyzed by NAD+ kinase (NADK). Here, we show that NADK is upregulated in metastatic breast cancer cells enabling de novo production of NADP(H) and the expansion of the NADP(H) pools thereby increasing the ability of these cells to adapt to the challenges of the metastatic cascade and efficiently metastasize. Mechanistically, we found that metastatic signals lead to a histone H3.3 variant-mediated epigenetic regulation of the NADK promoter, resulting in increased NADK levels in cells with metastatic ability. Together, our work presents a previously uncharacterized role for NADK and de novo NADP(H) production as a contributor to breast cancer progression and suggests that NADK constitutes an important and much needed therapeutic target for metastatic breast cancers.

Perivascular tenascin C triggers sequential activation of macrophages and endothelial cells to generate a pro-metastatic vascular niche in the lungs.

Disseminated cancer cells frequently lodge near vasculature in secondary organs. However, our understanding of the cellular crosstalk invoked at perivascular sites is still rudimentary. Here, we identify intercellular machinery governing formation of a pro-metastatic vascular niche during breast cancer colonization in the lung. We show that specific secreted factors, induced in metastasis-associated endothelial cells (ECs), promote metastasis in mice by enhancing stem cell properties and the viability of cancer cells. Perivascular macrophages, activated via tenascin C (TNC) stimulation of Toll-like receptor 4 (TLR4), were shown to be crucial in niche activation by secreting nitric oxide (NO) and tumor necrosis factor (TNF) to induce EC-mediated production of niche components. Notably, this mechanism was independent of vascular endothelial growth factor (VEGF), a key regulator of EC behavior and angiogenesis. However, targeting both macrophage-mediated vascular niche activation and VEGF-regulated angiogenesis resulted in added potency to curb lung metastasis in mice. Together, our findings provide mechanistic insights into the formation of vascular niches in metastasis.

Tumor-Derived Lactic Acid Modulates Activation and Metabolic Status of Draining Lymph Node Stroma.

Communication between tumors and the stroma of tumor-draining lymph nodes (TDLN) exists before metastasis arises, altering the structure and function of the TDLN niche. Transcriptional profiling of fibroblastic reticular cells (FRC), the dominant stromal population of lymph nodes, has revealed that FRCs in TDLNs are reprogrammed. However, the tumor-derived factors driving the changes in FRCs remain to be identified. Taking an unbiased approach, we have shown herein that lactic acid (LA), a metabolite released by cancer cells, was not only secreted by B16.F10 and 4T1 tumors in high amounts, but also that it was enriched in TDLNs. LA supported an upregulation of Podoplanin (Pdpn) and Thy1 and downregulation of IL7 in FRCs of TDLNs, making them akin to activated fibroblasts found at the primary tumor site. Furthermore, we found that tumor-derived LA altered mitochondrial function of FRCs in TDLNs. Thus, our results demonstrate a mechanism by which a tumor-derived metabolite connected with a low pH environment modulates the function of fibroblasts in TDLNs. How lymph node function is perturbed to support cancer metastases remains unclear. The authors show that tumor-derived LA drains to lymph nodes where it modulates the function of lymph node stromal cells, prior to metastatic colonization.

Diverted total synthesis leads to the generation of promising cell-migration inhibitors for treatment of tumor metastasis: in vivo and mechanistic studies on the migrastatin core ether analog.

A significantly simpler analog of the natural product migrastatin, termed migrastatin ether (ME), has been prepared and evaluated. Both in vivo and in vitro studies indicate that ME exhibits a concentration-dependent inhibitory effect on migration of breast cancer cells.

Stress-induced metastatic niches in breast cancer.

Interactions between disseminated cancer cells and the microenvironment in secondary organs are essential for the development of metastasis in most malignancies. Metastasis-initiating cells and their progeny can impose changes in the microenvironment leading to the formation of a metastatic niche that supports malignant growth at secondary sites. Our recent findings indicate that stress responses play a crucial role in generation of metastatic niches in breast cancer by modulating the extracellular matrix and promoting interactions with reactive fibroblasts.

Addicted to Acidic Microenvironment.

Acidic pH levels are often observed in growing tumors, with profound effects on cancer cells and surrounding microenvironment. In this issue of Developmental Cell, Funato et al. (2020) show that expression of oncogenic phosphatase of regenerating liver 3 (PRL3) shifts cellular preference for environmental pH, leading to acid addiction.

Metastasis-initiating cells induce and exploit a fibroblast niche to fuel malignant colonization of the lungs.

Metastatic colonization relies on interactions between disseminated cancer cells and the microenvironment in secondary organs. Here, we show that disseminated breast cancer cells evoke phenotypic changes in lung fibroblasts, forming a supportive metastatic niche. Colonization of the lungs confers an inflammatory phenotype in metastasis-associated fibroblasts. Specifically, IL-1α and IL-1ß secreted by breast cancer cells induce CXCL9 and CXCL10 production in lung fibroblasts via NF-κB signaling, fueling the growth of lung metastases. Notably, we find that the chemokine receptor CXCR3, that binds CXCL9/10, is specifically expressed in a small subset of breast cancer cells, which exhibits tumor-initiating ability when co-transplanted with fibroblasts and has high JNK signaling that drives IL-1α/ß expression. Importantly, disruption of the intercellular JNK-IL-1-CXCL9/10-CXCR3 axis reduces metastatic colonization in xenograft and syngeneic mouse models. These data mechanistically demonstrate an essential role for the molecular crosstalk between breast cancer cells and their fibroblast niche in the progression of metastasis.

Frequent Molecular Subtype Switching and Gene Expression Alterations in Lung and Pleural Metastasis From Luminal A-Type Breast Cancer.

PURPOSE: Conversion of tumor subtype frequently occurs in the course of metastatic breast cancer but is a poorly understood phenomenon. This study aims to compare molecular subtypes with subsequent lung or pleural metastasis. PATIENTS AND METHODS: In a cohort of 57 patients with breast cancer and lung or pleural metastasis (BCLPM), we investigated paired primary and metastatic tissues for differential gene expression of 269 breast cancer genes. The PAM50 classifier was applied to identify intrinsic subtypes, and differential gene expression and cluster analysis were used to further characterize subtypes and tumors with subtype conversion. RESULTS: In primary breast cancer, the most frequent molecular subtype was luminal A (lumA; 49.1%); it was luminal B (lumB) in BCLPM (38.6%). Subtype conversion occurred predominantly in lumA breast cancers compared with other molecular subtypes (57.1% v 27.6%). In lumA cancers, 62 genes were identified with differential expression in metastatic versus primary disease, compared with only 10 differentially expressed genes in lumB, human epidermal growth factor receptor 2 (HER2)-enriched, and basal subtypes combined. Gene expression changes in lumA cancers affected not only the repression of the estrogen receptor pathway and cell cycle-related genes but also the WNT pathway, proteinases (MME, MMP11), and motility-associated cytoskeletal proteins (CK5, CK14, CK17). Subtype-switched lumA cancers were further characterized by cell proliferation and cell cycle checkpoint gene upregulation and dysregulation of the p53 pathway. This involved 83 notable gene expression changes. CONCLUSION: Our results indicate that gene expression changes and subsequent subtype conversion occur on a large scale in metastatic luminal A-type breast cancer compared with other molecular subtypes. This underlines the significance of molecular changes in metastatic disease, especially in tumors of initially low aggressive potential.

Stress signaling in breast cancer cells induces matrix components that promote chemoresistant metastasis.

Metastatic progression remains a major burden for cancer patients and is associated with eventual resistance to prevailing therapies such as chemotherapy. Here, we reveal how chemotherapy induces an extracellular matrix (ECM), wound healing, and stem cell network in cancer cells via the c-Jun N-terminal kinase (JNK) pathway, leading to reduced therapeutic efficacy. We find that elevated JNK activity in cancer cells is linked to poor clinical outcome in breast cancer patients and is critical for tumor initiation and metastasis in xenograft mouse models of breast cancer. We show that JNK signaling enhances expression of the ECM and stem cell niche components osteopontin, also called secreted phosphoprotein 1 (SPP1), and tenascin C (TNC), that promote lung metastasis. We demonstrate that both SPP1 and TNC are direct targets of the c-Jun transcription factor. Exposure to multiple chemotherapies further exploits this JNK-mediated axis to confer treatment resistance. Importantly, JNK inhibition or disruption of SPP1 or TNC expression sensitizes experimental mammary tumors and metastases to chemotherapy, thus providing insights to consider for future treatment strategies against metastatic breast cancer.