A DNA Replication-Independent Function of the pre-Replication Complex 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 over-expressed 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 functions cell-autonomously in the G1-arrested AC to promote invasion, independently of its 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 acts in the non-proliferating AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.


Introduction
Cell invasion, defined as the movement of cells across compartment boundaries formed by basement membranes (BM), is an essential process during normal development. Yet, cell invasion can have fatal consequences during cancer progression [1]. During metastasis, individual cells of a primary tumor adopt an invasive phenotype, resulting in metastatic tumor spreading through the vasculature. Despite the different outcomes, developmental and cancerous cell invasion have many common features. Both types of invasion involve a phenotypic switch called epithelial to mesenchymal transition (EMT). Invasive cells adopt a mesenchymal-like phenotype characterized by the formation of actin-rich protrusions called invadopodia, the loss of cadherin-mediated cell adhesion and the expression of metalloproteinases (MMPs) that break down the BM. This phenotypic switch can be stimulated by extracellular signals, such as TGF-ß, EGF and Wnt, and is regulated by conserved zinc finger transcription factors of the Twist, Snail and Krüppel family as well as by the Jun/Fos (AP-1) transcription factors that activate characteristic, pro-invasive transcriptional programs [2].
Here, we have used C. elegans vulval development as an in vivo model to investigate the regulation of cell invasion. During the development of the vulva, the egg-laying organ of the hermaphrodite, the anchor cell (AC) in the ventral uterus breaches two BMs and invades the underlying vulval epithelium to establish direct contact between the developing uterus and vulva [3,4]. In the wild-type, AC invasion occurs during a specific time period, beginning in mid-L3 larvae after the first round of vulval cell divisions (the Pn.p two cell stage) and ending after the second round of divisions has been completed (Pn.p four cell stage). The Netrin homologue UNC-6 secreted by neurons in the ventral nerve cord, together with an unknown cue released by the primary (1°) vulval precursor cells (VPCs) that are nearest to the AC, polarizes and guides the invading AC ventrally [5]. Activation of the UNC-40/DCC receptor in the AC induces the recruitment of several actin regulators and the -integrin complex to the ventral AC cortex, thereby creating an invasive membrane [6]. At the same time, the expression of pro-invasive genes is activated by the AP-1 transcription factor FOS-1A and egl- 43, which encodes a zinc finger transcription factor homologous to the mammalian Evi1 proto-oncogene [7][8][9]. Known target genes of FOS-1A and EGL-43/EVI1 in the AC include metalloproteinase zmp-1, protocadherin cdh-3 and hemicentin him-4 [7].
Another important aspect of cell invasion is the dichotomy between the proliferative and invasive state of a cell. The AC must remain arrested in the G1 phase of the cell cycle in order to activate pro-invasive gene expression and breach the BMs [8]. The histone deacetylase hda-1, the homolog of mammalian HDAC1, the nuclear hormone receptor nhr-67 and egl-43 are critical to establish the G1 arrest of the AC [9,10].
Likewise, the invasiveness of human cancer cells often correlates with proliferation arrest. For example, during metastatic progression melanoma cells can undergo multiple rounds of phenotype switching between a proliferative and an invasive state [11]. However, the mechanisms by which cell cycle regulators affect cell invasion are poorly understood.
The conserved pre-replication complex (pre-RC) plays an essential role during the G1 phase of the cell cycle to license the DNA replication origins that will be used in the following S-phase [12][13][14]. The pre-RC assembles during the late M and early G1 phase of the cell cycle by the sequential recruitment of the origin recognition complex proteins ORC-1-ORC-6 to DNA replication origins, followed by the CDC-6 protein together with the chromatin licensing and replication factor CDT-1, and finally the DNA helicase complex formed by the mini-chromosome maintenance proteins MCM2 to MCM-7 [12,15]. After the pre-RC has been assembled in the G1 phase, the origins are licensed to initiate DNA replication during the following S-phase. Thereafter, CDK/Cyclin activity in the late G1 and early S-phase induces the disassembly and partial degradation of the pre-RC to prevent the re-licensing of replication origins.
Using C. elegans AC invasion as a model to screen for genes controlling cell invasion, we have found that multiple components of the pre-RC are required for BM breaching by the AC. In addition, we have identified the PI3K pathway as one critical downstream target of the pre-RC promoting BM breaching by the AC. Together, these data point to a replication-independent function of the pre-RC in regulating cell invasion during the G1 phase of the cell cycle.

Identification of cell invasion regulators among differentially expressed genes in invasive melanoma cells
In order to identify genes that are functionally involved in regulating cell invasion, we investigated candidate genes whose orthologs were upregulated in human melanoma cells that had been stimulated with TGF-β, exposed to hypoxia, or which overexpressed the transcription factor SOX9, and included genes upregulated in migrating neural crest cells that had undergone EMT [16][17][18]. This resulted in a list of 104 differentially expressed human genes that were upregulated under at least two conditions and for which a C. elegans ortholog could be identified (suppl . Table S1).
RNAi-mediated knock-down of 32 of the 104 C. elegans orthologs resulted in a protruding vulva (Pvl) phenotype, which is characteristic of defects in vulval morphogenesis or in AC invasion. In order to specifically identify regulators of AC invasion, we scored BM breaching by the AC after RNAi-mediated knock-down. AC invasion was analyzed in L3 larvae after the 1° VPCs had divided twice, at the VPC four-cell (Pn.pxx) stage of vulval development. If viability and fertility permitted, we scored the F1 progeny of RNAi-treated mothers, or else we scored the P0 animals that had been exposed to the dsRNA-producing bacteria from the L1 stage on. To visualize BM breaching by the invading AC, we used a LAM-1::GFP reporter that labels the two BMs separating the AC from the underlying vulval cells (Fig. 1A) [5]. Among the 104 candidates tested, RNAi knock-down of 18 genes perturbed AC invasion (suppl . Table   S1). Notably, three genes required for normal AC invasion encode cell cycle regulators; cdc-6 encodes an essential component of the DNA pre-replication complex (pre-RC) [12,15], cyd-1 encodes the only Cyclin D homolog in C. elegans [19] and cdk-12 encodes the Cyclin-dependent kinase required for activation of RNA polymerase II [20]. In particular, Cyclin D was shown to be a driver for invasion in a study investigating the development of thin melanoma [21]. After RNAi knockdown of cdc-6 the AC failed to invade in 32% of the cases (Fig. 1A).
In the following, we focused our experiments on studying the role of the pre-RC genes during cell invasion.

Several components of the pre-Replication Complex regulate AC invasion
Since the AC remains arrested in the G1 phase of the cell cycle while it breaches the BMs [8], the identification of the pre-RC component CDC-6 as a regulator of AC invasion was unexpected. We therefore tested if other pre-RC components are also required for BM breaching by the AC. Besides cdc-6, RNAi-mediated knockdown of several orc genes, as well as of cdt-1 and mcm-7 led to BM breaching defects ( Fig. 1A and Table 1). The penetrance of the invasion defects ranged from 15% for cdt-1 to 51% upon orc-2 RNAi (not taking into account the single cases observed after orc-3 and orc-4 RNAi). Among the mcm genes, only mcm-7 RNAi resulted in an invasion defect in 34% of the animals (Fig. 1A and Tab. 1). However, the absence of AC invasion defects for other mcm genes tested could be due to low efficiencies of the RNAi knock-down.
Thus, several components of the pre-RC are involved in BM breaching by the AC. In addition to the AC invasion defects, RNAi of pre-RC complex components induced a proliferation arrest in different cells of the larvae, notably in the dividing VPCs adjacent to the AC (Fig. 1B).

The DNA replication pre-Initiation Complex is not required for AC invasion
At the onset of the S-phase, the pre-RC transitions into the preinitiation complex (pre-IC) through the recruitment of accessory proteins of the GINS complex (PSF-1, PSF-2, PSF-3, SLD-5) and additional factors, such as CDC-45, MCM-10, CDC-7 and PCN-1, that are necessary to initiate DNA replication [12,15,22]. In order to test if the initiation of DNA replication is required for AC invasion, we examined the AC invasion phenotype of a psf-1 null (lf) allele and performed RNAi against different pre-IC components. Except for a single case in cdc-45 RNAi treated animals, we did not observe any invasion defects after RNAi knock-down or in mutants of pre-IC genes ( Fig. 1A bottom row and suppl. Table S2). It should be noted that the gap the AC created in the BMs of psf-1(lf) mutants could not be expanded because the underlying VPCs had ceased to proliferate (Fig. 1A bottom row) [23]. Therefore, components of the pre-IC are not necessary for AC invasion.

VPC proliferation is not required for AC invasion
Invasion by the post-mitotic AC is linked to the differentiation and proliferation of the underlying 1° VPCs that produce guidance signals, which attract the AC ventrally [7].
We therefore tested whether the AC invasion defects observed after down-regulation of pre-RC components are caused by a proliferation arrest of the VPCs. We first examined on an animal per animal basis whether the AC invasion defects caused by mcm-7 RNAi correlated with the proliferation arrest of the VPCs. An analysis of 110 animals revealed no correlation, as mcm-7i caused invasion defects occurred independently of whether the VPC had divided or not (Fig. 1B,C). Next, we blocked the cell cycle of 1° VPC P6.p in the G1 phase by over-expressing the CDK inhibitor cki-1 under control of the 1° lineage-specific egl-17 promoter (zhEx334[egl-17>cki-1::gfp; myo-2::mCherry] [24] and used a LAM-1::mCherry reporter to score BM breaching. In all egl-17>cki-1::gfp; lam-1>lam-1::mCherry animals with an undivided P6.p cell at the P5/7.p four-cell (n=16) or eight-cell stage (n=16) the BMs were breached, indicating that AC invasion does not depend on the division of the 1° VPCs ( Fig. 1D).
We conclude that the AC invasion defects caused by mcm-7 RNAi are not due to a proliferation arrest of the 1° VPCs.

mcm-7 is expressed in the AC prior to invasion
In order to determine the expression pattern of the pre-RC components during AC invasion we focused on mcm-7, because it encodes a core subunit of the MCM complex that is the last component recruited to the pre-RC. We used CRISPR/Cas9 engineering to insert a gfp tag upstream of the translational start codon in the mcm-7 locus along with two flippase (FLP) recognition target (FRT) sites within the gfp cassette. This permitted the tissue-specific inactivation of mcm-7 by FLP-induced recombination (zh118[frt::gfp::mcm-7], suppl. Fig. S1) [25,26]. zh118[frt::gfp::mcm-7] animals exhibited GFP::MCM-7 expression in the nuclei of proliferating cells, including many uterine cells and the VPCs ( Fig. 2A). In mid-L2 larvae, GFP::MCM-7 was expressed at comparably lower levels in the newly specified AC. During the late L2 and early L3 stage, GFP::MCM-7 expression in the AC further declined but was still detectable in mid-L3 larvae just prior to invasion. In late L3 larvae, after the BM had been breached, no GFP::MCM-7 expression was detectable in the AC ( Fig. 2A).
Since GFP::MCM-7 expression levels were highest in the newly formed AC, we tested whether mcm-7 is required for the specification or function of the AC. We first examined the expression of the b-HLH transcription factor HLH-2, whose expression onset in early L2 larvae instructs the AC fate [27]. mcm-7 RNAi had no significant effect on HLH-2::GFP expression levels in the AC of mid-L3 larvae (suppl. Fig.  S2A,B). The differentiated AC then expresses the proto-cadherin cdh-3 [28] and the EGF homolog lin-3, which induces the differentiation of the adjacent VPCs [4,29]. A transcriptional cdh-3>gfp reporter was not changed after mcm-7 RNAi, while lin-3>gfp expression was reduced (suppl. Fig. S2C-F). However, the lin-3 levels in the AC after mcm-7 RNAi were sufficient to induce vulval differentiation, as the 1° vulval cell fate marker egl-17>gfp [30] was expressed in the 1° VPCs (suppl. Fig. S2G). Thus, MCM-7 is not necessary for the specification of the AC fate or for the normal function of the AC during the induction of vulval differentiation.

mcm-7 acts in the AC to promote BM breaching
The observation that a proliferation arrest of the VPCs did not impair BM breaching raised the possibility that mcm-7 acts cell-autonomously in the AC to promote invasion, besides its established role in replication origin licensing in proliferating cells. To test whether the low GFP::MCM-7 expression levels observed in the AC of L3 larvae promote invasion, we first performed Pn.p cell and AC-specific RNAi of mcm-7 [31][32][33]. Notably, 16% of the animals treated with AC-specific mcm-7 RNAi showed a BM breaching defect, whereas none of the control RNAi treated or Pn.p cell-specific mcm-7 RNAi animals exhibited BM breaching defects (Fig. 2B). This analysis suggested a cell-autonomous role of mcm-7 in the post-mitotic AC. To independently confirm these findings, we performed FLP/FRT-induced mosaic analysis in the zh118[frt::gfp::mcm-7] strain [26]. The FLPase was expressed under control of the heat-shock inducible hs- 16-48 promoter (hs>flp) and induced by a single heat-shock at the late L1/early L2 stage. This protocol resulted in the development of mosaic animals that had lost GFP::MCM-7 expression either in most uterine cells, in the VPCs or in both lineages ( Fig. 2C). Among the VPC mosaics, we focused on animals showing loss of GFP::MCM-7 expression in the 1° lineage because the 1° VPCs are necessary to induce AC invasion [7]. Around 29% of animals with a loss of GFP::MCM-7 expression in most uterine cells and 38% of animals lacking expression in both the uterus and the 1° VPCs showed a BM breaching defect ( Fig. 2C and white bars in 2D). Except for the AC, other uterine cells are not required for BM breaching [34]. By contrast, only 3.1% of the 1° VPC mosaics and 2.7% of the cases without an apparent loss of GFP::MCM-7 expression in any of the two tissues exhibited a BM breaching defect.
Taken together, the tissue-specific RNAi experiments and the mosaic analysis indicated that mcm-7 acts cell-autonomously in the post-mitotic AC to positively regulate BM breaching. However, the highest penetrance of the BM breaching defect after FLP/FRT-mediated inactivation of mcm-7 was 38%, suggesting that pre-RC activity may not be absolutely required for AC invasion.

The DNA helicase activity of the pre-RC is required for AC invasion
The MCM protein complex provides the helicase activity of the pre-RC that is necessary for DNA unwinding during the formation of a replication fork. To test if the MCM helicase activity is necessary for AC invasion, we introduced four point These results indicated that the MCM helicase activity is not only required for the initiation of DNA replication, but also for BM breaching by the AC.

mcm-7 controls BM breaching in the G1-arrested AC
It was previously reported that the AC must remain arrested in the G1 phase of the cell cycle in order to adopt the invasive cell fate [8]. We therefore examined whether the pre-RC controls the cell cycle state of the AC by examining the expression of cell cycle reporters. A transcriptional reporter for the G1 phase marker cyd-1 cyclinD (zhIs131[cyd-1>gfp]) was expressed in the AC of mcm-7 RNAi treated animals at similar levels as in control animals (Fig. 3A). Conversely, the expression of the S phase marker rnr-1::gfp was undetectable in the AC of control and of mcm-7 RNAi treated animals ( Fig. 3B) [38]. Consistent with these findings, the histone deacetylase hda-1 that is induced in the AC upon G1 arrest and is required for the invasive fate remained expressed after mcm-7 knock-down (suppl. Fig. S4) [8].
In addition to the analysis of cell cycle markers, we made use of a CDK activity sensor to directly quantify CDK activity in the AC [39]. This sensor consists of a portion of the DNA Helicase B containing four CDK phosphorylation sites, a nuclear export and a nuclear import signal, N-terminally fused to an eGFP tag. Upon phosphorylation by CDKs, the sensor translocates from the nucleus to the cytoplasm. Thus, a low cytoplasmic to nuclear ratio of the eGFP signal indicates weak CDK activity. For our purpose, we expressed the CDK sensor specifically in the AC under control of the lin-3 ACEL enhancer/pes-10 minimal promoter fragment (zhIs130[ACEL>cdk sensor::egfp]). In control animals, the average cytoplasmic to nuclear fluorescence ratio in the AC was 0.51±0.12 (Fig. 3C,D). mcm-7 RNAi did not cause a change in sensor activity (average fluorescence ratio 0.54±0.12), indicating that the pre-RC is not required for the G1 arrest of the AC. By contrast, RNAi-mediated knock-down of the nuclear hormone receptor nhr-67, which is required for the G1 phase arrest of the AC [8], resulted in a significant increase in CDK activity (average ratio 0.84±0.14) (Fig.   3C,D). Moreover, reducing nhr-67 levels resulted in the formation of multiple ACs that failed to invade, as reported previously [8], and expressed high levels of GFP::MCM-7 characteristic of proliferating cells (Fig. 3E).
Hence, the downregulation of mcm-7 did not affect the G1 arrest of the AC. Low expression levels of MCM-7 are associated with G1 arrest and AC invasion, whereas high MCM-7 expression levels accompany the loss of the invasive phenotype in proliferating ACs.

mcm-7 regulates invadopodia formation and extracellular matrix gene expression in the AC
Prior to BM breaching, the AC is polarized ventrally and extends actin-rich protrusions, (invadopodia), towards the 1° VPCs, which are aligned at the ventral midline [5]. To assess whether the pre-RC controls invadopodia formation, we quantified the polarity of an F-actin reporter after RNAi knock-down of mcm-7. The probe consisted of the Factin-binding domain of moesin fused to mCherry and was expressed under control of the AC-specific mk62-63 enhancer element of cdh-3 (mCherry::moeABD) [6]. In control animals, the mCherry::moeABD signal was polarized towards the ventral side and enriched at the tip of the invasive protrusion, where the AC breaches the BM (Fig.   4A,B). Upon mcm-7 RNAi treatment, the F-actin reporter was de-polarized and no properly organized invasive membrane domain was formed by the AC (Fig. 4A,B).
The invasive AC phenotype is characterized by the expression of several pro-invasive genes, such as the matrix metalloproteinase zmp-1 and the hemicentin him-4, which is secreted by the AC to remodel the BM [7]. RNAi knock-down of mcm-7 decreased expression of the him-4>ΔSP::gfp and zmp-1>cfp reporters in the AC (Fig. 4C-F). The reduction in pro-invasive gene expression was not limited to the pre-RC component mcm-7, but was also observed after RNAi knock-down of cdc-6 ( Fig. 4G,H). Since  Fig. S5).
In summary, the analysis of AC-specific gene expression indicated that the pre-RC positively regulates the expression of specific extracellular matrix genes that facilitate AC invasion.

Expression profiling of human cancer cells identifies several PI3K pathway genes as candidate pre-RC targets
To systematically identify the changes in gene expression caused by inhibiting the pre-RC, we turned to human cancer cell lines. Since the pre-RC genes were originally selected based on their differential expression in human melanoma cells, we examined the changes in gene expression caused by MCM7 depletion in the A375 melanoma cell line, and to extend our study to another cancer type, included the A549 lung cancer cell line in the expression analysis. Moreover, to exclude possible effects caused by changes in cell cycle progression and focus on the replication-independent functions of MCM7, we analyzed gene expression in cells arrested at the G1/S boundary. For this purpose, we generated A375 and A549 cells stably expressing an MCM7 shRNA or a scrambled shRNA as negative control, each under control of the doxycycline-(Dox) inducible enhancer/promoter. Addition of 1 µg/ml Dox to induce shMCM7 expression resulted in a 90 % to 80% reduction in MCM7 protein levels in A375 and A549 cells, respectively (suppl. Fig. 6C-E). Cell cycle analysis revealed that MCM7 depletion caused a slight enrichment of cells in the G1 phase (suppl. Fig. 6A,B). Using a double thymidine (Thy) synchronization protocol to arrest cells at the G1/S boundary [41], we obtained cell populations, in which over 95% of the cells were arrested in the G1 phase of the cell cycle (suppl. Fig. 6A,B). From these synchronized cell populations mRNA was extracted to obtain global expression profiles by RNAseq for both cell lines.
RNAseq analysis revealed widespread changes in the mRNA expression profile of MCM7-depleted and G1/S phase-arrested cells (suppl. Fig. 7A,B and suppl. Table   S6). Clustering of the MCM7-regulated genes revealed an enrichment of genes encoding extracellular matrix components and cell junction and plasma membrane proteins among the up-regulated genes (suppl. Fig. 7A,B). To identify a core set of MCM7 target genes, we determined the overlap between the significantly up-and down-regulated genes in A375 and A549 cells (≥1.5-fold change with p<0.01 and FDR<0.05). This analysis identified 259 genes up-regulated and 327 genes downregulated after MCM7 depletion in both cell lines (suppl. Fig. 7C,D). GO enrichment analysis of the molecular functions and biological processes of these core MCM7 target genes indicated that many of the up-regulated genes encode extracellular matrix, integrin, actin and ß-catenin interacting proteins, while many of the down-regulated genes encode enzymes required for mitochondrial respiration and ribosomal RNAbinding proteins (suppl. Fig. 7E,F).
In particular, we observed several components of the 1-phosphatidylinositol-3-kinase (PI3K) signaling pathway among the most strongly downregulated genes ( Fig. 5A and suppl. Table S6). Expression of the regulatory PIK3R2 (p85β), and PIK3R3 (p55γ) subunits as well as the AKT3 kinase that acts downstream of PI3K was reduced upon MCM7 knock-down in G1/S arrested A375 and A459 cells, and the PIK3CD (p110δ) catalytic PI3K subunit was also significantly reduced in A549, but not in A375 cells (Fig. 5A). To examine whether the decreased expression of PI3K pathway components manifests in a diminished activity of the PI3K pathway, we quantified the levels of phosphorylated, activated AKT kinase using a phospho-specific AKT antibody for Western blot analysis. Insulin or EGF stimulation of serum-starved A375 and A549 cells that had previously been depleted of MCM7 resulted in an attenuated activation of the PI3K pathway (Fig. 5 B and suppl. Fig. 6C-E, note that A375 cells did not respond to EGF stimulation).
In summary, expression profiling of two human cancer cell lines identified several components of the PI3K/AKT pathway as candidate MCM7 targets. Since the analysis was performed with cells arrested at the G1/S boundary, the regulation of the PI3K pathway by the pre-RC appears to occur independently of cell cycle progression. In untreated or empty vector-treated control RNAi animals, GFP::AGE-1 and AAP-1::GFP were both expressed in the AC and the surrounding cells at the Pn.pxx stage before and during invasion (Fig. 5C,E). Interestingly, the GFP::AGE-1 and AAP-1::GFP proteins were asymmetrically localized in the AC and appeared polarized towards the invasive membrane. The levels of the catalytic PI3K subunit AGE-1 decreased in the AC upon mcm-7 RNAi (Fig. 5C-F). By contrast, we did not detect a significant change in the mean expression levels of the regulatory subunit AAP-1, but expression was more variable after mcm-7 RNAi (Fig. 5F, p<0.0001 in an F-test for variance). Moreover, the asymmetric distribution of AAP-1::GFP and GFP::AGE-1 in the AC was lost in most mcm-7 RNAi treated animals (Fig. 5G). By contrast, expression and localization of both PI3K subunits remained unchanged after fos-1 RNAi (suppl. Fig. S8).

PI3K signaling in C. elegans promotes BM breaching by the AC
Finally, we tested whether over-activation of the PI3K pathway could rescue the invasion defects caused by down-regulation of mcm-7. For this purpose, we performed mcm-7 RNAi in animals carrying a gain-of-function mutation in the AKT homolog akt-1 [43] or a deletion allele of daf-18, which encodes the homolog of the PTEN tumor suppressor that antagonizes PI3K activity [42], and scored BM breaching using the LAM-1::GFP marker. The akt-1(mg144gf) and daf-18(ok480lf) mutations partially suppressed the invasion defects caused by mcm-7 RNAi, while the egl-43 RNAi invasion defects were not affected by akt-1(gf) or daf-18(lf) (Fig. 5H).
Taken together, these data suggest that the positive regulation of the PI3K pathway by the pre-RC contributes to the invasive phenotype of the AC. Since hyper-activation of PI3K signaling only partially suppressed the mcm-7 invasion defects, the PI3K pathway is likely one of several critical processes regulated by the pre-RC in the AC.

Discussion
Using invasion, remained unchanged [8,28,40]. Taken together, these observations strongly support a DNA replication-independent function of the pre-RC during AC invasion (Fig. 6).
Expression profiling of two human cancer cell lines arrested at the G1/S-phase boundary identified several PI3K pathway components as candidate pre-RC targets.
Many PI3K pathway genes are involved in the malignant progression of various cancer types [45]. Especially, increased expression of the regulatory subunit PIK3R2 has consistently been associated with an advanced tumor stage and enhanced metastasis formation [46]. Moreover, a link between MCM7 and the PI3K pathway has previously been reported for human melanoma [47] and esophagal squamous carcinoma [48], and an inhibitory role of the MCM7 locus on the PI3K antagonist PTEN was observed in a transgenic mouse model [49]. Likewise, depletion of mcm-7 in C. elegans also lead to reduced expression of the catalytic PI3K subunit age-1, while increasing PI3K pathway activity partially suppressed the AC invasion defect caused by mcm-7 depletion. The PI3K pathway may be activated in the AC by the INA-2/PAT-2 -integrin complex on the invasive membrane [6] and signal via MIG-10/Lamellipodin to promote invasion, analogous to the interaction between AGE-1 and MIG-10 during axon guidance [50].
A replication-independent role of the pre-RC in the transcriptional regulation of specific targets has first been observed in budding yeast during the silencing of matingtype genes [51,52]. Also, the mammalian MCM proteins negatively regulate the transcriptional activity of the hypoxia inducible factor HIF-1 in quiescent cells [53], while MCM5 is required for Stat1-mediated transcriptional activation [54]. The genome-wide mapping of replication origins in C. elegans and in human cells revealed an enrichment of active, "firing" origins near transcription factor binding sites in the vicinity of highly transcribed genes [55,56]. It thus appears that the pre-RC has adopted a new function in the post-mitotic AC to license the expression of pro-invasive genes ( Fig. 6). It should be noted that even a global inactivation of mcm-7 by FLP/FRTmediated excision only caused a partially penetrant BM breaching defect. Hence, other factors may compensate for a loss of pre-RC activity to facilitate the switch towards an invasive fate.
Interestingly, the expression levels of MCM-7 in the AC of L3 larvae were lower than in the adjacent uterine and vulval cells that continued to proliferate, and ectopic expression of mcm-7 using a strong AC-specific enhancer element led to relatively low MCM-7 protein levels, suggesting that MCM-7 is down-regulated in the G1-arrested AC at the post-transcriptional level. Dividing cells usually express an excess of MCM proteins that assemble not only on active replication origins but are spread over the entire chromatin at sites that do not overlap with sites of DNA replication, a phenomenon termed "MCM paradox" [14]. It has been proposed that the excess of MCM complexes in dividing cells are used to license a large number of dormant origins that could be used as reserve in case of DNA replication stress [57]. The relatively low MCM-7 levels in the G1-arrested AC may confer a certain specificity to the MCM complex by allowing it to bind only to a limited number of high-affinity sites near its target genes, while being insufficient to permit the progression through S-phase.
Several components of the pre-RC are overexpressed in tumor cells and have therefore been used as biomarkers [13,14]. The pre-RC has been predominantly studied in the context of cell cycle progression and genomic instability in human cancer cells.
However, there exists a handful of reports associating pre-RC genes with cancer progression and increased cell migration [48,[58][59][60][61]. Notably, Lau et al. observed reduced migration and invasion of human medulloblastoma cells after inhibition of MCM proteins [62]. Our data using a developmental model for cell invasion provide direct evidence for a cell-cycle-independent function of the pre-RC during cell invasion.
In summary, the pro-invasive function of the pre-RC may be conserved between C.
elegans and mammalian cells. The changes in extracellular matrix and PI3K gene expression after down-regulating mcm-7 in the C. elegans AC and in human cancer cells suggest that the pre-RC controls cell adhesion and BM remodeling to facilitate cell invasion. Likewise, cells entering S-phase turn over their focal adhesions, while mitotic cells must completely detach from the matrix to undergo cytokinesis [63].
Therefore, the pre-RC may coordinate cell adhesion with DNA replication in dividing cells, while in invasive cells such as the AC the regulation of cell adhesion and BM breaching by the pre-RC has been uncoupled from its function in replication origin licensing.

C. elegans culture and maintenance
C. elegans strains were maintained at 20°C on standard nematode growth plates as described [64]. The wild-type strain was C. elegans Bristol, variety N2. We refer to translational protein fusions with a :: symbol between the gene name and the tag, while transcriptional fusions are indicated with a > symbol between the promoter/enhancer and the tag. The genotypes of the strains used in this study are listed in suppl. Table   S3. Except for strain PS4444, which was provided by CGC, all strains were generated in this study.

Scoring AC Invasion
AC invasion was scored at the Pn.pxx (four-cell) stage as described [8]. We monitored the continuity of the BM by fluorescence microscopy using the qyIs10[lam-1>lam-

RNA interference
RNAi by feeding dsRNA-producing E. coli was performed in strains carrying the indicated markers in the rrf-3(pk1426) RNAi hypersensitive background as described in [65,66]. Synchronized populations of L1 larvae were obtained by hypochlorite treatment of gravid adults. If the RNAi treatment did not cause lethality or sterility, then the F1 generation was scored after 5 days, or else the P0 animals were analyzed after 30-36 hours of treatment. RNAi clones targeting genes of interest were obtained from the C. elegans genome-wide RNAi library [65]

AC polarity and fluorescence-intensity measurements
Fluorescence intensities of the different reporters were quantified using Fiji software with the built-in z-projection and measurement tools [67]. To measure the intensity of the different reporters, background subtracted summed z-projections spanning the width of the AC labelled with an mCherry marker were quantified. Quantification of mCherry::moeABD polarity in the AC was done using deconvolved wide-field or confocal image stacks spanning the width of the AC. Polarity plots were calculated from the summed z-projections of the AC as described in [68]. Quantification of the CKD sensor was done according to [39]; a mid-sagital section of the AC was filtered with Gaussian-blur (sigma=50) to reduce camera noise, and the average intensities in three randomly selected areas, each in the AC nucleus and cytoplasm, were used to calculate the cytoplasmic to nuclear intensity ratios.

Generation of reporter and rescue transgenes
For all constructs, fragments were PCRs amplified with Phusion DNA Polymerase (New England Biolabs) and were assembled by T4 ligase (New England Biolabs) or if Gibson assembly was used with Gibson assembly kit #E2611 (New England Biolabs).
Details on the construction of the plasmids used to create the different transgenes are shown in suppl. Table S5. The sequences of the oligonucleotide primers used can be found in suppl. Table S4. All plasmids were verified by DNA sequencing. For each reporter, single-copy transgene insertions were created according to the MosSCI protocol [37] by microinjection of 50 ng/µl of the respective reporter plasmid together with 50 ng/µl pJL43.1 (mos-1 transposase) and 2.5 ng/µl pCFJ90 (myo-2>mCherry), 5 ng/µl pcFJ104 (myo-3>mCherry) and 10 ng/µl pGH8 (rap-3>mCherry) as co-injection markers. The streamlined CRISPR/CAS9 method described [25] was used to generate endogenously tagged reporters. The 5' and 3' homology arm were amplified from N2 genomic DNA with primers indicated in suppl. Table S5. These fragments were cloned into pMW75 digested with ClaI to create the repair templates pTD16, pTD60 and co-injection markers. The selection of the integrated CRISPR lines was carried out according to [25].

Generation of Dox-inducible shMCM7 and shControl cells
The human cell lines A375 and A549 were tested to be free of mycoplasm and cultured

Cell synchronization and cell cycle analysis
Cell synchronization with double thymidine block [41] was performed by growing cells in medium supplemented with 2 mM thymidine for 17 hours, followed by an 8 hour release in medium without thymidine, after which cells were kept arrested in medium containing 2 mM thymidine during the analysis. Cell cycle analysis by DAPI and EdU double labelling was done using the Click-iT assay (Thermo Fischer C10420) as described [69] and analyzed by flow cytometry (BD LSR II Fortessa).

RNAseq analysis of Dox-inducible shMCM7cells
Total RNA from Dox-inducible shMCM7 or parental A375 and A549 cells,

EGF and Insulin Stimulation
28'000 cells were seeded into 12-well plates and treated after 24 hours throughout the experiment with 1 µg/ml Dox where indicated. 48 hours after seeding, cells were additionally arrested at the G1/S boundary by growing cells in medium supplemented with 2 mM thymidine for 17 hours, followed by an 8 hour release in medium without thymidine [38]. Thereafter, growth medium was replaced with DMEM containing 2 mM thymidine and lacking FCS. After 15 hours of starvation, cells were stimulated for 10 minutes with 100 ng/ml human EGF (E9644 Sigma) or with 10 nM human Insulin (I9278 Sigma) before they were lysed and prepared for western blot analysis. prevented BM breaching (see Table 1     Statistical significance was determined with a two-tailed t-test for independent samples and is indicated as n.s. for p>0.05, * for p<0.05, ** for p<0.01 and *** for p< 0.001.
The scale bars are 5 µm.