Emerging Biological Principles of Metastasis

Post-translational modification by covalent attachment of isoprenoid lipids (prenylation) regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a CAAX motif at the C-terminal that serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, thus facilitating membrane association. C17orf37 (chromosome 17 open reading frame 37), also known as C35/Rdx12/MGC14832, located in the 17q12 amplicon, is overexpressed in human cancer, and its expression correlates with the migratory and invasive phenotype of cancer cells. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I (GGTase-I). Geranylgeranylation of C17orf37 at the CAAX motif facilitates association of the protein to the inner leaflet of plasma membrane, enhances migratory phenotype of cells by inducing increased filopodia formation, and potentiates directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of tail vein-injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the post-translational modification may functionally regulate metastatic progression of disease.

Hematogenous dissemination of lung cancer cells during surgery: quantitative detection by flow cytometry and prognostic significance

Tumor cell intravasation is an essential step in the metastatic cascade, but its exact mechanism is not completely understood. We have previously shown that the direct physical association of a tumor cell over-expressing Mena, a Tie2hi/Vegfhi macrophage and an endothelial cell, creates a micro-anatomic doorway called “tumor microenvironment of metastasis” (TMEM). TMEM are responsible for cancer cell intravasation and dissemination to distant sites. The density of TMEM doorways is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that TMEM doorways create increased localized vascular permeability which cancer cells utilize to intravasate, the precise molecular mechanisms relating TMEM-doorway function and intravasation has not been elucidated. Active TMEM doorways are found in pre-invasive and invasive ductal breast carcinoma as well as in metastatic foci in lymph nodes and lungs, indicating that TMEM-mediated cancer cell dissemination occurs not only at the primary tumor site but also at metastatic sites, which may perpetuate metastatic dissemination even after removal of the primary tumor. Thus it is essential to understand the exact molecular mechanism of TMEM-doorway function so that specific targeted therapies can be developed to intercept systemic cancer cell dissemination. We outline here the exact molecular mechanism of TMEM-doorway functions. TMEM doorway endothelial cell-secreted Ang2 (a Tie2 ligand) stimulates VEGF expression and production by the Tie2hi TMEM macrophage. Subsequently, the TMEM doorway tumor cell- secreted CSF1 stimulates local secretion of VEGF from the Tie2hi TMEM macrophages, leading to dissociation of endothelial adherens and tight junctions near TMEM and cancer cell intravasation. In addition, we show that acute blockage of CSF1R and Tie2-Ang2 signaling by inhibitors and blocking antibodies both in vitro and in mammary tumors leads to decreased macrophage VEGF production and secretion, decreased tumor cell trans-endothelial migration, and decreased TMEM-dependent vascular permeability, tumor cell dissemination and circulating tumor cells. This is the first description of the molecular mechanisms regulating TMEM doorway function and thus represents a major step in defining new biomarkers and targets for the treatment of metastatic tumors. Citation Format: Chinmay Surve, Allison S. Harney, Mary Chen, Yarong Wang, Xianjun Ye, Yu Lin, Ved Sharma, Richard Stanley, Maja H. Oktay, John S. Condeelis. Regulation of breast tumor metastasis by the dynamic interaction between the TMEM doorway macrophage, tumor and endothelial cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-10.

One of the hallmarks of lung cancers is the earlier metastasis resulting from the dissemination of cancer cells. Although accumulating evidence suggests that bacterial infection may be involved in the development of the metastasis of lung cancer, few studies have explored the molecular mechanisms of bacterial infection in the dissemination of lung cancer cells. A series of studies have indicated that certain Gram-negative bacteria are able to hijack antigen-presenting cells (APCs) via interaction with DC-SIGN (CD209) receptors to facilitate the dissemination of pathogens, including viruses, bacteria, fungi, and parasites. Therefore, in the present work, it was hypothesized that bacterial infection may promote the dissemination of cancer cells via the utilization of a similar mechanism. It was first discovered that human lung cancer tissues contain a very high diversity of bacterial DNAs, indicating the co-existence of lung cancer tissues and microbial organisms. It was then found that lung cancer tissues express DC-SIGN, leading to binding with a Gram-negative bacterium, Shigella sonnei. Further, this bacterium was found to be able not only to induce the expression of DC-SIGN on macrophages but also to enhance the migration ability of lung cancer cells in vitro. The in vivo experiments supported these observations, showing that in wild-type (WT) mice, Shigella sonnei infection significantly increased tumor size, weight, and metastatic nodules compared to SIGNR1 knockout (KO) mice. These observations were associated with increasing DC-SIGN expression in WT mice. Finally, these results suggest that bacterial infections could play a significant role in promoting lung cancer progression and metastasis via DC-SIGN-mediated mechanisms.

Metastatic lung cancer is a major cause of death worldwide. Dissemination of cancer cells can be facilitated by various agonists within the tumor microenvironment, including by lysophosphatidic acid (LPA). We postulate that Rho guanine nucleotide exchange factors (RhoGEFs), which integrate signaling cues driving cell migration, are critical effectors in metastatic cancer. Specifically, we addressed the hypothetical role of ARHGEF17, a RhoGEF, as a potential effector of Gβγ in metastatic lung cancer cells responding to LPA. Here, we show that ARHGEF17, originally identified as a tumor endothelial marker, is involved in tumor growth and metastatic dissemination of lung cancer cells in an immunocompetent murine model. Gene expression–based analysis of lung cancer datasets showed that increased levels of ARHGEF17 correlated with reduced survival of patients with advanced-stage tumors. Cellular assays also revealed that this RhoGEF participates in the invasive and migratory responses elicited by Gi protein–coupled LPA receptors via the Gβγ subunit complex. We demonstrate that this signaling heterodimer promoted ARHGEF17 recruitment to the cell periphery and actin fibers. Moreover, Gβγ allosterically activates ARHGEF17 by the removal of inhibitory intramolecular restrictions. Taken together, our results indicate that ARHGEF17 may be a valid potential target in the treatment of metastatic lung cancer.

<div>AbstractPurpose:<p>Anastomotic leak (AL) is a major complication in colorectal cancer surgery and consists of the leakage of intestinal content through a poorly healed colonic wound. Colorectal cancer recurrence after surgery is a major determinant of survival. We hypothesize that AL may allow cancer cells to escape the gut and lead to cancer recurrence and that improving anastomotic healing may prevent local implantation and metastatic dissemination of cancer cells.</p>Experimental Design:<p>We investigated the association between AL and postoperative outcomes in patients with colorectal cancer. Using mouse models of poor anastomotic healing, we assessed the processes of local implantation and dissemination of cancer cells. The effect of dietary supplementation with inulin and 5-aminosalicylate (5-ASA), which activate PPAR-γ in the gut, on local anastomotic tumors was assessed in mice undergoing colonic surgery. Inulin and 5-ASA were also assessed in a mouse model of liver metastasis.</p>Results:<p>Patients experiencing AL displayed lower overall and oncologic survival than non-AL patients. Poor anastomotic healing in mice led to larger anastomotic and peritoneal tumors. The microbiota of patients with AL displays a lower capacity to activate the antineoplastic PPAR-γ in the gut. Modulation of gut microbiota using dietary inulin and 5-ASA reinforced the gut barrier and prevented anastomotic tumors and metastatic spread in mice.</p>Conclusions:<p>Our findings reinforce the hypothesis that preventing AL is paramount to improving oncologic outcomes after colorectal cancer surgery. Furthermore, they pave the way toward dietary targeting of PPAR-γ as a novel way to enhance healing and diminish cancer recurrence.</p></div>

Anastomotic leak (AL) is a major complication in colorectal cancer surgery and consists of the leakage of intestinal content through a poorly healed colonic wound. Colorectal cancer recurrence after surgery is a major determinant of survival. We hypothesize that AL may allow cancer cells to escape the gut and lead to cancer recurrence and that improving anastomotic healing may prevent local implantation and metastatic dissemination of cancer cells. We investigated the association between AL and postoperative outcomes in patients with colorectal cancer. Using mouse models of poor anastomotic healing, we assessed the processes of local implantation and dissemination of cancer cells. The effect of dietary supplementation with inulin and 5-aminosalicylate (5-ASA), which activate PPAR-γ in the gut, on local anastomotic tumors was assessed in mice undergoing colonic surgery. Inulin and 5-ASA were also assessed in a mouse model of liver metastasis. Patients experiencing AL displayed lower overall and oncologic survival than non-AL patients. Poor anastomotic healing in mice led to larger anastomotic and peritoneal tumors. The microbiota of patients with AL displays a lower capacity to activate the antineoplastic PPAR-γ in the gut. Modulation of gut microbiota using dietary inulin and 5-ASA reinforced the gut barrier and prevented anastomotic tumors and metastatic spread in mice. Our findings reinforce the hypothesis that preventing AL is paramount to improving oncologic outcomes after colorectal cancer surgery. Furthermore, they pave the way toward dietary targeting of PPAR-γ as a novel way to enhance healing and diminish cancer recurrence.

<div>AbstractPurpose:<p>Anastomotic leak (AL) is a major complication in colorectal cancer surgery and consists of the leakage of intestinal content through a poorly healed colonic wound. Colorectal cancer recurrence after surgery is a major determinant of survival. We hypothesize that AL may allow cancer cells to escape the gut and lead to cancer recurrence and that improving anastomotic healing may prevent local implantation and metastatic dissemination of cancer cells.</p>Experimental Design:<p>We investigated the association between AL and postoperative outcomes in patients with colorectal cancer. Using mouse models of poor anastomotic healing, we assessed the processes of local implantation and dissemination of cancer cells. The effect of dietary supplementation with inulin and 5-aminosalicylate (5-ASA), which activate PPAR-γ in the gut, on local anastomotic tumors was assessed in mice undergoing colonic surgery. Inulin and 5-ASA were also assessed in a mouse model of liver metastasis.</p>Results:<p>Patients experiencing AL displayed lower overall and oncologic survival than non-AL patients. Poor anastomotic healing in mice led to larger anastomotic and peritoneal tumors. The microbiota of patients with AL displays a lower capacity to activate the antineoplastic PPAR-γ in the gut. Modulation of gut microbiota using dietary inulin and 5-ASA reinforced the gut barrier and prevented anastomotic tumors and metastatic spread in mice.</p>Conclusions:<p>Our findings reinforce the hypothesis that preventing AL is paramount to improving oncologic outcomes after colorectal cancer surgery. Furthermore, they pave the way toward dietary targeting of PPAR-γ as a novel way to enhance healing and diminish cancer recurrence.</p></div>

While anaesthetics are frequently used on cancer patients during surgical procedures, their consequence on cancer progression remains to be elucidated. In this study, we sought to investigate the influence of local anesthetics on lung cancer cell dissemination in vitro and in vivo. A549 human non-small lung cancer cells were treated with various local anaesthetics including ropivacaine, lidocaine, levobupivacaine and bupivacaine. Cell barrier property was assessed using an electric cell-substrate impedance sensing (ECIS) system. The epithelial-to-mesenchymal transition (EMT) of treated cells was studied by immunofluorescence staining. In vitro and in vivo cancer cell dissemination were investigated.Gene expression microarray and quantitative real-time PCR (qrt-PCR) assays were used to identify the genes responsible for levobupivacaine-mediated cancer cell dissemination.The results illustrated that only levobupivacaine induced EMT in the treated cells and also caused the dissemination of cancer cells in vitro. In addition, after intravenous injection, levobupivacaine encouraged cancer cell dissemination in vivo. Gene expression microarray, qrt-PCR and immunoblotting revealed that after levobupivacaine treatment, the hypoxia-inducible factor (HIF)- 2α gene was upregulated in cancer cells. Our findings suggest that levobupivacaine may induce A549 lung cancer cell dissemination both in vitro and in vivo. More specifically, HIF-2α signaling possibly contributes to levobupivacaine-mediated A549 lung cancer cell dissemination.

Metastatic dissemination of cancer cells from primary to distant sites often occurs early and it has been shown that their detection is linked to poor outcomes. Eradication of disseminated cancer cells (DCC) has therefore become the primary goal of adjuvant therapies. Since the phenotype of DCC is largely unknown, we set out to search, isolate, and molecularly characterize these candidate metastasis founder cells from bone marrow of breast cancer patients, years before manifestation of metastasis. We screened more than 200 bone marrow samples of breast cancer patients with no evident metastasis (UICC stage M0) for EpCAM-positive cells. EpCAM positive cells in the bone marrow of healthy donors were used as a negative control. In addition, we isolated DCC from breast cancer patients with manifest metastasis (UICC stage M1). We then performed single cell RNA sequencing after whole transcriptome amplification of the collected DCC and the healthy donor (HD) control cells. To confirm the malignant origin of picked DCCs, we projected the transcriptome data into the bone marrow atlas, inferred copy number alterations, and searched for mutations shared with the matched primary tumor. EpCAM-positive cells from control patients mostly comprised plasma cells. In contrast, DCCs derived from M0-stage breast cancer patient formed a unique, non-overlapping cluster in the bone marrow atlas. UMAP-clustering placed M1-stage DCC clearly separate from M0 stage DCC. Among the collected M0-stage DCCs, we identified at least three separate subclusters. Strikingly, M0-stage DCC displayed much higher stemness and potency scores than M1-stage DCCs, reminiscent of embryonic cells. DCCs, identified by EpCAM, display very high transcriptional stemness scores when isolated before metastatic manifestation. Upon expansion and proliferation, stemness scores are reduced indicating epithelial re-differentiation. The embryonic phenotype of M0-stage DCCs may reflect an early adaptive mechanism that enables DCC to survive in an ectopic environment and may impact on the metastatic potential of the DCC. Citation Format: Elia Raya, Huiqin Koerkel-Qu, Laura Rudhart, Lisa-Marie Köhler, Anna Damboeck, Christoph Irlbeck, Catherine Botteron, Melanie Werner-Klein, Stephan Seitz, Christoph Klein. EpCAM+ DCCs isolated from the bone marrow of breast cancer patients display high stemness and potency scores [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3788.

Increasing evidence shows that cancer cells can disseminate from early evolved primary lesions much earlier than the classical metastasis models predicted. Here, we reveal at a single-cell resolution that mesenchymal-like (M-like) and pluripotency-like programs coordinate dissemination and a long-lived dormancy program of early disseminated cancer cells (DCCs). The transcription factor ZFP281 induces a permissive state for heterogeneous M-like transcriptional programs, which associate with a dormancy signature and phenotype in vivo. Downregulation of ZFP281 leads to a loss of an invasive, M-like dormancy phenotype and a switch to lung metastatic outgrowth. We also show that FGF2 and TWIST1 induce ZFP281 expression to induce the M-like state, which is linked to CDH1 downregulation and upregulation of CDH11. We found that ZFP281 not only controls the early dissemination of cancer cells but also locks early DCCs in a dormant state by preventing the acquisition of an epithelial-like proliferative program and consequent metastases outgrowth.

Disseminated cancer cells in breast cancer: Mechanism of dissemination and dormancy and emerging insights on therapeutic opportunities.

BackgroundRFA is a safe and effective procedure for treating unresectable primary or secondary liver malignancies, but it is not without complications. The most common reported complications include abdominal hemorrhage, bile leakage, biloma formation, hepatic abscesses, and neoplastic seeding.The aim of this study is to evaluate the feasibility of percutaneous use of surgical sealant with a new coaxial bilumen catheter, to prevent the perihepatic bleeding and dissemination of cancer cells through the needle-electrode (neoplastic seeding) or along the needle track.MethodsWe designed a novel dual-lumen catheter to facilitate the optimal application of fibrin sealant after diagnostic and therapeutic percutaneous procedures. Percutaneous RFA has been performed using mask ventilation or neuroleptanalgesia. The main aims of this study, after the ablation procedure, in the treatment of unresectable liver cancer were to prevent major adverse events: a) the perihepatic bleeding; b) dissemination of cancer cells through the needle-electrode and or needle track.ResultsA total of 181 patients were evaluated for this study at National Cancer Institute of Naples from January 2012 to January 2014. The association of blood loss (≤1 g/dl; ≥1 g/dl) with age, gender, histological diagnosis were analyzed. No statistical significance was observed between bleeding and age (p = 0.840), gender (p = 0.607) and histological diagnosis (p = 0,571), respectively.ConclusionsThis study demonstrated that fibrin sealant or other surgical sealant injection, after any locoregional procedure such as biopsy or ablation, could make adverse events even more rare.

Ovarian cancer is a gynecological malignancy with a high mortality rate. Patients with ovarian cancer are diagnosed at an advanced stage of the disease with massive peritoneal dissemination. However, the underlying mechanism by which ovarian cancer cells preferentially colonize the omentum is still poorly understood. In this study, we have identified the factors and mechanisms which promote ovarian cancer dissemination by regulating peritoneal immune system. First, we established a mouse ovarian cancer cell line, ID8GO2, with an increased ability to disseminate in the peritoneal cavity by in vivo passage of ID8 cells in B6J mice. To determine the candidate genes that drive the dissemination, we examined the differences in gene expression profiles between two cell lines. Expression of the candidate genes in human ovarian cancer tissues and their correlation with the clinical outcomes of patients were also analyzed using public database. We found that pigment epithelium-derived factor (PEDF) was the most relevant gene associated with poor prognosis. To clarify whether PEDF contributes to peritoneal dissemination, ID8 cells were retrovirally transduced with mouse PEDF (ID8-PEDF) or empty vector (ID8-EV) and transplanted into the syngeneic mouse peritoneal cavity. All mice transplanted with ID8-PEDF cells developed intraperitoneal dissemination more rapidly and the overall survival time was significantly shorter than that in mice transplanted with ID8-EV cells. Moreover, ID8-PEDF cells survived longer in the peritoneal cavity. We further found that ID8 cells originally consisted of clones with varied PEDF expression. Transplantation of the PEDF-high clones resulted in faster development of intraperitoneal dissemination and shorter overall survival than that of the PEDF-low clones. These findings suggest that PEDF contributes to peritoneal dissemination of ovarian cancer cells, and cells with high initial PEDF expression are selected during dissemination. Next, to clarify the mechanism underlying the role of PEDF in peritoneal dissemination, intraperitoneal immune cell subsets in tumor bearing mice were analyzed. Peritoneal macrophages in mice transplanted with ID8-PEDF cells displayed alternative activated macrophage phenotype (F4/80+ CD206+) and high IL10 expression. Interestingly, at later time point, regulatory T and regulatory B cells were induced in the peritoneal cavity. Collectively, these findings suggest that PEDF plays an important role in the peritoneal dissemination of ovarian cancer cells potentially by regulating macrophages and regulatory lymphocytes to evade the peritoneal immune system. Citation Format: Sayaka Ueno, Eiji Sugihara, Tamotsu Sudo, Hideyuki Saya. Pigment epithelium-derived factor promotes tumor dissemination of ovarian cancer cells through an interaction with peritoneal immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5202.

Postoperative Peritoneal Dissemination of Ovarian Cancer Cells is not Promoted by Carbon-dioxide Pneumoperitoneum at Low Intraperitoneal Pressure in a Syngenic Mouse Laparoscopic Model with Controlled Respiratory Support: A Pilot Study