Mesenchymal stem cells within tumour stroma promote breast cancer metastasis.

Mesenchymal stem cells have been recently described to localize to breast carcinomas, where they integrate into the tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived human mesenchymal stem cells, when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour xenograft. The breast cancer cells stimulate de novo secretion of the chemokine CCL5 (also called RANTES) from mesenchymal stem cells, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion and metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by eliciting reversible changes in the phenotype of cancer cells.

The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy.

Patients undergoing surgical resection of primary breast tumors confront a risk for metastatic recurrence that peaks sharply 12 to 18 months after surgery. The cause of early metastatic relapse in breast cancer has long been debated, with many ascribing these relapses to the natural progression of the disease. Others have proposed that some aspect of surgical tumor resection triggers the outgrowth of otherwise-dormant metastases, leading to the synchronous pattern of relapse. Clinical data cannot distinguish between these hypotheses, and previous experimental approaches have not provided clear answers. Such uncertainty hinders the development and application of therapeutic approaches that could potentially reduce early metastatic relapse. We describe an experimental model system that definitively links surgery and the subsequent wound-healing response to the outgrowth of tumor cells at distant anatomical sites. Specifically, we find that the systemic inflammatory response induced after surgery promotes the emergence of tumors whose growth was otherwise restricted by a tumor-specific T cell response. Furthermore, we demonstrate that perioperative anti-inflammatory treatment markedly reduces tumor outgrowth in this model, suggesting that similar approaches might substantially reduce early metastatic recurrence in breast cancer patients.

Enumeration of the simian virus 40 early region elements necessary for human cell transformation.

While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.

Neutrophils Suppress Intraluminal NK Cell-Mediated Tumor Cell Clearance and Enhance Extravasation of Disseminated Carcinoma Cells.

UNLABELLED: Immune cells promote the initial metastatic dissemination of carcinoma cells from primary tumors. In contrast to their well-studied functions in the initial stages of metastasis, the specific roles of immunocytes in facilitating progression through the critical later steps of the invasion-metastasis cascade remain poorly understood. Here, we define novel functions of neutrophils in promoting intraluminal survival and extravasation at sites of metastatic dissemination. We show that CD11b(+)/Ly6G(+) neutrophils enhance metastasis formation via two distinct mechanisms. First, neutrophils inhibit natural killer cell function, which leads to a significant increase in the intraluminal survival time of tumor cells. Thereafter, neutrophils operate to facilitate extravasation of tumor cells through the secretion of IL1ß and matrix metalloproteinases. These results identify neutrophils as key regulators of intraluminal survival and extravasation through their cross-talk with host cells and disseminating carcinoma cells. SIGNIFICANCE: This study provides important insights into the systemic contributions of neutrophils to cancer metastasis by identifying how neutrophils facilitate intermediate steps of the invasion-metastasis cascade. We demonstrate that neutrophils suppress natural killer cell activity and increase extravasation of tumor cells. Cancer Discov; 6(6); 630-49. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561.

Immortalization and transformation of primary human airway epithelial cells by gene transfer.

One critical step in the development of a cancerous cell is its acquisition of an unlimited replicative lifespan, the process termed immortalization. Experimental model systems designed to study cellular transformation ex vivo have relied to date on the in vitro selection of a subpopulation of cells that have become immortalized through treatment with chemical or physical mutagens and the selection of rare clonal variants. In this study, we describe the direct immortalization of primary human airway epithelial cells through the successive introduction of the Simian Virus 40 Early Region and the telomerase catalytic subunit hTERT. Cells immortalized in this way are now responsive to malignant transformation by an introduced H-ras or K-ras oncogene. These immortalized human airway epithelial cells, which have been created through the stepwise introduction of genetic alterations, provide a novel experimental model system with which to study further the biology of the airway epithelial cell and to dissect the molecular basis of lung cancer pathogenesis.

An integrin-linked machinery of cytoskeletal regulation that enables experimental tumor initiation and metastatic colonization.

Recently extravasated metastatic cancer cells use the Rif/mDia2 actin-nucleating/polymerizing machinery in order to extend integrin ß1-containing, filopodium-like protrusions (FLPs), which enable them to interact productively with the surrounding extracellular matrix; this process governs the initial proliferation of these cancer cells. Here, we identify the signaling pathway governing FLP lifetime, which involves integrin-linked kinase (ILK) and ß-parvin, two integrin:actin-bridging proteins that block cofilin-mediated actin-filament severing. Notably, the combined actions of Rif/mDia2 and ILK/ß-parvin/cofilin pathways on FLPs are required not only for metastatic outgrowth but also for primary tumor formation following experimental implantation. This provides one mechanistic explanation for how the epithelial-mesenchymal transition (EMT) program imparts tumor-initiating powers to carcinoma cells, since it enhances FLP formation through the activation of ILK/ß-parvin/cofilin pathway.

The outgrowth of micrometastases is enabled by the formation of filopodium-like protrusions.

UNLABELLED: Disseminated cancer cells that have extravasated into the tissue parenchyma must interact productively with its extracellular matrix components to survive, proliferate, and form macroscopic metastases. The biochemical and cell biologic mechanisms enabling this interaction remain poorly understood. We find that the formation of elongated integrin ß(1)-containing adhesion plaques by cancer cells that have extravasated into the lung parenchyma enables the proliferation of these cells via activation of focal adhesion kinase. These plaques originate in and appear only after the formation of filopodium-like protrusions (FLP) that harbor integrin ß(1) along their shafts. The cytoskeleton-regulating proteins Rif and mDia2 contribute critically to the formation of these protrusions and thereby enable the proliferation of extravasated cancer cells. Hence, the formation of FLPs represents a critical rate-limiting step for the subsequent development of macroscopic metastases. SIGNIFICANCE: Although the mechanisms of metastatic dissemination have begun to be uncovered, those involved in the establishment of extravasated cancer cells in foreign tissue microenvironments remained largely obscure. We have studied the behavior of recently extravasated cancer cells in the lungs and identified a series of cell biologic processes involving the formation of filopodium-like protrusions and the subsequent development of elongated, mature adhesion plaques, which contribute critically to the rapid proliferation of the micrometastatic cells and thus are prerequisites to the eventual lung colonization by these cells.

Telomerase maintains telomere structure in normal human cells.

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.

Telomerase contributes to tumorigenesis by a telomere length-independent mechanism.

Once immortalized, human cells are susceptible to transformation by introduction of an oncogene such as ras. Several lines of evidence now suggest that the maintenance of telomere length is a major determinant of replicative lifespan in human cells and thus of the immortalized state. The majority of human tumor cells acquire immortality through expression of the catalytic subunit of telomerase (hTERT), whereas others activate an alternative mechanism of telomere maintenance (ALT) that does not depend on the actions of telomerase. We have examined whether ALT could substitute for telomerase in the processes of transformation in vitro and tumorigenesis in vivo. Expression of oncogenic H-Ras in the immortal ALT cell line GM847 did not result in their transformation. However, subsequent ectopic expression of hTERT in these cells imparted a tumorigenic phenotype. Indeed, this outcome was also observed after introduction of a mutant hTERT that retained catalytic activity but was incapable of maintaining telomere length. These studies indicate that hTERT confers an additional function that is required for tumorigenesis but does not depend on its ability to maintain telomeres.