Tina El Rayes

Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance.

The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We show that within a predominantly epithelial primary tumour, a small proportion of tumour cells undergo EMT. Notably, lung metastases mainly consist of non-EMT tumour cells that maintain their epithelial phenotype. Inhibiting EMT by overexpressing the microRNA miR-200 does not affect lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment owing to reduced proliferation, apoptotic tolerance and increased expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.The role of epithelial to mesenchymal transition (EMT) in metastasis is a longstanding source of controversy, largely due to an inability to monitor transient and reversible EMT phenotypes in vivo. We established an EMT lineage tracing system to monitor this process, using a mesenchymal-specific Cre-mediated fluorescent marker switch system in spontaneous breast-to-lung metastasis models. We confirmed that within a predominantly epithelial primary tumor, a small portion of tumor cells undergo EMT. Strikingly, lung metastases mainly consisted of non-EMT tumor cells maintaining their epithelial phenotype. Inhibiting EMT by overexpressing miR-200 did not impact lung metastasis development. However, EMT cells significantly contribute to recurrent lung metastasis formation after chemotherapy. These cells survived cyclophosphamide treatment due to reduced proliferation, apoptotic tolerance, and elevated expression of chemoresistance-related genes. Overexpression of miR-200 abrogated this resistance. This study suggests the potential of an EMT-targeting strategy, in conjunction with conventional chemotherapies, for breast cancer treatment.

Lung inflammation promotes metastasis through neutrophil protease-mediated degradation of Tsp-1

Significance Lungs are highly susceptible to inflammation. However, molecular insights into how external inflammation enhances metastatic outgrowth in the lungs remain lacking. Clinically, approaches are required to block tumor outgrowth in secondary organs for effective treatment of metastatic cancers. We demonstrate a previously unidentified mechanism of thrombospondin-1 (Tsp-1) regulation by inflammatory neutrophil proteases in the metastatic organ. Our findings suggest the potential of using neutrophil protease inhibitors as antimetastatic therapies. Inflammation is inextricably associated with primary tumor progression. However, the contribution of inflammation to tumor outgrowth in metastatic organs has remained underexplored. Here, we show that extrinsic inflammation in the lungs leads to the recruitment of bone marrow-derived neutrophils, which degranulate azurophilic granules to release the Ser proteases, elastase and cathepsin G, resulting in the proteolytic destruction of the antitumorigenic factor thrombospondin-1 (Tsp-1). Genetic ablation of these neutrophil proteases protected Tsp-1 from degradation and suppressed lung metastasis. These results provide mechanistic insights into the contribution of inflammatory neutrophils to metastasis and highlight the unique neutrophil protease–Tsp-1 axis as a potential antimetastatic therapeutic target.

Bone Marrow-Derived Gr1+ Cells Can Generate a Metastasis-Resistant Microenvironment Via Induced Secretion of Thrombospondin-1

UNLABELLED Metastatic tumors have been shown to establish permissive microenvironments for metastases via recruitment of bone marrow-derived cells. Here, we show that metastasis-incompetent tumors are also capable of generating such microenvironments. However, in these situations, the otherwise prometastatic Gr1(+) myeloid cells create a metastasis-refractory microenvironment via the induction of thrombospondin-1 (Tsp-1) by tumor-secreted prosaposin. Bone marrow-specific genetic deletion of Tsp-1 abolished the inhibition of metastasis, which was restored by bone marrow transplant from Tsp-1(+) donors. We also developed a 5-amino acid peptide from prosaposin as a pharmacologic inducer of Tsp-1 in Gr1(+) bone marrow cells, which dramatically suppressed metastasis. These results provide mechanistic insights into why certain tumors are deficient in metastatic potential and implicate recruited Gr1(+) myeloid cells as the main source of Tsp-1. The results underscore the plasticity of Gr1(+) cells, which, depending on the context, promote or inhibit metastasis, and suggest that the peptide could be a potential therapeutic agent against metastatic cancer. SIGNIFICANCE The mechanisms of metastasis suppression are poorly understood. Here, we have identified a novel mechanism whereby metastasis-incompetent tumors generate metastasis-suppressive microenvironments in distant organs by inducing Tsp-1 expression in the bone marrow–derived Gr1+myeloid cells. A 5-amino acid peptide with Tsp-1–inducing activity was identified as a therapeutic agent against metastatic cancer.

Identification of Reprogrammed Myeloid Cell Transcriptomes in NSCLC

Lung cancer is the leading cause of cancer related mortality worldwide, with non-small cell lung cancer (NSCLC) as the most prevalent form. Despite advances in treatment options including minimally invasive surgery, CT-guided radiation, novel chemotherapeutic regimens, and targeted therapeutics, prognosis remains dismal. Therefore, further molecular analysis of NSCLC is necessary to identify novel molecular targets that impact prognosis and the design of new-targeted therapies. In recent years, tumor “activated/reprogrammed” stromal cells that promote carcinogenesis have emerged as potential therapeutic targets. However, the contribution of stromal cells to NSCLC is poorly understood. Here, we show increased numbers of bone marrow (BM)-derived hematopoietic cells in the tumor parenchyma of NSCLC patients compared with matched adjacent non-neoplastic lung tissue. By sorting specific cellular fractions from lung cancer patients, we compared the transcriptomes of intratumoral myeloid compartments within the tumor bed with their counterparts within adjacent non-neoplastic tissue from NSCLC patients. The RNA sequencing of specific myeloid compartments (immature monocytic myeloid cells and polymorphonuclear neutrophils) identified differentially regulated genes and mRNA isoforms, which were inconspicuous in whole tumor analysis. Genes encoding secreted factors, including osteopontin (OPN), chemokine (C-C motif) ligand 7 (CCL7) and thrombospondin 1 (TSP1) were identified, which enhanced tumorigenic properties of lung cancer cells indicative of their potential as targets for therapy. This study demonstrates that analysis of homogeneous stromal populations isolated directly from fresh clinical specimens can detect important stromal genes of therapeutic value.

Development of a prosaposin-derived therapeutic cyclic peptide that targets ovarian cancer via the tumor microenvironment

A cyclic prosaposin-derived peptide targets ovarian cancer cells through the fatty acid translocase CD36. Running rings around ovarian cancer Although approved drugs for ovarian cancer are available, this remains a difficult disease to overcome, and most ovarian cancer patients cannot be successfully treated, particularly in the setting of advanced disease. Wang et al. determined that prosaposin, a naturally occurring protein with antimetastatic properties, can promote regression of ovarian cancer because of its effects on thrombospondin, another antitumorigenic protein, which targets a receptor called CD36. The authors generated a cyclic peptide modeled on the active site of prosaposin and showed that the new peptide is very effective in treating mice with patient-derived xenografts of metastatic ovarian cancer, suggesting that this peptide is a candidate for future testing in human patients. The vast majority of ovarian cancer–related deaths are caused by metastatic dissemination of tumor cells, resulting in subsequent organ failure. However, despite our increased understanding of the physiological processes involved in tumor metastasis, there are no clinically approved drugs that have made a major impact in increasing the overall survival of patients with advanced, metastatic ovarian cancer. We identified prosaposin (psap) as a potent inhibitor of tumor metastasis, which acts via stimulation of p53 and the antitumorigenic protein thrombospondin-1 (TSP-1) in bone marrow–derived cells that are recruited to metastatic sites. We report that more than 97% of human serous ovarian tumors tested express CD36, the receptor that mediates the proapoptotic activity of TSP-1. Accordingly, we sought to determine whether a peptide derived from psap would be effective in treating this form of ovarian cancer. To that end, we developed a cyclic peptide with drug-like properties derived from the active sequence in psap. The cyclic psap peptide promoted tumor regression in a patient-derived tumor xenograft model of metastatic ovarian cancer. Thus, we hypothesize that a therapeutic agent based on this psap peptide would have efficacy in treating patients with metastatic ovarian cancer.

Regulation of Tumor Progression and Metastasis by Bone Marrow-Derived Microenvironments

Activating mutations in driver oncogenes and loss-of-function mutations in tumor suppressor genes contribute to tumor progression and metastasis. Accordingly, therapies targeting key tumor cell-intrinsic signaling pathways are being used in clinical trials, and some have met FDA approval. However, these treatments benefit only a small proportion of patients harboring key driver mutations, and acquired resistance to these therapies presents a major impediment to effective treatment. More recently, the contribution of the tumor microenvironment (TME) has been an area of active investigation and has begun to provide critical insights into carcinogenesis. The host stromal cells in the TME coevolve with tumors and contribute to carcinogenesis in several ways. Among the host cells, bone marrow (BM)-derived cells constitute a significant fraction and directly contribute to proliferation, invasion, intravasation, extravasation, and outgrowth at the metastatic site. While the tumor-reprogrammed BM cells constitute attractive targets for anticancer therapy, recent studies have also begun to unravel their role as prognostic and predictive molecular markers of the disease.

Inflammation and Lung Cancer: The Link to Angiogenesis

Emerging studies have begun to strengthen the link between chronic inflammation and cancer. Inflammation is now accepted as an underlying or enabling characteristic that contributes to key hallmarks of cancer, and nonsteroidal anti-inflammatory drugs (NSAID) have shown a reduction in the risk of several cancers. In lung cancer patients, pulmonary disorders, such as chronic obstructive pulmonary disease (COPD) and emphysema, constitute comorbid conditions and comprise an independent risk factor for lung cancer. Despite the clinical association, the mechanistic link between COPD and lung cancer is not completely understood and constitutes an area of intense investigation. Notably, chronic inflammation appears to be a pivotal pathological feature in both COPD and lung cancer. The inflammatory microenvironment encountered in COPD/emphysema may contribute to tumorigenesis via several possible signaling pathways, including angiogenesis. Accumulating evidence suggests that angiogenesis is closely linked to inflammation, with regulators of angiogenesis playing key roles in various inflammatory conditions and vice versa. Inflammatory cells, namely neutrophils, mast cells, monocytes/macrophages, and lymphocytes, play an active role in enhancing tumor angiogenesis by secreting chemokines, inflammatory cytokines, and proteases into the local microenvironment that control endothelial cell (EC) activation by virtue of regulating proliferation, survival and apoptosis, and migration. Therefore, targeting the inflammatory and angiogenic pathways provides unique opportunities for both prevention and treatment of lung cancer.

Abstract 2073: Neutrophil serine proteases, cathepsin-G and elastase, promote metastasis through cleavage of thrombospondin-1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Inflammation is known to be inextricably associated with tumor progression. However, the role of inflammation in metastatic initiation and outgrowth remains largely unexplored. In our study, we show that CXCR2-CXCL2-dependent recruitment of neutrophils supported a pro-metastatic phenotype in acutely inflamed lungs. The increased influx of Ly6G+ neutrophils into inflamed lungs was accompanied by a loss of thrombospondin-1 (Tsp-1) protein. In vitro, the degranulation of neutrophils was marked by an increase in the activity of the neutrophil proteases elastase and cathepsin-G, leading to degradation of Tsp-1, which was protected by using the protease inhibitor Sivelestat. Whereas genetic ablation of either cathepsin-G or elastase in vivo partially impaired metastasis, pharmacological inhibition of both neutrophil proteases, using the small molecule drug Sivelestat, dramatically suppressed metastasis. We have uncovered a novel pathway in which the serine proteases, cathepsin-G and elastase produced by degranulating neutrophils, promote a metastasis-conducive microenvironment through the cleavage of Tsp-1. Our results provide mechanistic insights into how inflammatory components of the premetastatic niche can support metastatic outgrowth, and they suggest that the neutrophil protease-Tsp-1 axis presents a therapeutic target against metastatic cancers. Citation Format: Tina El Rayes, Raul Catena, Sharrell Lee, Dingcheng Gao, Marcin Stawowczyk, Nasser Altorki, Vivek Mittal. Neutrophil serine proteases, cathepsin-G and elastase, promote metastasis through cleavage of thrombospondin-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2073. doi:10.1158/1538-7445.AM2014-2073

Abstract 4584: Bone marrow-derived Gr1+ cells can generate a metastasis-resistant microenvironment via induced secretion of thrombospondin-1.

Previous studies have shown that metastatic tumors establish premetastatic niches comprised of recruited bone marrow (BM)-derived cells in potential distant target metastatic organs. These premetatatic niches serve as permissive hubs for supporting future metastases. However, little is known about whether tumors that lack metastatic potential establish such niches, or generate niches that inhibit metastasis. Here, we show that metastasis-incompetent tumors are also capable of systemically generating BM-derived niches in the metastatic organs. However, by secreting the glycoprotein prosaposin these tumors systemically stimulate expression of antitumorigenic factor thrombospondin-1 (Tsp-1) in the Gr1+ myeloid cells converting them it into a metastasis-refractory niche. Genetic deletion of Tsp-1 abolished the inhibition of metastasis, which was restored by BM transplant from Tsp-1+ donors. We developed a 5-amino acid peptide from prosaposin as a pharmacological inducer of Tsp-1 in Gr1+ BM cells. Strikingly, the peptide suppresses metastasis. These results provide mechanistic insights into why certain tumors are deficient in metastatic potential and implicate recruited Gr1+ myeloid cells as the main source of Tsp-1, contrary to previous notion. These results also underscore the plasticity of Gr1+ cells, which, depending on the context and the nature of paracrine factor/s can either promote or inhibit metastasis. Clinically, examination of a tumor tissue microarray (TMA) compiled from 103 prostate cancer patients with long and complete follow up, revealed that prosaposin expression positively correlated with increased overall survival, suggesting that the prosaposin peptide may be a potential therapeutic agent against metastatic cancer. Citation Format: Raul Catena, Nandita Bhattacharya, Tina El Rayes, Hyejin Choi, Suming Wang, Dingcheng Gao, Natasha Joshi, Seongho Ryu, Diane Bielenberg, Sharrell Lee, Svein Haukaas, Karsten Gravdal, Ole Halvorsen, Lars Akslen, Randolph Watnick, Vivek Mittal. Bone marrow-derived Gr1+ cells can generate a metastasis-resistant microenvironment via induced secretion of thrombospondin-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4584. doi:10.1158/1538-7445.AM2013-4584