Anchor Cell Invasion Research Articles

De novo lipid synthesis and polarized prenylation drive cell invasion through basement membrane.

To breach the basement membrane, cells in development and cancer use large, transient, specialized lipid-rich membrane protrusions. Using live imaging, endogenous protein tagging, and cell-specific RNAi during Caenorhabditis elegans anchor cell (AC) invasion, we demonstrate that the lipogenic SREBP transcription factor SBP-1 drives the expression of the fatty acid synthesis enzymes POD-2 and FASN-1 prior to invasion. We show that phospholipid-producing LPIN-1 and sphingomyelin synthase SMS-1, which use fatty acids as substrates, produce lysosome stores that build the AC's invasive protrusion, and that SMS-1 also promotes protrusion localization of the lipid raft partitioning ZMP-1 matrix metalloproteinase. Finally, we discover that HMG-CoA reductase HMGR-1, which generates isoprenoids for prenylation, localizes to the ER and enriches in peroxisomes at the AC invasive front, and that the final transmembrane prenylation enzyme, ICMT-1, localizes to endoplasmic reticulum exit sites that dynamically polarize to deliver prenylated GTPases for protrusion formation. Together, these results reveal a collaboration between lipogenesis and a polarized lipid prenylation system that drives invasive protrusion formation.

Cell cycle perturbation uncouples mitotic progression and invasive behavior in a post-mitotic cell

The acquisition of the post-mitotic state is crucial for the execution of many terminally differentiated cell behaviors during organismal development. However, the mechanisms that maintain the post-mitotic state in this context remain poorly understood. To gain insight into these mechanisms, we used the genetically and visually accessible model of C. elegans anchor cell (AC) invasion into the vulval epithelium. The AC is a terminally differentiated uterine cell that normally exits the cell cycle and enters a post-mitotic state before initiating contact between the uterus and vulva through a cell invasion event. Here, we set out to identify the set of negative cell cycle regulators that maintain the AC in this post-mitotic, invasive state. Our findings revealed a critical role for CKI-1 (p21CIP1/p27KIP1) in redundantly maintaining the post-mitotic state of the AC, as loss of CKI-1 in combination with other negative cell cycle regulators—including CKI-2 (p21CIP1/p27KIP1), LIN-35 (pRb/p107/p130), FZR-1 (Cdh1/Hct1), and LIN-23 (β-TrCP)—resulted in proliferating ACs. Remarkably, time-lapse imaging revealed that these ACs retain their ability to invade. Upon examination of a node in the gene regulatory network controlling AC invasion, we determined that proliferating, invasive ACs do so by maintaining aspects of pro-invasive gene expression. We therefore report that the requirement for a post-mitotic state for invasive cell behavior can be bypassed following direct cell cycle perturbation.

The Caenorhabditis elegans anchor cell transcriptome: ribosome biogenesis drives cell invasion through basement membrane.

Cell invasion through basement membrane (BM) barriers is important in development, immune function and cancer progression. As invasion through BM is often stochastic, capturing gene expression profiles of actively invading cells in vivo remains elusive. Using the stereotyped timing of Caenorhabditis elegans anchor cell (AC) invasion, we generated an AC transcriptome during BM breaching. Through a focused RNAi screen of transcriptionally enriched genes, we identified new invasion regulators, including translationally controlled tumor protein (TCTP). We also discovered gene enrichment of ribosomal proteins. AC-specific RNAi, endogenous ribosome labeling and ribosome biogenesis analysis revealed that a burst of ribosome production occurs shortly after AC specification, which drives the translation of proteins mediating BM removal. Ribosomes also enrich near the AC endoplasmic reticulum (ER) Sec61 translocon and the endomembrane system expands before invasion. We show that AC invasion is sensitive to ER stress, indicating a heightened requirement for translation of ER-trafficked proteins. These studies reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration.

Reevaluating the relationship between EGL-43 (EVI1) and LIN-12 (Notch) during C. elegans anchor cell invasion.

Development of the Caenorhabditis elegans reproductive tract is orchestrated by the anchor cell (AC). This occurs in part through a cell invasion event that connects the uterine and vulval tissues. Several key transcription factors regulate AC invasion, such as EGL-43, HLH-2, and NHR-67. Specifically, these transcription factors function together to maintain the post-mitotic state of the AC, a requirement for AC invasion. Recently, a mechanistic connection has been made between loss of EGL-43 and AC cell-cycle entry. The current model states that EGL-43 represses LIN-12 (Notch) expression to prevent AC proliferation, suggesting that Notch signaling has mitogenic effects in the invasive AC. To reexamine the relationship between EGL-43 and LIN-12, we first designed and implemented a heterologous co-expression system called AIDHB that combines the auxin-inducible degron (AID) system of plants with a live cell-cycle sensor based on human DNA helicase B (DHB). After validating AIDHB using AID-tagged GFP, we sought to test it by using AID-tagged alleles of egl-43 and lin-12. Auxin-induced degradation of either EGL-43 or LIN-12 resulted in the expected AC phenotypes. Lastly, we seized the opportunity to pair AIDHB with RNAi to co-deplete LIN-12 and EGL-43, respectively, which revealed that LIN-12 is not required for AC proliferation following loss of EGL-43.

Localized glucose import, glycolytic processing, and mitochondria generate a focused ATP burst to power basement-membrane invasion.

Invasive cells use transient, energy-consuming protrusions to breach basement membrane (BM) barriers. Using the ATP sensor PercevalHR during anchor cell (AC) invasion in Caenorhabditis elegans, we show that BM invasion is accompanied by an ATP burst from mitochondria at the invasive front. RNAi screening and visualization of a glucose biosensor identified two glucose transporters, FGT-1 and FGT-2, which bathe invasive front mitochondria with glucose and facilitate the ATP burst to form protrusions. FGT-1 localizes at high levels along the invasive membrane, while FGT-2 is adaptive, enriching most strongly during BM breaching and when FGT-1 is absent. Cytosolic glycolytic enzymes that process glucose for mitochondrial ATP production cluster with invasive front mitochondria and promote higher mitochondrial membrane potential and ATP levels. Finally, we show that UNC-6 (netrin), which polarizes invasive protrusions, also orients FGT-1. These studies reveal a robust and integrated energy acquisition, processing, and delivery network that powers BM breaching.

A DNA replication-independent function of pre-replication complex genes during cell invasion in C. elegans.

Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.

The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo.

Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and chromatin regulating factors that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis.

Reciprocal EGFR signaling in the anchor cell ensures precise inter-organ connection during Caenorhabditis elegans vulval morphogenesis.

ABSTRACTDuring Caenorhabditis elegans vulval development, the uterine anchor cell (AC) first secretes an epidermal growth factor (EGF) to specify the vulval cell fates and then invades the underlying vulval epithelium. By doing so, the AC establishes direct contact with the invaginating primary vulF cells and attaches the developing uterus to the vulva. The signals involved and the exact sequence of events joining these two organs are not fully understood. Using a conditional let-23 EGF receptor (EGFR) allele along with novel microfluidic short- and long-term imaging methods, we discovered a specific function of the EGFR in the AC during vulval lumen morphogenesis. Tissue-specific inactivation of let-23 in the AC resulted in imprecise alignment of the AC with the primary vulval cells, delayed AC invasion and disorganized adherens junctions at the contact site forming between the AC and the dorsal vulF toroid. We propose that EGFR signaling, activated by a reciprocal EGF cue from the primary vulval cells, positions the AC at the vulval midline, guides it during invasion and assembles a cytoskeletal scaffold organizing the adherens junctions that connect the developing uterus to the dorsal vulF toroid. Thus, EGFR signaling in the AC ensures the precise alignment of the two developing organs.

Microfluidic-based imaging of complete Caenorhabditis elegans larval development.

ABSTRACTSeveral microfluidic-based methods for Caenorhabditis elegans imaging have recently been introduced. Existing methods either permit imaging across multiple larval stages without maintaining a stable worm orientation, or allow for very good immobilization but are only suitable for shorter experiments. Here, we present a novel microfluidic imaging method that allows parallel live-imaging across multiple larval stages, while maintaining worm orientation and identity over time. This is achieved through an array of microfluidic trap channels carefully tuned to maintain worms in a stable orientation, while allowing growth and molting to occur. Immobilization is supported by an active hydraulic valve, which presses worms onto the cover glass during image acquisition only. In this way, excellent quality images can be acquired with minimal impact on worm viability or developmental timing. The capabilities of the devices are demonstrated by observing the hypodermal seam and P-cell divisions and, for the first time, the entire process of vulval development from induction to the end of morphogenesis. Moreover, we demonstrate feasibility of on-chip RNAi by perturbing basement membrane breaching during anchor cell invasion.

To Divide or Invade: A Look Behind the Scenes of the Proliferation-Invasion Interplay in the Caenorhabditis elegans Anchor Cell.

Cell invasion is defined by the capability of cells to migrate across compartment boundaries established by basement membranes (BMs). The development of complex organs involves regulated cell growth and regrouping of different cell types, which are enabled by controlled cell proliferation and cell invasion. Moreover, when a malignant tumor takes control over the body, cancer cells evolve to become invasive, allowing them to spread to distant sites and form metastases. At the core of the switch between proliferation and invasion are changes in cellular morphology driven by remodeling of the cytoskeleton. Proliferative cells utilize their actomyosin network to assemble a contractile ring during cytokinesis, while invasive cells form actin-rich protrusions, called invadopodia that allow them to breach the BMs. Studies of developmental cell invasion as well as of malignant tumors revealed that cell invasion and proliferation are two mutually exclusive states. In particular, anchor cell (AC) invasion during Caenorhabditis elegans larval development is an excellent model to study the transition from cell proliferation to cell invasion under physiological conditions. This mini-review discusses recent insights from the C. elegans AC invasion model into how G1 cell-cycle arrest is coordinated with the activation of the signaling networks required for BM breaching. Many regulators of the proliferation-invasion network are conserved between C. elegans and mammals. Therefore, the worm may provide important clues to better understand cell invasion and metastasis formation in humans.

The Caenorhabditis elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion.

Cell invasion allows cells to migrate across compartment boundaries formed by basement membranes. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. Anchor cell (AC) invasion in C. elegans is an excellent in vivo model to study the regulation of cell invasion during development. Here, we have examined the function of egl-43, the homolog of the human Evi1 proto-oncogene (also called MECOM), in the invading AC. egl-43 plays a dual role in this process, firstly by imposing a G1 cell cycle arrest to prevent AC proliferation, and secondly, by activating pro-invasive gene expression. We have identified the AP-1 transcription factor fos-1 and the Notch homolog lin-12 as critical egl-43 targets. A positive feedback loop between fos-1 and egl-43 induces pro-invasive gene expression in the AC, while repression of lin-12 Notch expression by egl-43 ensures the G1 cell cycle arrest necessary for invasion. Reducing lin-12 levels in egl-43 depleted animals restored the G1 arrest, while hyperactivation of lin-12 signaling in the differentiated AC was sufficient to induce proliferation. Taken together, our data have identified egl-43 Evi1 as an important factor coordinating cell invasion with cell cycle arrest.

A developmental gene regulatory network for C. elegans anchor cell invasion.

Cellular invasion is a key part of development, immunity and disease. Using an in vivo model of Caenorhabditis elegans anchor cell invasion, we characterize the gene regulatory network that promotes cell invasion. The anchor cell is initially specified in a stochastic cell fate decision mediated by Notch signaling. Previous research has identified four conserved transcription factors, fos-1 (Fos), egl-43 (EVI1/MEL), hlh-2 (E/Daughterless) and nhr-67 (NR2E1/TLX), that mediate anchor cell specification and/or invasive behavior. Connections between these transcription factors and the underlying cell biology that they regulate are poorly understood. Here, using genome editing and RNA interference, we examine transcription factor interactions before and after anchor cell specification. Initially, these transcription factors function independently of one another to regulate LIN-12 (Notch) activity. Following anchor cell specification, egl-43, hlh-2 and nhr-67 function largely parallel to fos-1 in a type I coherent feed-forward loop with positive feedback to promote invasion. Together, these results demonstrate that the same transcription factors can function in cell fate specification and differentiated cell behavior, and that a gene regulatory network can be rapidly assembled to reinforce a post-mitotic, pro-invasive state.

Adaptive F-Actin Polymerization and Localized ATP Production Drive Basement Membrane Invasion in the Absence of MMPs

Matrix metalloproteinases (MMPs) are associated with decreased patient prognosis but have failed as anti-invasive drug targets despite promoting cancer cell invasion. Through time-lapse imaging, optical highlighting, and combined genetic removal of the five MMPs expressed during anchor cell (AC) invasion in C.elegans, we find that MMPs hasten invasion by degrading basement membrane (BM). Though irregular and delayed, AC invasion persists in MMP- animals via adaptive enrichment of the Arp2/3 complex at the invasive cell membrane, which drives formation of an F-actin-rich protrusion that physically breaches and displaces BM. Using a large-scale RNAi synergistic screen and a genetically encoded ATP FRET sensor, we discover that mitochondria enrich within the protrusion and provide localized ATP that fuels F-actin network growth. Thus, without MMPs, an invasive cell can alter its BM-breaching tactics, suggesting that targeting adaptive mechanisms will be necessary to mitigate BM invasion in human pathologies.

Long-term C. elegans immobilization enables high resolution developmental studies in vivo.

Live-imaging of C. elegans is essential for the study of conserved cellular pathways (e.g. EGFR/Wnt signaling) and morphogenesis in vivo. However, the usefulness of live imaging as a research tool has been severely limited by the need to immobilize worms prior to and during imaging. Conventionally, immobilization is achieved by employing both physical and chemical interventions. These are known to significantly affect many physiological processes, and thus limit our understanding of dynamic developmental processes. Herein we present a novel, easy-to-use microfluidic platform for the long-term immobilization of viable, normally developing C. elegans, compatible with image acquisition at high resolution, thereby overcoming the limitations associated with conventional worm immobilization. The capabilities of the platform are demonstrated through the continuous assessment of anchor cell (AC) invasion and distal tip cell (DTC) migration in larval C. elegans and germ cell apoptosis in adult C. elegans in vivo for the first time.

Cell Invasion In Vivo via Rapid Exocytosis of a Transient Lysosome-Derived Membrane Domain

Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in Caenorhabditis elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume by more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion, which enables protrusion growth. Together these studies define a netrin-dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.

Live-cell confocal microscopy and quantitative 4D image analysis of anchor-cell invasion through the basement membrane in Caenorhabditis elegans.

Cell invasion through basement membrane (BM) barriers is crucial in development, leukocyte trafficking and the spread of cancer. The mechanisms that direct invasion, despite their importance in normal and disease states, are poorly understood, largely because of the inability to visualize dynamic cell-BM interactions in vivo. This protocol describes multichannel time-lapse confocal imaging of anchor-cell invasion in live Caenorhabditis elegans. Methods presented include outline-slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min) and quantitative analysis (variable timing). The acquired images enable direct measurement of invasive dynamics including formation of invadopodia and cell-membrane protrusions, and removal of BM. This protocol can be combined with genetic analysis, molecular-activity probes and optogenetic approaches to uncover the molecular mechanisms underlying cell invasion. These methods can also be readily adapted by any worm laboratory for real-time analysis of cell migration, BM turnover and cell-membrane dynamics.

Abstract A18: G1 cell cycle arrest is required for invasive behavior

Abstract The ability for a cell to breach the surrounding basement membrane and adopt an invasive phenotype is a critical, but poorly understood step in the progression of cancer. Like many aspects of cancer cell behavior, the genetic programs that regulate invasive behavior are likely shared with normal cell biological processes, as invasion occurs during embryonic development and immune surveillance. Due to difficulties of studying this dynamic behavior in vivo, elucidating the genetic and epigenetic controls of cell invasive activity has been difficult. Our laboratory uses a unique in vivo model to examine cell invasion that combines functional genomic and genetic tools with single-cell high resolution visual analyses. We examine anchor cell (AC) invasion into the vulval epithelium during the larval development of the model roundworm, C. elegans. Our data functionally links G1 cell cycle arrest to acquisition of invasive behavior. Previously we have identified that a single transcription factor, the conserved nuclear hormone receptor nhr-67 (NR2E1/TLX) is required in the AC to prevent the invasive AC from entering the cell cycle. NHR-67 maintains the AC in G1 cell cycle arrest, in part through upregulation of the cyclin-dependent kinase inhibitor cki-1 (p21CIP1/p27Kip1). Loss of nhr-67 results in non-invasive mitotic ACs that fail to express matrix metalloproteinases (MMPs) and actin regulators or form invadopodia, F-actin rich membrane-localized protrusions that are required for invasion. Strikingly, AC invasion can be rescued through induction of G1 arrest, preventing cell division and promoting differentiation. Downstream of G1 arrest, the AC requires the activity of the conserved histone deacetylase HDA-1, a key regulator of cell differentiation, to regulate the expression of pro-invasive genes and localize invadopodia. Through loss-of-function RNA interference (RNAi) screening, we have identified new cell cycle regulatory components (lin-35/RB, skr-2/SKP1, cdc-14/CDC14A, cul-1/ Cullin-1 and cul-4/ Cullin-4B) and epigenetic modifiers (let-418/Mi-2/CHD3 and Swi/snf-components) that function with cki-1 and hda-1, respectively, to maintain G1 cell cycle arrest and differentiate the invasive phenotype. Together our results suggest that the acquisition of the invasive phenotype is a post-mitotic differentiated state, which may help explain the paradoxical reports that the invasive fronts of many metastatic cancers are non-proliferative. Citation Format: Abraham Q. Kohrman, Mana Chandhok, Wan Zhang, David Q. Matus. G1 cell cycle arrest is required for invasive behavior. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A18.

SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane.

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression

Despite critical roles in development and cancer, the mechanisms that specify invasive cellular behavior are poorly understood. Through a screen of transcription factors in Caenorhabditis elegans, we identified G1 cell-cycle arrest as a precisely regulated requirement of the anchor cell (AC) invasion program. We show that the nuclear receptor nhr-67/tlx directs the AC into G1 arrest in part through regulation of the cyclin-dependent kinase inhibitor cki-1. Loss of nhr-67 resulted in non-invasive, mitotic ACs that failed to express matrix metalloproteinases or actin regulators and lack invadopodia, F-actin-rich membrane protrusions that facilitate invasion. We further show that G1 arrest is necessary for the histone deacetylase HDA-1, a key regulator of differentiation, to promote pro-invasive gene expression and invadopodia formation. Together, these results suggest that invasive cell fate requires G1 arrest and that strategies targeting both G1-arrested and actively cycling cells may be needed to halt metastatic cancer.