Ras drives malignancy through stem cell crosstalk with the microenvironment.

Squamous cell carcinomas are triggered by marked elevation of RAS-MAPK signalling and progression from benign papilloma to invasive malignancy1-4. At tumour-stromal interfaces, a subset of tumour-initiating progenitors, the cancer stem cells, obtain increased resistance to chemotherapy and immunotherapy along this pathway5,6. The distribution and changes in cancer stem cells during progression from a benign state to invasive squamous cell carcinoma remain unclear. Here we show in mice that, after oncogenic RAS activation, cancer stem cells rewire their gene expression program and trigger self-propelling, aberrant signalling crosstalk with their tissue microenvironment that drives their malignant progression. The non-genetic, dynamic cascade of intercellular exchanges involves downstream pathways that are often mutated in advanced metastatic squamous cell carcinomas with high mutational burden7. Coupling our clonal skin HRASG12V mouse model with single-cell transcriptomics, chromatin landscaping, lentiviral reporters and lineage tracing, we show that aberrant crosstalk between cancer stem cells and their microenvironment triggers angiogenesis and TGFß signalling, creating conditions that are conducive for hijacking leptin and leptin receptor signalling, which in turn launches downstream phosphoinositide 3-kinase (PI3K)-AKT-mTOR signalling during the benign-to-malignant transition. By functionally examining each step in this pathway, we reveal how dynamic temporal crosstalk with the microenvironment orchestrated by the stem cells profoundly fuels this path to malignancy. These insights suggest broad implications for cancer therapeutics.

Tracking the Cartoon mouse phenotype: Hemopexin domain-dependent regulation of MT1-MMP pericellular collagenolytic activity.

Following ENU mutagenesis, a phenodeviant line was generated, termed the "Cartoon mouse," that exhibits profound defects in growth and development. Cartoon mice harbor a single S466P point mutation in the MT1-MMP hemopexin domain, a 200-amino acid segment that is thought to play a critical role in regulating MT1-MMP collagenolytic activity. Herein, we demonstrate that the MT1-MMPS466P mutation replicates the phenotypic status of Mt1-mmp-null animals as well as the functional characteristics of MT1-MMP-/- cells. However, rather than a loss-of-function mutation acquired as a consequence of defects in MT1-MMP proteolytic activity, the S466P substitution generates a misfolded, temperature-sensitive mutant that is abnormally retained in the endoplasmic reticulum (ER). By contrast, the WT hemopexin domain does not play a required role in regulating MT1-MMP trafficking, as a hemopexin domain-deletion mutant is successfully mobilized to the cell surface and displays nearly normal collagenolytic activity. Alternatively, when MT1-MMPS466P-expressing cells are cultured at a permissive temperature of 25 °C that depresses misfolding, the mutant successfully traffics from the ER to the trans-Golgi network (ER → trans-Golgi network), where it undergoes processing to its mature form, mobilizes to the cell surface, and expresses type I collagenolytic activity. Together, these analyses define the Cartoon mouse as an unexpected gain-of-abnormal function mutation, wherein the temperature-sensitive mutant phenocopies MT1-MMP-/- mice as a consequence of eliciting a specific ER → trans-Golgi network trafficking defect.

E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine.

Cadherin-based intercellular adhesions are essential players in epithelial homeostasis, but their dynamic regulation during tissue morphogenesis and remodeling remain largely undefined. Here, we characterize an unexpected role for the membrane-anchored metalloproteinase MT2-MMP in regulating epithelial cell quiescence. Following co-immunoprecipitation and mass spectrometry, the MT2-MMP cytosolic tail was found to interact with the zonula occludens protein-1 (ZO-1) at the apical junctions of polarized epithelial cells. Functionally, MT2-MMP localizes in the apical domain of epithelial cells where it cleaves E-cadherin and promotes epithelial cell accumulation, a phenotype observed in 2D polarized cells as well as 3D cysts. MT2-MMP-mediated cleavage subsequently disrupts apical E-cadherin-mediated cell quiescence resulting in relaxed apical cortical tension favoring cell extrusion and re-sorting of Src kinase activity to junctional complexes, thereby promoting proliferation. Physiologically, MT2-MMP loss of function alters E-cadherin distribution, leading to impaired 3D organoid formation by mouse colonic epithelial cells ex vivo and reduction of cell proliferation within intestinal crypts in vivo Taken together, these studies identify an MT2-MMP-E-cadherin axis that functions as a novel regulator of epithelial cell homeostasis in vivo.

Functional roles of MMP14 and MMP15 in early postnatal mammary gland development.

During late embryogenesis, mammary epithelial cells initiate migration programs that drive ductal invasion into the surrounding adipose-rich mesenchyme. Currently, branching morphogenesis is thought to depend on the mobilization of the membrane-anchored matrix metalloproteinases MMP14 (MT1-MMP) and MMP15 (MT2-MMP), which drive epithelial cell invasion by remodeling the extracellular matrix and triggering associated signaling cascades. However, the roles that these proteinases play during mammary gland development in vivo remain undefined. Here, we characterize the impact of global Mmp14 and Mmp15 targeting on early postnatal mammary gland development in mice. Unexpectedly, both Mmp14-/- and Mmp15-/- mammary glands retain the ability to generate intact ductal networks. Although neither proteinase is required for branching morphogenesis, transcriptome profiling reveals a key role for MMP14 and MMP15 in regulating mammary gland adipocyte differentiation. Whereas MMP14 promotes the generation of white fat depots crucial for energy storage, MMP15 differentially controls the formation of thermogenic brown fat. Taken together, these data not only indicate that current paradigms relevant to proteinase-dependent morphogenesis need be revisited, but also identify new roles for the enzymes in regulating adipocyte fate determination in the developing mammary gland.

GOLPH3 modulates mTOR signalling and rapamycin sensitivity in cancer.

Genome-wide copy number analyses of human cancers identified a frequent 5p13 amplification in several solid tumour types, including lung (56%), ovarian (38%), breast (32%), prostate (37%) and melanoma (32%). Here, using integrative analysis of a genomic profile of the region, we identify a Golgi protein, GOLPH3, as a candidate targeted for amplification. Gain- and loss-of-function studies in vitro and in vivo validated GOLPH3 as a potent oncogene. Physically, GOLPH3 localizes to the trans-Golgi network and interacts with components of the retromer complex, which in yeast has been linked to target of rapamycin (TOR) signalling. Mechanistically, GOLPH3 regulates cell size, enhances growth-factor-induced mTOR (also known as FRAP1) signalling in human cancer cells, and alters the response to an mTOR inhibitor in vivo. Thus, genomic and genetic, biological, functional and biochemical data in yeast and humans establishes GOLPH3 as a new oncogene that is commonly targeted for amplification in human cancer, and is capable of modulating the response to rapamycin, a cancer drug in clinical use.

Divergent regulation of basement membrane trafficking by human macrophages and cancer cells.

Macrophages and cancer cells populations are posited to navigate basement membrane barriers by either mobilizing proteolytic enzymes or deploying mechanical forces. Nevertheless, the relative roles, or identity, of the proteinase -dependent or -independent mechanisms used by macrophages versus cancer cells to transmigrate basement membrane barriers harboring physiologically-relevant covalent crosslinks remains ill-defined. Herein, both macrophages and cancer cells are shown to mobilize membrane-anchored matrix metalloproteinases to proteolytically remodel native basement membranes isolated from murine tissues while infiltrating the underlying interstitial matrix ex vivo. In the absence of proteolytic activity, however, only macrophages deploy actomyosin-generated forces to transmigrate basement membrane pores, thereby providing the cells with proteinase-independent access to the interstitial matrix while simultaneously exerting global effects on the macrophage transcriptome. By contrast, cancer cell invasive activity is reliant on metalloproteinase activity and neither mechanical force nor changes in nuclear rigidity rescue basement membrane transmigration. These studies identify membrane-anchored matrix metalloproteinases as key proteolytic effectors of basement membrane remodeling by macrophages and cancer cells while also defining the divergent invasive strategies used by normal and neoplastic cells to traverse native tissue barriers.

Osteoclast-mediated bone resorption is controlled by a compensatory network of secreted and membrane-tethered metalloproteinases.

Osteoclasts actively remodel both the mineral and proteinaceous components of bone during normal growth and development as well as pathologic states ranging from osteoporosis to bone metastasis. The cysteine proteinase cathepsin K confers osteoclasts with potent type I collagenolytic activity; however, cathepsin K-null mice, as well as cathepsin K-mutant humans, continue to remodel bone and degrade collagen by as-yet-undefined effectors. Here, we identify a cathepsin K-independent collagenolytic system in osteoclasts that is composed of a functionally redundant network of the secreted matrix metalloproteinase MMP9 and the membrane-anchored matrix metalloproteinase MMP14. Unexpectedly, whereas deleting either of the proteinases individually leaves bone resorption intact, dual targeting of Mmp9 and Mmp14 inhibited the resorptive activity of mouse osteoclasts in vitro and in vivo and human osteoclasts in vitro. In vivo, Mmp9/Mmp14 conditional double-knockout mice exhibited marked increases in bone density and displayed a highly protected status against either parathyroid hormone- or ovariectomy-induced pathologic bone loss. Together, these studies characterize a collagenolytic system operative in mouse and human osteoclasts and identify the MMP9/MMP14 axis as a potential target for therapeutic interventions for bone-wasting disease states.

Divergent Matrix-Remodeling Strategies Distinguish Developmental from Neoplastic Mammary Epithelial Cell Invasion Programs.

Metastasizing breast carcinoma cells have been hypothesized to mobilize tissue-invasive activity by co-opting the proteolytic systems employed by normal mammary epithelial cells undergoing branching morphogenesis. However, the critical effectors underlying morphogenesis remain unidentified, and their relationship to breast cancer invasion programs is yet to be established. Here, we identify the membrane-anchored matrix metalloproteinase, Mmp14/MT1-MMP, but not the closely related proteinase Mmp15/MT2-MMP, as the dominant proteolytic effector of both branching morphogenesis and carcinoma cell invasion in vivo. Unexpectedly, however, epithelial cell-specific targeting of Mmp14/MT1-MMP in the normal mammary gland fails to impair branching, whereas deleting the proteinase in carcinoma cells abrogates invasion, preserves matrix architecture, and completely blocks metastasis. By contrast, in the normal mammary gland, extracellular matrix remodeling and morphogenesis are ablated only when Mmp14/MT1-MMP expression is specifically deleted from the periductal stroma. Together, these findings uncover the overlapping but divergent strategies that underlie developmental versus neoplastic matrix remodeling programs.

Snail/Slug binding interactions with YAP/TAZ control skeletal stem cell self-renewal and differentiation.

Bone-marrow-derived skeletal stem/stromal cell (SSC) self-renewal and function are critical to skeletal development, homeostasis and repair. Nevertheless, the mechanisms controlling SSC behaviour, particularly bone formation, remain ill-defined. Using knockout mouse models that target the zinc-finger transcription factors Snail or Slug, or Snail and Slug combined, a regulatory axis has been uncovered wherein Snail and Slug cooperatively control SSC self-renewal, osteoblastogenesis and bone formation. Mechanistically, Snail/Slug regulate SSC function by forming complexes with the transcriptional co-activators YAP and TAZ in tandem with the inhibition of the Hippo-pathway-dependent regulation of YAP/TAZ signalling cascades. In turn, the Snail/Slug-YAP/TAZ axis activates a series of YAP/TAZ/TEAD and Runx2 downstream targets that control SSC homeostasis and osteogenesis. Together, these results demonstrate that SSCs mobilize Snail/Slug-YAP/TAZ complexes to control stem cell function.

Snail1-dependent p53 repression regulates expansion and activity of tumour-initiating cells in breast cancer.

The zinc-finger transcription factor Snail1 is inappropriately expressed in breast cancer and associated with poor prognosis. While interrogating human databases, we uncovered marked decreases in relapse-free survival of breast cancer patients expressing high Snail1 levels in tandem with wild-type, but not mutant, p53. Using a Snail1 conditional knockout model of mouse breast cancer that maintains wild-type p53, we find that Snail1 plays an essential role in tumour progression by controlling the expansion and activity of tumour-initiating cells in preneoplastic glands and established tumours, whereas it is not required for normal mammary development. Growth and survival of preneoplastic as well as neoplastic mammary epithelial cells is dependent on the formation of a Snail1/HDAC1/p53 tri-molecular complex that deacetylates active p53, thereby promoting its proteasomal degradation. Our findings identify Snail1 as a molecular bypass that suppresses the anti-proliferative and pro-apoptotic effects exerted by wild-type p53 in breast cancer.

Proinvasion metastasis drivers in early-stage melanoma are oncogenes.

Clinical and genomic evidence suggests that the metastatic potential of a primary tumor may be dictated by prometastatic events that have additional oncogenic capability. To test this "deterministic" hypothesis, we adopted a comparative oncogenomics-guided function-based strategy involving: (1) comparison of global transcriptomes of two genetically engineered mouse models with contrasting metastatic potential, (2) genomic and transcriptomic profiles of human melanoma, (3) functional genetic screen for enhancers of cell invasion, and (4) evidence of expression selection in human melanoma tissues. This integrated effort identified six genes that are potently proinvasive and oncogenic. Furthermore, we show that one such gene, ACP5, confers spontaneous metastasis in vivo, engages a key pathway governing metastasis, and is prognostic in human primary melanomas.

Developmental ECM sculpting: laying it down and cutting it up.

In mammals, proteolytic remodeling of the embryonic extracellular matrix (ECM) controls morphogenesis, but the key players remain elusive. Two recent reports identify new roles for metalloproteinases belonging to the MT-MMP and ADAMTS families in branching morphogenesis and interdigital web regression.