extfieldsofstudy: Biology Medicine

text:
Translational HIV-1 research: from routine diagnostics to new virology insights in Amsterdam, the Netherlands during 1983-2013

An HIV-1 diagnostic laboratory was established in the Academic Medical Center (AMC) of the University of Amsterdam after the discovery of human immunodeficiency virus (HIV) as the cause of the acquired immunodeficiency syndrome (AIDS). The first AIDS patients were diagnosed here in 1981 and since 1983 we have tested the samples of 50992 patients using a variety of assays that greatly improved over the years. We will describe some of the basic results from this diagnostic laboratory and then focus on the spin-off in terms of the development of novel virus assays to detect super-infections and ultra-sensitive assays to measure the intracellular HIV-1 RNA load. We also review several original research findings in the field of HIV-1 virology that stem from initial observations made in the diagnostic unit. This includes the study of genetic defects in the HIV-1 genome and time trends of the replication fitness over 30 years of viral evolution, but also the description of novel HIV-1 variants in difficult-to-diagnose clinical specimen.

Introduction
The acquired immunodeficiency syndrome (AIDS) was recognized by the Centers for Disease Control (Atlanta, USA) in 1981 [1]. The causative agent, the human immunodeficiency virus (HIV) was isolated first by Barré-Sinoussi et al and described as lymphadenopathyassociated virus (LAV) in 1983 [2], and also by Gallo et al, who named it human T lymphotropic virus type III, HTLV-III [3]. The complete nucleotide sequence of this novel retrovirus was reported almost two years later [4,5]. The development of antiretroviral compounds in the next decade changed AIDS from a lethal infectious disease to a chronic condition. The first antiretroviral drug, azidothymidine (AZT, zidovudine), a nucleoside analogue targeting the reverse transcriptase (RT) enzyme, was approved by the US Food and Drug Administration in March 1987 for clinical use in AIDS patients [6], and many others rapidly followed. AZT was administered to Dutch AIDS patients since mid-1987 as monotherapy [7,8]. After that, serial monotherapy with novel RT inhibitors was the standard of care, until the development of protease (PR) inhibitors enabled combination therapy to be established [9]. Large scale introduction of HAART (Highly Active AntiRetroviral Therapy), now designated as combination antiretroviral therapy or cART, into the clinical practice in the Netherlands started in June 1996, when the PR inhibitors indinavir, ritonavir, and saquinavir became widely available [10].
From the early days on, it was appreciated that the main feature of AIDS is a steady decline of CD4+ T-cells circulating in peripheral blood, causing a significant change in the CD4+/CD8+ T-cell ratio (see: [11]). CD4+ (and CD8+) T-cell counts were hence used to monitor disease progression. Furthermore, individuals infected with HIV have benefited tremendously from the advances in molecular techniques that became available in the 1980s, which markedly increased the virus assays available for patient care. Plasma viral RNA load (pVL) was found to be another predictor of disease progression [12], and pVL determination became part of routine patient care. After the introduction of antiretroviral drugs, determination of drug-resistance mutations in patient-derived viral genomes by PCR amplification and sequencing of the protease-reverse transcriptase genes (PR/RT genotyping) became yet another standard clinical test [13,14].
This review focuses on the HIV-1 epidemic in the Netherlands and the diagnostics and research performed at the AMC hospital of the University of Amsterdam. However, we should point out that the HIV research field developed rapidly through international efforts that were conducted simultaneously by many centers of clinical care and research. In fact, competition between these international centers has been an important driver of scientific advancement in the area of HIV research.

Review
Basic numbers of HIV-1 diagnostics at the AMC hospital The first three diagnosed Dutch AIDS patients; all hospitalized in the AMC in Amsterdam were described in March 1983 [15][16][17], two months before the discovery of HIV was published. Two of these patients had been seen by clinicians for AIDS-related complaints since 1981, the third was first seen in 1982 and was probably infected outside the Netherlands [16].
After the discovery of HIV in 1983, serum antibody ELISA and Western blot tests were developed that could diagnose HIV-infected individuals [18]. The total number individuals tested in the AMC from 1984-2012 for antibodies against HIV (both with positive and negative outcome) is plotted in Figure 1A. The number of individuals tested has increased substantially from the 1980s till recent years, but is levelling off in recent years. Figure 1B shows the number of HIV antibody tests performed in relation with the most likely transmission route according to the risk group: men having sex with men (MSM) (homoor bisexual men), heterosexual contacts (mainly immigrants), (intravenous) drug users (DU), recipients of blood products (haemophiliacs, needle accidents ) and motherto-child (vertical) transmission (MTCT). In most cases, the risk group is not registered, so that patient numbers shown are much lower than in Figure 1A. Peaks in 1984/ 1985 and 1998/1999 in numbers of MSM correspond with active recruitment for the Amsterdam Cohort Studies on HIV/AIDS (see below). The AIDS epidemic was initially recognized as being confined to the MSM risk group, patients with haemophilia who received blood products and intravenous drug users, so testing of other risk groups occurred infrequently in the early years of the epidemic. After some years it was realized that other groups such as heterosexuals and children born to infected mothers were also at risk, and the testing policy was expanded accordingly. In 2001, the Municipal Health Service started to offer HIV testing to pregnant women in the Amsterdam area [19]. However, as most refused the test, opt-out testing (patients may elect to decline or defer testing after being notified that the test is routinely performed) was applied in 2003 [19]. Since 2004, all pregnant women in the Netherlands are being tested for HIV using this opt-out policy [20]. Since then, MTCT rates of HIV have decreased in the Netherlands from 5-10 per year to less than 1 per year ( Figure 1B). In fact, all children that tested HIV-positive after 2003 were immigrants from non-Western countries.
Testing for HIV-1 is done mainly at outpatient clinics for sexually transmitted infections. These centers include the Municipal Health Service in Amsterdam, where >20.000 HIV tests were performed per year in recent years [21]. Since 2005, HIV rapid tests -based on HIV-antibody detection in saliva (for a review, see [22]) -have been introduced that dramatically increased the number of tests performed [21]. In addition, HIV-tests are requested by general practitioners (GPs), but numbers are much lower. Approximately 11 HIV-tests/10.000 patients per year were GP-requested in 2009 [23]. Self-testing is another way to determine one's HIV status. A survey under internet users in the Netherlands showed that 71/4416 (1.6%) respondents had ever performed an HIV/AIDS self-test, of which only 9% had used a true home-test [24].
HIV screening assays performed at the AMC are mainly done on patients visiting the outpatient clinics, hospitalized patients, on patients visiting GPs in the neighbourhood and on individuals participating in the Amsterdam Cohort Studies (see below) [25].
HIV-1 infections in the Netherlands are mainly found in MSM. Of the 16,169 HIV-infected patients in clinical care in June 2012, 80% are men of which at least 74% have been infected through homosexual contact according to the HIV Monitoring Foundation Report 2012 [26]. However, HIV-testing, both at the Municipal Health Service and as requested by GPs mostly involves heterosexuals, as high-risk MSM often opt-out [21,23]. It has been estimated that 20-40% of HIV-infected individuals in the Netherlands are unaware of their HIV status [21].
The numbers of HIV-1/2 screening antibody tests and confirmatory Western blots and other confirmatory assays performed in the AMC since 1996 are plotted in Figure 2A. Antigen tests (HIV-1 p24) became available in 1986 and have been used as a prognostic marker [27,28]. pVL assays became part of the standard patient care around 1996 ( Figure 2B). The early peak in the number of assays performed around 1998-1999, reflects the intense testing of the few available patients using a variety of assay formats. Subsequent peaks reflect the increase in the number of patients, usually being tested with a single specific test that screens for both HIV antigen and anti-HIV antibodies.
After the introduction of antiretroviral regimens that contained multiple drugs from 1997 onwards, drugresistance testing was implemented in patient care to monitor the development of drug-resistance mutations, which is usually due to low compliance. This test also allowed one to investigate the pre-existence of drugresistance mutations in plasma virus before the start of treatment, so that drug prescription could be adjusted accordingly. Numbers of sequence tests performed are shown in Figure 2C. In recent years, due to the introduction of antiretroviral drugs targeting viral proteins other than PR or RT, sequencing of integrase (IN) and envelope (ENV) gene fragments was performed for a limited number of patients.
Overall, we witnessed a steady increase over time of the number of assays performed, but also a more recent drop. This drop mainly reflects decreasing clinical budgets in this economically challenging period. Consequently, fewer assays are being requested.

The Amsterdam Cohort Studies
Subsequent analysis of a cohort of homosexual men participating in a hepatitis B virus (HBV) vaccine efficacy trial between 1980 and 1982 suggested that HIV was introduced in the homosexual population in Amsterdam at the end of the 1970s [29]. The Amsterdam Cohort Studies on HIV/AIDS (ACS) were started in October 1984, initially as a two-year prospective study of HIV incidence in MSM [30,31], then as an open, prospective cohort study on HIV. Later, DUs were also included in this cohort, but the HIV-1 incidence in drug users has declined steadily in the Netherlands from 1986 onwards to less than 1.0/100 person-years in 2010 with no new seroconversions after 2009 [32,33]. From 2003-2010, adult patients with primary HIV-1 infection (PHI) from 13 Dutch treatment centres were recruited in a cohort study coordinated by the AMC to analyse the effect of early cART treatment on disease progression [34]. Patient samples from both the ACS and the PHI cohort were subsequently used for in-depth studies of HIV infection characteristics that will be summarized below.
Similar cohorts for the study of HIV infection and AIDS were recruited in other countries, such as the Multicenter  [35] and the Swiss HIV Cohort study (SHCS) that was established in 1988 in Switzerland [36]. Also, many cohort studies have been or are being performed in African countries, often in association with western universities.

Drug-resistance mutations and HIV-1 evolution
After the introduction of the first antiretroviral compounds into the clinic in 1987, it was realized that HIV can rapidly become resistant to single-or double-drug regimens. RT genotyping became part of the standard patient care followed by PR sequencing around 1995. It was implemented for patients failing on therapy or starting treatment, the latter such that optimal regimens could be installed. Also, there was a concern that drugresistant HIV-1 strains might be transmitted to become stable components of the epidemic. An in-house sequencing assay was used until 2001, when a commercial genotyping assay became available (Applied Biosystems ViroSeq HIV-1 Genotyping System [13,14]).
In the AMC, a total of 5266 PR/RT sequences from 4226 patients were determined between 1995 and 2012 that could be retrieved from the database. For most of the patients (84.3%) a single PR/RT test was performed, but several patients (10.6%) were tested repeatedly. Due to the early use of monotherapy combined with suboptimal patient adherence, drug-resistance mutations, estimated according to the Stanford University HIV drug resistance database [37], were observed in up to 88% of RT gene sequences in 1996 ( Figure 3A). The numbers of HIV-1 sequences containing drug-resistance mutations, gradually decreased after the introduction of cART in 1996, and the decline continued after that time probably due to the introduction of better tolerated drug regimens and increased patient adherence.
The number of key mutations in PR ( Figure 3B) and RT ( Figure 3C) was plotted. HIV-1 sequences with multiple key mutations were observed mainly from 1998-2007 for RT and from 2000-2007 for PR, roughly coinciding with the clinical introduction of these drugs. However, data from more recent years (>2009) indicate that sequences with more than 2 (PR) or 5 (RT) key mutations are observed less frequently, probably due to the development of optimized drug-regimens that improve therapy adherence and consequently plasma drug levels and antiviral activity. It thus seems that HIV-1 variants within the Amsterdam epidemic are not able to preserve these drug-related mutations, which may imply that there is a significant fitness cost involved and that compensating changes are rare [38]. We also described drug-dependent HIV-1 variants that will rapidly disappear in an epidemic upon transmission in the absence of the drug [39].
A changing epidemic through import of novel HIV-1 variants Subtype B has always been the most prevalent HIV-1 subtype that circulated in the Netherlands [40], most likely imported through drug users from the USA to initiate the epidemic [41]. However, since the late 1990s several non-B subtypes have been detected, usually linked to immigration from HIV-endemic areas [42]. Subtype B continues to dominate the epidemic, but substantial numbers of patients are infected with CRF01_AE, CRF02_AG and subtype C ( Figure 4A). The latter two strains are mainly detected in heterosexual immigrants, while the former is present in MSM. Infections with untypable HIV-1 strains, including complex mosaic recombinant strains, are also on the rise from 2004 onwards with a steep ascend in 2008-2010 ( Figure 4A). Subtype B strains are most prevalent in MSM, with very low percentages below 4% in other risk groups ( Figure 4B). Of the non-B subtypes identified, 48% are carried by MSM, 34% by the heterosexual risk group, and 12% by children in the MTCT group ( Figure 4B). HIV-1 subtypes differ in replication kinetics [43], e.g. basal transcription levels vary [44], as well in the induction of disease progression [45,46]. It is therefore important for patient monitoring to analyze changes in subtype distribution over time.
A remarkable case was identified in our diagnostic unit in 2000. An HIV-1 infected patient from the Netherlands yielded an unusual HIV-1 genotype that did not cluster with subtype B or any of the other subtypes [47]. The patient had worked as sailor from 1985 till 1989 and was probably infected via a heterosexual contact in Africa or perhaps Asia. We presented the full-length genome sequence of this exotic HIV-1 variant and determined that the genetic distance with known subtypes fell within the range of intersubtype and intersubsubtype distances. Thus, we could not conclude on this isolate being a novel subtype, a subsubtype or an outlier variant of the most closely (See figure on previous page.) Figure 3 HIV drug resistance testing in the AMC. A. Percentage of key drug resistance mutations PR/RT in sequences from 1995-2012. Percentages of A (green), C (blue), U (red) and G (black) nucleotides are also shown. B. The number of key drug resistance mutations in PR from 1995-2012. The size of the circles is correlated with the number of sequences in that category. C. The number of key drug resistance mutations in RT per sequence from 1995-2012. The size of the circles is correlated with the number of sequences in that category; colours were added for clarity. related subtype K [47]. Similar sequences of other patients are needed to draw more firm conclusions and at least two epidemiologically unrelated patients with a similar HIV-1 variant are required to formally designate a new HIV-1 subtype. We also probed some biological properties of this virus, but did not reveal any particular assets. Because the patient was prescribed antiretroviral therapy, we were also able to monitor the appearance of drugresistance mutations, which did not reveal major differences with the regular subtype B viruses.
Overall, our sample set has become more enriched for exotic HIV-1 variants, which is illustrated by the steep increase in the number of untypable sequences in the regular PR/RT genotyping performed. Likewise, we see a steady increase in the number of non-B subtypes in Amsterdam.

A changing epidemic, but is the virus changing?
We described that the Dutch epidemic is changing with respect to the influx of new HIV-1 variants and subtypes. But is the virus that has spread in Amsterdam since 1983 also evolving? At the population level, one can monitor temporal trends in the pVL and CD4+ Tcell counts to reveal changes in the replication capacity and/or virulence of the respective HIV-1 strains. These values are important as they correlate with disease progression and the chance of HIV-1 transmission [12,48]. In Amsterdam, the set-point HIV-1 pVL of subtype B infected individuals was found to be higher in patients infected in more recent calendar years, with an accompanying decrease in CD4+ T-cell count [49,50]. Other studies have described similar trends [51][52][53], whereas

Non-B subtypes
Non-B subtype n = 108 others found no evidence for such temporal changes [54,55].
To address this experimentally, we have measured the in vitro replication capacity or fitness of early versus late Amsterdam HIV-1 isolates. Changes in virulence and fitness of viral pathogens in epidemics are common. For HIV-1, increases in viral fitness have been reported within patients, while bottlenecks occurring during transmission can reduce the fitness [56,57]. Early replication studies suggested that HIV-1 attenuates over time within an epidemic, probably due to repeated serial transmission bottlenecks [58]. However, one should carefully match the early and late samples for viral subtype and duration of the infection. Twenty-five early and late HIV-1 subtype B isolates, carefully matched for seroconversion time, were tested in replication and competition assays on peripheral blood mononuclear cells [59]. We observed a clear trend of increasing fitness over time in the HIV-1 epidemic of Amsterdam. This in vitro virus characteristic may constitute one of the determinants of clinical virulence, but this will be hard to assess in this era of antiretroviral therapy.
We have also focused on the molecular description of genetic defects in viruses of patients with a particular low pVL and slow disease progression. Such genetic markers that correlate with delayed disease progression have been identified in several HIV-1 proteins (see references in [60]). The Amsterdam patient under study had a low pVL of less than 1000 copies/ml for 2 years after seroconversion [60], which is observed in less than 1% of the individuals in the ACS cohort. This may suggest that the patient was infected with a poorly replicating virus. Indeed, a unique sequence insertion in the dimerization initiation site (DIS) of the untranslated 5' region of the HIV-1 RNA genome was found that abolished RNA dimerization, one of the molecular hallmarks of retroviral replication [60]. Subsequent changes over time restored this important RNA function, in line with an increase in the pVL to 50.000 copies/ml. The pVL measured at set-point [61] may be influenced by host genetic factors [62,63], but can clearly also be influenced by viral genetic factors. The mutations that influence pVL at set-point can be hidden in every part of the HIV-1 genome. More dramatic mutations and deletions can be detected by sequencing and comparing complete genomes from other patients.
Ongoing HIV-1 evolution, but a stable nucleotide count Most virus genomes have a signature nucleotide composition, and HIV-1 is no exception, as its RNA genome is rich in A-nucleotides (approximately 35%) and poor in Cnucleotides (approximately 18%) [64][65][66]. Recently, we have described the extreme stability of this nucleotide composition over 30 years of evolution in the worldwide HIV-1 epidemic [67]. We plotted the nucleotide composition of PR and RT, which are sensitive to antiretroviral drug pressure, from patient samples sequenced in Amsterdam between 1995 and 2012 ( Figure 3A). The percentages of all four nucleotides were found to be extremely stable in this genomic region over this period, and similar to older HIV-1 sequences. This suggests that during the Dutch epidemic HIV-1 is changing its virulence, but not its overall nucleotide composition. A recent study indicated that HIV-1 is able to replicate efficiently in cell culture after recoding parts of the gag or protease gene to reflect human codon usage [68]. These pilot results may suggest that changes in nucleotide composition do not always interfere with HIV replication capacity and can be tolerated. The major factor contributing to genome composition stability in HIV-1 remains to be elucidated.

HIV-1 superinfections
The large number of recombinant strains in the HIV-1 epidemic suggested already in the early years that dual infections with different HIV-1 strains should occur. Dual infections were supposed to occur before seroconversion, because the first HIV-1 infection could protect against subsequent infections. It took until 2002 before the first HIV-1 superinfections (= infection with a different strain after seroconversion) were reported [69][70][71]. We reported the first triple HIV-1 infection in 2005 [72]. This subtype B infected patient was superinfected with a second subtype B strain and subsequently by a CRF01_AE strain. All three strains could be detected for several years in blood and seminal plasma, but the two superinfecting strains replicated to a higher level than the original virus [73].
We developed several methods to identify superinfections in clinical cohorts. The first method focused on patients with a sudden rise in pVL of at least 10-fold, which was found to predict superinfection in 2 of 14 patients [74]. Second, we described the use of degenerate base codes in the PR/RT genotyping sequence, which is a population sequence performed in routine clinical care. Multiple degenerate base code counts of ≥34 were found to be a strong indicator of superinfection [75].
We were interested to study the clinical outcome of dually infected patients. Several pieces of evidence, based on in vitro virus replication studies, infection of animal models and mathematical modelling, suggest that superinfection leads to increased virulence and a higher pVL [76]. Virus competition experiments with HIV-1 subtype B strains from two superinfected patients suggested that the superinfecting strain has a significant replicative advantage over the initial strain [77]. Follow-up in a small cohort of patients suggested that dual infection is significantly associated with increased CD4+ T-cell decline (p = 0.0001 in a multivariate analysis), and earlier start of antiretroviral therapy (p < 0.0001) [78].

The incidence of HIV-1 dual infections is increasing in
Amsterdam from an absence in early years to 1.0-2.4% in 2003-2007 [79,80]. The clinically unfavourable outcome of dual infection should translate into preventive strategies, in particular because dual infection is associated with risk-taking behaviour that has been reported to increase in recent years [81].

More sensitive HIV-1 detection
Although pVL is traditionally used as the biomarker of HIV-1 replication [82], the introduction of suppressive cART necessitated the development of alternative virological biomarkers for monitoring the therapy response, as pVL is undetectable by commercial assays in most of the cART-treated patients. In the ideal situation, such markers should be predictive of cART complications (e.g. therapy failure due to suboptimal adherence) and indicate the need for intervention, e.g. a change in drug-regimen. Quantitation of cell-associated (CA) HIV biomarkers provides an alternative to the measurements of pVL [83], but to be able to measure minute amounts of HIV nucleic acids in limited biological material from patients on suppressive cART, very sensitive assays are required.
We have developed sensitive and precise assays based on innovative semi-nested real-time PCR technology for quantitation of CA HIV unspliced (us) and multiply spliced RNA and total CA HIV DNA [84]. The addition of a limited-cycle preamplification step before the realtime PCR resulted in large improvements in both assay sensitivity and accuracy (especially in the lower quantitative range). This semi-nested real-time PCR is superior to the conventional single-step real-time PCR in the power of detection and quantitation of HIV-1 RNA and DNA [84]. Importantly, because we use only 15 cycles in the first PCR, preamplification does not interfere with the linearity of the standard curves throughout the whole range of serial dilutions (R 2 > 0.99). The assay allows routine detection and quantitation of HIV RNA and DNA in the vast majority of PBMC samples of patients on cART with undetectable pVL [84][85][86]. Notably, this technology dramatically reduces the necessary amount of starting biologic material.
Using the semi-nested real-time PCR technology, we have shown that: (i.) in untreated patients during the asymptomatic phase of HIV infection with steady-state plasma viremia, the levels of HIV-1 usRNA in PBMC increased significantly in time [87]; (ii.) in patients on suppressive cART, the levels of HIV-1 usRNA in PBMC were predictive for therapy failure [85], an observation that was recently confirmed by an independent study that used a completely different assay for usRNA [88]; (iii.) in patients on ART with long-term virological success, modest nonadherence to ART caused significant longitudinal increase in the levels of usRNA in the absence of virological rebound in plasma [86]; (iv.) usRNA is a correlate of natural HIV control, as levels of usRNA in natural controllers were significantly lower than in patients on ART, while, importantly, T-cell proliferative responses to Gag and Pol peptides were higher [89].
These encouraging observations suggest that CA HIV-1 RNA is a significant and sensitive marker that allows monitoring the size and dynamics of the "active viral reservoir" in patients under cART. By contrast, total viral DNA is a marker of the "total viral reservoir", which consists mostly of infected, non-producing cells. Moreover, our results show that CA HIV-1 RNA is a significantly more sensitive correlate of viral control than pVL, at least when the latter is measured by an assay with a detection limit of 40-50 copies/ml.

HIV-diagnostics in resource-limited settings
The above described viral quantification method is a further example of the thriving merger between biomedical science and patient care in the Western world. However, assays like this are hardly applicable to resource-limited settings where most HIV-infected patients are living [90]. To be useful in these settings, methods should be low cost, low complexity and preferably avoid cold-storage requirements. HIV rapid tests that employ saliva to detect antibodies against HIV have greatly simplified HIV screening and early diagnosis in developing countries [22,90]. However, now that ART availability is expanding in the developing world, affordable methods to monitor treatment efficacy and virological failure are needed to avoid the spread of drug-resistant HIV strains [91]. Although portable CD4+ T cell counters are on the market for use in resource-limited settings, the CD4+ T cell count is not the most optimal parameter to monitor treatment failure (see: [90]). Viral load measurements are superior to CD4+ T-cell counts, but as blood plasma requires cold storage, dried blood spot-based technologies would be the method of choice (see: [90,92]). For instance, a simple real-time PCR method using dried blood spots has been developed and tested by the Affordable Resistance Test for Africa (ARTA) program, in which the AMC participates [93]. Dried blood spots can also be analysed for drug-resistance mutations [94], preferably using simplified methods that can be used locally [95].

A single case of seroreversion
As a retrovirus, HIV-1 induces a chronic infection that leads to a broad and well-sustained antibody response against multiple viral proteins. Seroconversion therefore occurs in all HIV-1 infected individuals, and antibody levels are maintained until the terminal AIDS phase in the majority of subjects. Seroreversion can be observed in noninfected children born to HIV-infected mothers that gradually loose their maternal antibodies [96,97] and also in exceptional AIDS patients with little remaining B-cell activity [98], but not in the bulk of HIV-1 infected patients [99].
However, we reported HIV-1 seroreversion in a patient that was treated with cART during acute infection and with a fully reactive Western blot [100]. Another seroreversion case was reported in 2005 that was also treated with cART during primary infection. In this case, an incomplete HIV-1 antibody evolution pattern was revealed, suggesting that treatment was started before the antibody repertoire was completed [101]. Larger studies involving adult and young patients treated early in the infection suggested that HIV-1 seroreversion is not uncommon [102,103]. In contrast, seroreversion is rare in patients treated with cART later in infection, even when long-term undetectable pVL is achieved. One study reported no seroreversions in 80 patients tested [104] and another study found 1 out of 84 patients to have seroreverted [105].

Conclusions
The HIV-1 epidemic has triggered an evolution in patient care with the implementation of molecular biology techniques, which over 30 years have been optimized considerably. The careful collection of thousands of patient samples for these clinical tests has benefitted basic scientific research enormously, as illustrated for the AMC hospital in this review. Among the 50992 patients analyzed, we described unique HIV-1 variants in individual patients, including exotic mutants and unfamiliar strains. We performed studies to probe virus evolution, showing a rapidly changing pathogen (virulence, drug resistance, complex mosaic strains or undetermined subtypes), but at the same time very constant properties (genomic nucleotide composition). In return, molecular virology research allowed the development of novel diagnostics, including the ultra-sensitive detection of HIV-1 RNA in cells instead of plasma.


text:
Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila

Lysosomal degradation and recycling of sequestered autophagosome content is crucial to maintain proper functioning of the fly nervous system.

Introduction
Autophagy ensures degradation and recycling of intracellular material, including macromolecules and even whole organelles in eukaryotic cells. Autophagy has a role in a wide range of physiological and pathological settings, such as cellular adaptation to stress, starvation, and protection from aging, cancer, neurodegeneration, and invading pathogens (Mizushima et al., 2008). Cytoplasmic cargo sequestered into double-membrane autophagosomes by phagophore cisterns (also called isolation membranes) is transported to lysosomes for degradation and reuse to support biosynthetic and energy production pathways (Tooze and Yoshimori, 2010). Several different sources such as ER, Golgi, mitochondria, endosomes, and plasma membrane have been suggested to supply membranes for the phagophore (Axe et al., 2008;Tooze and Yoshimori, 2010). Regardless of the membrane source, all autophagosomes fuse with late endosomes and lysosomes to generate amphisomes and autolysosomes, respectively, whereas phagophores do not. Therefore, autophagosomes must gain competence for specific fusion through acquiring the required molecular machinery during their maturation process.
Vesicle fusion events are usually mediated by the action of different SNARE proteins that assemble into complexes in a combinatorial fashion. A SNARE complex contains four parallel SNARE helix bundles, supplied by a Qa SNARE and either separate Qb and Qc SNAREs or a SNAP protein that contains both Qb and Qc SNARE domains, all located on the membrane of the first vesicle, and an R SNARE anchored to the second vesicle (Hong, 2005). Yeast SNARE proteins Vam3, Vam7, Vti1, and Ykt6 have all been suggested to play a role in fusion of autophagosomes with the vacuole, the equivalent of metazoan lysosomes (Dilcher et al., 2001;Ishihara et al., 2001;Ohashi and Munro, 2010). Vam3 and Vam7 have no clear homologues in metazoa. Moreover, we and others showed that autophagosomes accumulate if formation of late endosomes is blocked in Drosophila melanogaster and mammals, indicating that amphisomes play a critical role in autophagosome clearance in these cells, unlike in yeast (Filimonenko et al., 2007;Rusten et al., 2007;Juhász et al., 2008). Mammalian VAMP7, VAMP8, and D uring autophagy, phagophores capture portions of cytoplasm and form double-membrane autophagosomes to deliver cargo for lysosomal degradation. How autophagosomes gain competence to fuse with late endosomes and lysosomes is not known. In this paper, we show that Syntaxin17 is recruited to the outer membrane of autophagosomes to mediate fusion through its interactions with ubisnap (SNAP-29) and VAMP7 in Drosophila melanogaster. Loss of these genes results in accumulation of autophagosomes and a block of autolysosomal degradation during basal, starvation-induced, and developmental autophagy. Viable Syntaxin17 mutant adults show largescale accumulation of autophagosomes in neurons, severe locomotion defects, and premature death. These mutant phenotypes cannot be rescued by neuron-specific inhibition of caspases, suggesting that caspase activation and cell death do not play a major role in brain dysfunction. Our findings reveal the molecular mechanism underlying autophagosomal fusion events and show that lysosomal degradation and recycling of sequestered autophagosome content is crucial to maintain proper functioning of the nervous system. Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila and VAMP7 loss-of-function cells compared with adjacent control fat body cells, both in starved (Fig. 2, A-C and M) and well-fed larvae (Fig. S1, G-I and O). Levels of the specific cargo p62 inversely correlate with autophagic degradation in flies and mammals (Bjørkøy et al., 2009;Pircs et al., 2012). p62 aggregates accumulated in Syx17, usnp, and VAMP7 lossof-function cells both in starved (Fig. 2, D-F and N) and wellfed larvae (Fig. S1, J-L and P), indicating impaired autophagic breakdown. Western blots also revealed increased p62 and autophagosome-associated, lipidated Atg8a-II levels in starved Syx17 and VAMP7 mutant larvae compared with controls ( Fig. 2 G). Larvae expressing usnp RNAi in all cells showed similar accumulation of Atg8a-II and p62 (Fig. S1 Q). Syx17 mutants are viable despite profound autophagy defects, similar to Atg7-and Atg8a-null mutants Simonsen et al., 2008). Levels of p62 and lipidated Atg8a-II also increased in well-fed Syx17 mutant adult flies compared with controls ( Fig. 2 H). Western blots using our novel polyclonal rat and guinea pig anti-Syx17 antisera detected two bands near the predicted molecular weight of this protein, both of which were missing from Syx17 mutants ( Our loss-of-function data suggested that Syx17, usnp, and VAMP7 function as part of the same SNARE complex. Human STX17 (Syntaxin17) was previously shown to bind to SNAP-29, but the significance of this interaction remained unknown (Steegmaier et al., 1998). We found that both HA-tagged usnp and VAMP7 coimmunoprecipitated with FLAG-tagged Syx17 in cultured Drosophila cells (Fig. 3 A). The amount of VAMP7 bound to beads increased when all three proteins were coexpressed, suggesting that Syx17 and usnp together bind more efficiently to VAMP7 than Syx17 alone (Fig. 3 A). We also produced anti-usnp antisera to detect interactions of endogenous proteins. These polyclonal rat antibodies specifically recognized endogenous usnp as a single 36-kD band, which was reduced by systemic expression of usnp RNAi transgenes in larvae (Fig. S1 Q). We could readily detect endogenous usnp in anti-Syx17 immunoprecipitates from lysates of adult flies (Fig. 3 B). Vice versa, endogenous Syx17 could be immunoprecipitated with anti-usnp antibodies from cultured cell lysates (Fig. 3 C).
These results established the identity of autophagosome fusion-specific SNARE complex subunits but did not reveal which ones are present on autophagosomes. As ubisnap/SNAP-29 has both Qb and Qc SNARE domains but no transmembrane domain or lipidation site, it is likely recruited to its target membrane through interaction with Syx17, a Qa SNARE. Drosophila Syx17 localized to the ER (Fig. S2, A and B), similar to human STX17 (Hong, 2005). Drosophila VAMP7 is predicted to be present in late endosomes and lysosomes (Hong, 2005). We thus reasoned that Syx17 could also localize to autophagosomes to Vti1b were all suggested to be involved in autophagosomal fusion events (Fader et al., 2009;Furuta et al., 2010). The accumulation of both autophagosomes and autolysosomes in Vti1b knockout mice indicates that this gene product is likely to function in later steps of autophagy (Atlashkin et al., 2003). Ykt6 and VAMP7 have also been implicated in the formation of phagophores in yeast and mammalian cells, respectively (Moreau et al., 2011;Nair et al., 2011).
In a screen for SNAREs involved in autophagy, we identified Syx17 (Syntaxin17, Qa), the SNAP-29 homologue ubisnap (usnp, Qbc), and VAMP7 (CG1599, R) as required for autophagosome fusion events. We showed that Syx17 is recruited to the outer membrane of autophagosomes to acquire fusion competence, and loss of Syx17 results in neuronal dysfunction, locomotion defects, and early death of adult flies.

Results and discussion
We performed a genetic screen for SNARE proteins involved in starvation-induced autophagy, by generating GFP-marked RNAi cell clones in mCherry-Atg8a-expressing Drosophila fat bodies. The mCherry-Atg8a reporter is bound to phagophores and autophagosomes through a lipid anchor on its C terminus. In addition, mCherry-Atg8a attached to the inner membrane of autophagosomes is selectively transported to autolysosomes, which are prominently labeled by this reporter as a result of large-scale accumulation of the protease-and low pH-resistant mCherry tag inside lysosomes (Kimura et al., 2007). Syx17, usnp, and VAMP7 knockdown cells showed a similar and very characteristic phenotype: small mCherry-Atg8a dots accumulated in the perinuclear region, unlike the evenly distributed, bigger, and brighter dots observed in neighboring control cells (Fig. 1, A-C and P; and Table S1 shows the results of our screen).
Depletion of Syx17, usnp, or VAMP7 resulted in a complete block of starvation-induced punctate LysoTracker staining, a dye commonly used to label autolysosomes in Drosophila fat body ( Fig. 1, D-F and Q). Starvation-induced LysoTracker red (LTR) staining was also impaired in Syx17 [LL] and VAMP7 [EP] mutant larvae that carry transposon insertions in the coding sequences of these genes 22 and 24 nucleotides downstream of the translation start site, respectively ( Fig. 1, G, H, J, and R), similar to an independent RNAi line targeting usnp (Fig. 1, K and Q). Transgenic expression of Syx17 restored punctate LysoTracker staining in Syx17 mutants, confirming that loss of Syx17 caused the mutant phenotype ( Fig. 1, I and R). Tandemtagged mCherry-GFP-Atg8a is commonly used for analyzing autophagic flux (Kimura et al., 2007). This reporter is normally transported to autolysosomes where GFP is rapidly quenched, but mCherry persists in control cells (Fig. 1, L and L). In contrast, structures positive for both GFP and mCherry accumulated in the perinuclear region of Syx17, usnp, and VAMP7 RNAi cells, indicating a block of GFP inactivation (Fig. 1, M-O). Developmental autophagy of the fat body at the onset of metamorphosis was also impaired in Syx17, usnp, and VAMP7 RNAi cells (Fig. S1, A-F, M, and N).
Immunostainings revealed accumulation of endogenous Atg8a-positive dots representing autophagosomes in Syx17, usnp, Fig. 4 E). Finally, immuno-EM showed the presence of endogenous Syx17 in the outer membrane of autophagosomes (Fig. 4 F). Thus, Syx17 is recruited to autophagosomes to promote their fusion with late endosomes and lysosomes through its interactions with usnp and VAMP7.
Two papers relying on siRNA depletion of human STX17 were published while our manuscript was under review (Itakura et al., 2012;Hamasaki et al., 2013). Itakura et al. (2012) found that STX17, SNAP-29, and VAMP8 cooperate to mediate fusion of autophagosomes with late endosomes and lysosomes in mammalian cells. Also, GFP-STX17 localized to the outer membrane of autophagosomes. In contrast, Hamasaki et al. (2013) suggested that no autophagosomes form in STX17 siRNA-treated recruit usnp, and they mediate fusion of autophagosomes and endo/lysosomes by binding to VAMP7 located in the membranes of the degradative organelles. Endogenous Syx17 was indeed detected in 30.5% (61/200) of endogenous Atg8a-positive autophagosomes in starved animals and also colocalized with 43% (86/200) of GFP-Atg8a dots (Fig. 4, A and B). In contrast, Syx17 essentially did not colocalize with the phagophore marker Atg5 (2.5%, 5/200) or GFP-dLAMP-positive late endosomes and lysosomes (6.8%, 16/236; Fig. 4, C and D). Loss of Atg2 results in accumulation of stalled phagophores that already contain Atg8 homologues in worms and mammals (Lu et al., 2011;Velikkakath et al., 2012). Accordingly, Atg8a and Syx17 did not colocalize in Atg2 mutants (2.8%, 9/322;  2-d-old Syx17 mutants performed poorly in a climbing test (Fig. 5 H), an established measure of nervous system function . Again, transgenic expression of Syx17 rescued this locomotion defect (Fig. 5 H). We showed earlier that loss of Atg7 leads to widespread apoptosis in brains of 30-d-old mutant adults . On average, 25% of neurons were positive for active caspase 3 in the absence of Syx17 in mutant brains, unlike in similarly aged controls (Fig. S2, D-F). TUNEL assays showed that 20% of mutant cells contained fragmented DNA indicating cell death, whereas practically no TUNEL-positive neurons were identified in control or rescued 2-d-old adult brains (Fig. 5, I-K; and Fig. S2 G, quantification). This seemed specific to neurons, as no TUNEL-positive cells were detected in muscles of mutant adults (Fig. S2, H and I). Transgenic expression of the effector caspase inhibitor p35 in mutant neurons suppressed TUNEL staining (Fig. S2, G and J), but 85% of neurons (202/238, n = 3) still contained autophagosomes in brain sections similar to Syx17 mutants (Fig. S2 K). The potential contribution of caspase activation to mutant phenotypes was tested in epistasis analyses. Neuron-specific expression of p35 or the pancaspase inhibitor DIAP1 (Drosophila inhibitor of apoptosis protein 1) failed to rescue locomotion defects in 2-d-old Syx17 mutant adults (Fig. 5 H). In addition, although transgenic expression of Syx17 fully rescued the viability of Syx17 mutant adults (174/175 adults alive on day 4), cells. Our results support the findings of the former work with the exception of VAMP8, but flies appear to have only VAMP7 and lack the closely related VAMP8. According to Itakura et al. (2012), a pool of free cytosolic STX17 may be directly inserted into the outer membrane of autophagosomes. This is likely mediated by its two glycine-rich transmembrane domains and the C-terminal region (Itakura et al., 2012). Drosophila Syx17 shows a similar domain structure to human STX17, so it may also be transported to autophagosomes by this pathway (Fig. S2 C).
Depletion of syntaxin5 was recently shown to cause defects in autophagic protein degradation caused by impaired trafficking of hydrolases from ER to Golgi and thus lysosomes . Importantly, syntaxin5 seemed to be dispensable for the fusion of autophagosomes with endosomes and lysosomes, indicating that it functions at a later step during autophagy than Syx17.
Continuous basal autophagy is critical for the homeostasis of quiescent, nondividing cells, such as neurons. We and others reported earlier that autophagy deficiency results in accumulation of protein aggregates and progressive neurodegeneration in flies and mice (Hara et al., 2006;Komatsu et al., 2006;Juhász et al., 2007). Syx17 mutant adults were viable but unable to fly or climb properly, and all died within 4 d of eclosion (n = 351). We detected large-scale accumulation of Atg8apositive autophagosomes and p62 aggregates in Syx17 mutant brains (Fig. 5, A, B, and F). Neurons of well-fed Syx17 mutant adult flies contained vast numbers of autophagosomes: on average, 20% of total cytoplasm was enclosed within them (Fig. 5, D and G). In contrast, autophagic structures were rarely observed in ultrastructural images of control brains or mutants   shape and size in relevant fluorescent channels. For statistical analyses, original, unmodified images were imported in ImageJ (National Institutes of Health), and the intensity threshold for the relevant channel was set so that as many dots as possible were selected without the merging of clearly separable adjacent dots. In clonal analyses, mutant or RNAi cells were then manually encircled based on the GFP channel, and the number and size of dots in the relevant channel within that area were recorded. Adjacent control cells were selected randomly from the same image and analyzed. When comparing images from different control and mutant animals, a 300 × 300-pixel area was randomly selected from each image and analyzed as in clonal experiments. Images taken from the number of animals (n) per genotype as indicated in figure legends were evaluated for all experiments. Data were then imported into SPSS Statistics (IBM) and tested for normality of data distribution, and then, p-values were calculated with the appropriate statistical tests: two-tailed, two-sample, unequal variance t test or U test for pairwise comparison of normal or nonnormal distribution data, respectively, and analysis of variance or nonparametric Kruskal-Wallis tests for multiple comparisons of normal or nonnormal distribution data, respectively. For autophagic flux measurements with mCherry-GFP-Atg8a, primary images were imported into ImageJ and analyzed automatically using the colocalization plugin with the intensity correlation tool. Clones from 5-10 animals per genotype were evaluated with similar results, and one representative dot plot is shown in Fig. 1 (L-O) for each.

Cell culture and coimmunoprecipitations
Embryonic hemocyte-derived D.Mel-2 cells were maintained in Express-Five Serum-Free medium (Invitrogen) and transfected with UAS constructs and metallothionein-Gal4 plasmid using TransIT-2020 reagent (Mirus Bio LLC). 48 h later, protein expression was induced by adding 1 mM CuSO 4 for overnight incubation. Cultured cells were collected, washed twice in PBS, lysed on ice in lysis buffer (0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, and 20 mM Tris-HCl, pH 7.5) containing complete protease and phosphatase inhibitor cocktails (Sigma-Aldrich), and spun for 10 min at 10,000 g in a centrifuge (5430R; Eppendorf) at 4°C followed by the addition of anti-FLAG slurry (Sigma-Aldrich) to the cleared supernatant. After incubation at 4°C for 2 h, beads were collected by centrifugation at 5,000 g for 30 s at 4°C followed by extensive washes in lysis buffer and finally boiling in 30 µl Laemmli sample buffer. Coimmunoprecipitations were repeated using a different DNA clone for all constructs, with similar results. For endogenous interaction experiments, either 100 mg of adult flies was starved for 2 h or 150 mg of cultured cells was washed for 2 × 10 min in PBS and homogenized for 2 × 10 s on ice in 1 ml lysis buffer containing 1% Triton X-100, using an homogenizer (Ultra-Turret T10; IKA) equipped with a disperser (S10N-5G; IKA). Lysates were cleared by centrifugation at 30,130 g for 10 min at 4°C (adult supernatants were spun once more to completely all mutant flies expressing either p35 or DIAP1 still died within 4 d of eclosion (n = 90 and 105, respectively). These results suggest that neuronal caspase activation may not be a major cause for the locomotion defects and premature death of Syx17 mutant adult flies.
Collectively, we showed that a complex of SNARE proteins Syx17, usnp, and VAMP7 is required for autophagosome clearance and that autophagosomes gain competence to fuse with late endosomes and lysosomes by recruiting Syx17. The locomotion defects and early death of viable Syx17 mutants indicate that lysosomal degradation and recycling of sequestered autophagosomal cargo is crucial to maintain organismal health and proper brain functions in Drosophila.

Materials and methods
Fly strains and genetics Flies were maintained on standard yeast/cornmeal/agar media. In climbing tests, a total of 90 adults per genotype were recorded and scored in cohorts of three to six flies, after tapping them down in a plastic graduated cylinder . In clonal analyses, RNAi cells were generated spontaneously in larvae carrying hs-Flp; upstream activation sequence (UAS)-Dcr2; Actin>CD2>Gal4 UAS-RNAi (and UAS-GFP or UAS-LAMP1-GFP as a knockdown cell marker), and mutant cell clones (marked by lack of GFP expression) were generated by heat shocking 2-4-h embryos of the genotype hs-Flp; ubi-GFP FRT2A/Syx17[LL] FRT2A in a 38°C water bath for 1 h Pircs et al., 2012). Expression of mCherry-Atg8a was driven by a fat body-specific r4 promoter in our screen (transgenic flies were provided by T. Neufeld, University of Minnesota, Minneapolis, MN; Pircs et al., 2012   mutants, and SNARE loss-of-function strains listed in Table S1. Knockdown of usnp was induced by Actin-Gal4 for Western blots and collagen-Gal4 for EM in L3 stage larvae, and overexpression of UAS-p35 or UAS-DIAP1 in adult neurons was mediated by elav-Gal4 (all obtained from Bloomington Drosophila Stock Center).

Image analysis and statistics
Colocalization of puncta was counted manually on a computer situated in a darkroom, only taking into account overlapping structures with a similar


text:
WNT‐5A triggers Cdc42 activation leading to an ERK1/2 dependent decrease in MMP9 activity and invasive migration of breast cancer cells

An important role for WNT‐5A is implicated in a variety of tumors, including breast carcinoma. We previously showed that WNT‐5A signaling inhibits migration and metastasis of breast cancer cells, and that patients with primary breast cancer in which WNT‐5A was expressed have a better prognosis. Despite the fact that RhoGTPase Cdc42 is commonly associated with increased cell migration, we here show that recombinant WNT‐5A activates the Cdc42 in breast cancer cells (lines MDA‐MB468 and MDA‐MB231) in a time‐dependent manner. Activation of Cdc42 was also observed in MDA‐MB468 cells that were stably transfected with a WNT‐5A plasmid (MDA‐MB468‐5A). In all situations, increased Cdc42 activity was accompanied by decreased migration and invasion of the breast cancer cells. To explore these findings further we also investigated the effect of WNT‐5A signaling on ERK1/2 activity. Apart from an initial Ca2+‐dependent rWNT‐5A‐induced activation of ERK1/2, Cdc42 activity was inversely correlated with ERK1/2 activity in both rWNT‐5A‐stimulated parental MDA‐MB468 and MDA‐MB468‐5A cells. We also demonstrated increased ERK1/2 activity in MDA‐MB468‐5A cells following siRNA knockdown of Cdc42. Consistent with these results, breast cancer cells transfected with constitutively active Cdc42 exhibited reduced ERK1/2 activity, migration and invasion, whereas cells transfected with dominant negative Cdc42 had increased ERK1/2 activity in response to rWNT‐5A. To gain information on how ERK1/2 can mediate its effect on breast cancer cell migration and invasion, we next investigated and demonstrated that WNT‐5A signaling and constitutively active Cdc42 both decreased matrix metalloproteinase 9 (MMP9) activity. These data indicate an essential role of Cdc42 and ERK1/2 signaling and MMP9 activity in WNT‐5A‐impaired breast cancer cells.

Experiments performed on C57MG cells (an epithelial cell line derived from normal mouse mammary tissue) showed that unlike WNT-1, WNT-3A and WNT-7A, WNT-5A failed to induce cell transformation in these cells (Wong et al., 1994), and that the expression of exogenous WNT genes (e.g. WNT-1 and WNT-2) lead to a downregulation of WNT-5A and a more efficient cell transformation (Olson and Papkoff, 1994). Subsequent experiments revealed that ectopic expression of WNT-5A in tumorigenic uroepithelial cells resulted in suppressed anchorageeindependent growth and reduced tumorigenicity when inoculating the cells into athymic nude mice (Olson et al., 1997). In support of these findings, our group has shown that WNT-5A can reverse the effect on WNT-3Ainduced Topflash reporter activity in A2058 melanoma cells (Ekstrom et al., 2011).
The role of WNT-5A in tumor initiation and progression is complex as differences in cellular constitutions such as receptor repertoire can trigger diverse signaling pathways. This is probably why WNT-5A acts as a tumor suppressor in breast cancer, colon cancer, thyroid cancer, liver cancer, and lymphoid malignancies (Dejmek et al., 2005a;Jiang et al., 2013;Jonsson et al., 2002;Kremenevskaja et al., 2005;Liu et al., 2008;Roman-Gomez et al., 2007) but promotes tumor progression in malignant melanoma and gastric cancer (Da Forno et al., 2008;Kurayoshi et al., 2006;Weeraratna et al., 2002).
In primary invasive breast cancer tissue, we previously reported that low expression of WNT-5A protein is associated with a higher histological grade of tumors and earlier disease recurrence in patients (Jonsson et al., 2002), reflecting a more rapid development of distant metastases. In a different cohort of patients, low expression of WNT-5A protein in breast carcinomas was associated with reduced overall survival (Dejmek et al., 2005b). These findings indicate a suppressive role of the WNT-5A protein in breast cancer progression. Translational regulation of WNT-5A mRNA (Clark et al., 1993;Dejmek et al., 2005b;Leandersson et al., 2006;Sheehy et al., 2010) indicates that the WNT-5A mRNA level in human breast cancer tissue differs from the WNT-5A protein level in the same tissue sample. Therefore, it is important to be cautious when correlating WNT-5A mRNA levels to clinical outcome of patients.
These findings point to the importance of identifying the molecular mechanisms responsible for the tumor suppressing effect of the WNT-5A protein in breast cancer. Its expression in clinical breast cancer samples was not associated with the expression of the proliferation markers Ki-67, Cyclin D1, or Rb, suggesting that WNT-5A does not inhibit the proliferation of these tumors (Dejmek et al., 2005b). This conclusion is supported by in vitro studies on 4T1 breast cancer cells, in which WNT-5A had no effect on proliferation and apoptosis (Safholm et al., 2008).
Other in vitro studies of breast cancer cells have led to the suggestion that WNT-5A reduces the metastatic capability of invasive breast cancer by enhancing adhesion and decreasing migration Jiang et al., 2013;Jonsson and Andersson, 2001;Medrek et al., 2009;Roarty and Serra, 2007;. However, there are some reports that question the ability of WNT-5A to inhibit the migration of breast cancer cells and suggest that WNT-5A increases migration of breast cancer cells (Pukrop et al., 2006;Zhu et al., 2012). The proposal that WNT-5A increases breast cancer cell migration is compatible with its capacity to increase Cdc42 activity in fibroblasts (Schlessinger et al., 2007) and non-tumorigenic breast epithelial cells , since Cdc42 activity is commonly associated with increased cell migration (Fritz et al., 1999;Karlsson et al., 2009;Tang et al., 2008).
To explore these discrepancies, we investigated the precise role of Cdc42 in WNT-5A-mediated signaling in breast cancer cells. We employed two different experimental strategies. First, we evaluated the primary effects of WNT-5A by stimulating the cells with rWNT-5A. Second, we established a breast cancer cell line with stable expression of the WNT-5A protein to study its long-term effects. We also incorporated Matrigel invasion studies, as it is believed that this approach better reflects the in-vivo situation (Kleinman and Martin, 2005;Lee et al., 2008), by providing a more appropriate extracellular matrix environment.
In this study, we report that WNT-5A-mediated activation of Cdc42 leads to decreased breast cancer cell migration and invasion via reduced ERK1/2 and MMP-9 activities.

2.
Materials and methods

2.2.
Plasmid and siRNA transfections MDA-MB468 cells stably transfected with WNT-5A were obtained by plating 7.5 Â 10 5 cells in 100-mm-diameter cellculture dishes prior to lipofectamine transfection with either pcDNA3.1(þ)-WNT-5A plasmid or a pcDNA3.1 empty vector (Ying et al., 2008). Cells were transfected with 15 mg (12 mg in the case of empty vector) of plasmid DNA complex with 30 mL Lipofectamine 2000 transfection reagent and incubated with the transfection complex for 6 h. Cells were then gently rinsed with growth medium and fresh complete growth medium (DMEM supplemented with 10% FBS and 700 mg/mL Geneticin [G418]) was added to the dishes 24 h after transfection for selection of stable transfectants. Forty-eight hours after transfection, growth medium supplemented with 800 mg/ mL (as determined by kill curve) G418 was added for further selection of stable transfectants. The stable MDA-MB468 cells expressing WNT-5A (MDA-MB468-5A) were maintained in DMEM supplemented with 10% FBS and 700 mg/mL G418. Prior to assays (migration, invasion, and Western blot analysis) the cells were washed with PBS and grown for 48 h in G418-free DMEM supplemented with 2% FBS to condition the media with WNT-5A. Continued WNT-5A expression was verified by Western blot analysis performed on cell lysates as well as on conditioned cell-culture media. Transient transfections of MDA-MB468 cells with constitutively active Cdc42 (pRK5myc-Cdc42L61) or dominant negative Cdc42 (pRK5myc-Cdc42N17) were performed, using the pRK5myc empty vector as the control. When the MDA-MB468 cells were 70e75% confluent, transfections were performed in 6-well plates (Costar Ò , Corning Incorporated) in serum-free DMEM (2 mL/well) supplemented with 2 mg plasmid and 6 mL Lipofectamine 2000 transfection reagent. The transfection complex medium was exchanged after 6 h with fresh DMEM supplemented with 10% FBS. After 16 h, this media was exchanged again, but with DMEM supplemented with 2% FBS, in which the cells were allowed to grow for 48 h. After this period, the cells were harvested and used for invasion or migration studies and, in parallel; the levels of Cdc42 and pERK1/2 were analyzed by Western blot.
For Cdc42 knockdown, we used siRNA. We obtained Cdc42 siRNA(h), a pool of four target-specific 19e25 nt siRNAs from Santa Cruz Biotechnology, Inc. (sc-29256). This pool of Cdc42 siRNA has been shown to effectively and selectively abolish Cdc42 protein in primary human fibroblast cells (Pankov et al., 2005). Silencer Ò Select Negative control siRNA was purchased from Applied Biosystems. MDA-MB468-5A cells were transiently transfected with a transfection complex of 75 nM siRNA and Lipofectamine 2000 suspended in serum-free DMEM. After 6 h, the transfection complex was removed and the medium changed to fresh DMEM containing 10% FBS. After 16 h, cells were allowed to grow in DMEM supplemented with 2% FBS for 72 h before being used for Western blotting.

Active Cdc42 pull-down assay
For effective pull-down of active Cdc42, we used a kit with human p21 activated kinase 1 (PAK1)-Rac/Cdc (p21) binding domain (PBD) on agarose beads from Cell Biolabs (Cat. No. STA-404). The breast cancer cells were stimulated with 0.4 mg/mL of rWNT-5A for the stipulated time points. Prior to the rWNT-5A treatment, cells were incubated in DMEM containing 2% FBS for 16 h. Later stimulations with rWNT-5A were also carried out in the same medium. For the analysis of active Cdc42 in MDA-MB468-5A and MDA-MB468-EVcells, the cells were washed with PBS and grown in DMEM (containing 2% FBS) for 48 h to condition the media with WNT-5A. The cells were washed and lyzed in the lysis buffer provided in the kit. The lysates were centrifuged and the supernatants collected and adjusted to have equal protein concentrations after estimating their protein concentrations (Bradford Reagent, Sigma). A sample from each of the adjusted supernatants was kept for equal loading detection with an a-tubulin antibody (Santa Cruz Biotechnology Inc.) by Western blotting. Pull-downs were performed from the remaining supernatants (approximately 1 mg of protein per sample) by addition of PAK PBD beads, thorough mixing, and incubation for 1 h at 4 C with gentle agitation. After this incubation, the beads in each mixture were briefly centrifuged, washed, and resuspended in 2Â Lammeli buffer, Finally, samples were boiled for 5 min under reducing conditions and loaded onto a 15% SDS-PAGE gel. For these experiments addition of GTPgS or GDP (provided in the kit) to cell lysates were used as positive and negative controls. Following gel electrophoresis, the separated proteins were electrically transferred to PVDF membranes. Western blot analyses of these membranes were carried out as described below. Western blotting of all samples was run in parallel and analyzed for their relative content of total Cdc42 and a-tubulin (loading controls).

sFRP1 treatment
Recombinant Human sFRP1 was procured from R&D systems (Cat no. 5396-SF-025). MDA-MB468 cells were pre-incubated in DMEM with 2% FBS for 16 h. After this the cells incubated in the same medium for 4 h with sFRP1 (3.5 mg/ml) or rWNT-5A (0.4 mg/ml) alone or in combination. For combinatorial treatments, cells were pretreated with sFRP1 for 1 h, before addition of rWNT-5A. After stimulations, Cdc42 activities were analyzed as described before.

Cell migration
Cell culture inserts with a pore size of 8 mm were purchased from BD Labware and pre-coated by adding 10 mg/mL Collagen I (Cat no. 354236 obtained from BD Biosciences) in PBS to the upper chamber at room temperature for 2 h, as previously described (Senger et al., 2002). At the onset of each experiment, the cells were detached with Versene and resuspended as single cells in serum-free DMEM. For the migration experiments, we took 25,000 cells in 0.5 mL (of both cell lines) and diluted them in serum-low DMEM (supplemented with 2% FBS). Cells were added to the upper chamber in the absence or presence of rWNT-5A (0.4 mg/mL), and the lower chamber was filled with 0.70 mL DMEM supplemented with 10% FBS. The cells were allowed to migrate for 24 h at 37 C in a humidified atmosphere of 5% CO 2 . The experiment was terminated by discarding the medium and fixing the cells in the filter with 4% paraformaldehyde for 10 min. Non-invading cells on the upper side of the insert were removed by a cotton-tipped applicator. Staining of the cells on the bottom of the membrane was performed with DAPI (300 nM in PBS) for 5 min at room temperature and washed with PBS. Membranes were excised from the inserts and mounted on slides using Dako Fluorescent Mounting Medium. We captured five or more representative fields of each membrane at 400Â magnification with a fluorescent microscope (Nikon Eclipse 80i). Cells were either counted manually or with the help of the ImageJ software (National Institutes of Health, US).

Cell invasion
Cell invasion experiments were performed in BD Matrigelä invasion chambers (BD Biosciences) with a membrane pore diameter of 8 mm. For the invasion experiments, we used 50,000 MDA-MB468 cells per well or 25,000 of MDA-MB231 cells per well, suspended in 0.5 mL serum-low DMEM (supplemented with 2% FBS). The rest of the protocol was identical to that described in the migration assay section above.

Immunoblotting
Immunoblotting was performed on several different sets of samples. One set was from un-transfected cells, treated or untreated with rWNT-5A. Other sets were from cells stably transfected either with an empty vector (MDA-MB468-EV) or with WNT-5A containing vector (MDA-MB468-5A). Finally, immunoblotting was performed on MDA-MB468-5A cells transiently transfected with either constitutively active Cdc42 (pRK5myc-Cdc42L61) or dominant negative Cdc42 (pRK5myc-Cdc42N17). After termination of incubation, the cells were washed with PBS and lysed in ice-cold phosphorylation lysis buffer (PLB; 1 M Tris-HCl pH 7.5, 0.5 M NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 0.5 M EDTA, 1.5 mM MgCl 2 , 10% Glycerol, and 1% TritonX-100). The protein content of each sample was estimated using the Pierce Ò BCA Protein Assay kit (Thermo). After adjustments for differences in protein content, the samples were suspended in 4Â Lammeli buffer, boiled for 5 min, and loaded on SDS-PAGE. After transfer of the separated proteins to PVDF membranes, the membranes were probed with one of these antibodies: to WNT-5A from R&D Systems (at 1:500), to pERK and ERK (at 1:1000) from Cell Signaling Technology, to Cdc42 (at 1:1000) from BD Transductions Lab, or to atubulin (at 1:10000) from Santa Cruz Biotechnology, Inc. overnight at 4 C. After extensive washing, the membranes were incubated with either secondary goat anti-mouse/rabbit or rabbit anti-goat HRP-conjugated antibodies purchased from Dako. The separated protein bands were visualized by incubating the membranes with Chemiluminescence HRP substrate (Millipore), after which the blots were imaged and analyzed in the Chemi Docä imaging system from Bio-Rad.

Immunofluorescence
Breast cancer cells were grown on 13-mm glass cover-slips for 24 h at 37 C in a humidified atmosphere of 5% CO 2. Sixteen hours prior to stimulation with rWNT-5A or vehicle alone, the cells were suspended in DMEM with 2% FBS; stimulations and control experiments were carried out under the same media conditions. After the stimulations, cells were washed in PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.1% TritonX-100 for 3 min, all at room temperature. Next, the cells were washed in PBS and incubated with an anti-pERK antibody (Cell Signaling Technology) at a 1:200 dilution. After extensive washing, a goat anti-rabbit Alexa-488 secondary antibody (1:400 dilution; from Molecular Probesä, Oregon, USA) was added for 45 min at 4 C. The cells were also counter-stained with Phalloidin-TRITC (1:400; Sigma Aldrich) for 45 min at room temperature. Finally, the cells were rinsed with PBS and counter-stained with DAPI (300 nM in PBS) for 2 min. For Confocal microscopy (Olympus FV10i-O or Carl Zeiss LSM 700), the fixed and stained cells were mounted with the Dako Fluorescent Mounting Medium and viewed with a 60Â objective. Quantification of the immunofluorescent staining was performed with the ImageJ software (NIH, U.S.). Briefly, the original images were converted into 8-bit images by using the Split Channels. Plot profiles were generated using a line cross-sectioning approach for each individual cell. The profiles generated exhibited peaks depicting membranous and cytoplasmic intensities. The area under each curve was calculated using freehand selection and measure area tools in the software. Graphs were obtained showing mean fluorescence (a.u.) corresponding to membranous and cytoplasmic localization of the protein in each cell.

Gelatin zymography
Gelatin zymography was done using 10% pre-cast zymography gels according to the manufacturer's instructions (Cat no. 161-1167, Bio-Rad). After electrophoresis, SDS was removed by incubating the gels in a 2.5% TritonX-100 solution and the gelatinase activities were recovered by a subsequent incubation in a Tris-based buffer containing 10 mM calcium for 48 h. Finally, the gels were stained with Coomassie Brilliant Blue and the enzymatic activity was detected by observing the lack of gelatin protein in the sample lanes, represented by a clear band. The bands in the zymograms were inverted and integrated densitometric analyses were carried out with a Chemi Docä imaging system from Bio-Rad and the ChemiImager 4400 software (Alpha Innotech).

Statistical analysis
Data are expressed as the mean AE standard error of mean. Each experiment was repeated at least 3 times, as indicated in the figure legends. Statistical evaluations were performed either by a two-tailed Student's t-test ( p values <0.05 were considered significant), or by ANOVA together with Dunnett's Multiple Comparison test ( p < 0.01 was considered significant). All the statistical significances and plots were generated using the GraphPad Prism program, version 5.00 for Windows.

WNT-5A activates Cdc42 in breast cancer cells
We have previously demonstrated that rWNT-5A activated Cdc42 in non-cancerous ductal breast epithelial HB2 cells, where the WNT-5A protein level was low because the cells were WNT-5A antisense-transfected .
Moreover, we also reported that in resting HB2 cells expressing WNT-5A, Cdc42 activities were higher than in cells not expressing WNT-5A . In the current set of experiments, we stimulated MDA-MB468 breast cancer cells with rWNT-5A at a dose of 0.4 mg/mL at various times ( Figure 1A). Glutathione S-transferase (GST) PAK1 PBD beads were used to pull down GTP-bound Cdc42 (the active form). rWNT-5A significantly activated Cdc42 in MDA-MB468 cells in a timedependent manner ( Figure 1A). The results were verified by analyzing integrated densitometric values of active Cdc42 ( Figure 1B). In order to relate the rWNT-5A induced Cdc42 response quantitatively with maximal-receptor mediated response in these cells, we stimulated MDA-MB468 cells with 100 ng/ml of EGF (epidermal growth factor) for 1, 5, 15 and 30 min. We found that EGF induced a 3-fold increase in Cdc42-GTP within 1 min (as shown in Supplementary  Figure 1). To confirm the specificity of the rWNT-5A induced Cdc42 activation, we utilized a WNT-5A antagonist i.e. recombinant sFRP1. We observed that sFRP1 significantly neutralized the increased activity of Cdc42 that was mediated by rWNT-5A in MDA-MB468 cells ( Figure 1C). To further confirm that WNT-5A induces an increase in Cdc42 activity, we generated MDA-MB468 cells stably transfected with pcDNA3.1-WNT-5A plasmid. We detected significant increased levels of WNT-5A mRNA and protein in MDA-MB468 cells transfected with the pcDNA3.1-WNT-5A plasmid compared to empty pcDNA3.1 transfected cells (Supplementary Figure 2A). The release of WNT-5A from these cells was confirmed by Western blot analysis of the media (Supplementary Figure 2B). We observed significantly higher levels of active Cdc42 in MDA-MB468 cells that were stably expressing the WNT-5A protein (MDA-MB468-5A) than in the empty-vector transfected cells (MDA-MB468-EV; Figure 1D and E). To rule out the possibility that the increase in active Cdc42 induced by WNT-5A is a unique property of MDA-MB468 breast cancer cells, we employed MDA-MB231 in our experiments. rWNT-5A (0.4 mg/mL at 4 h) significantly activated Cdc42 also in MDA-MB231 cells ( Figure 1F). In MDA-MB231 cells transfected with the pcDNA3.1-WNT-5A plasmid, we detected an increased WNT-5A transcript level in transfected MDA-MB231 cells, but the lysates of these transfected cells showed little or no expression of the WNT-5A protein (Supplementary Figure 2C). Consequently, we could not study the effect of WNT-5A transfection on Cdc42 activity in this cell line.

WNT-5A inhibits breast cancer cell migration and invasion
It is well established that Cdc42, a member of the RhoGTPase family, positively regulates cancer cell migration (Heasman and Ridley, 2008;Ridley et al., 1999;Whale et al., 2011). Because our results indicated that WNT-5A comprehensively activates Cdc42 in breast cancer cells, we next performed Trans-well migration and invasion assays with breast cancer cells in the absence or presence of WNT-5A. For the Transwell migration assay, we employed a thin film of collagen by adding a collagen-I solution (10 mg/mL) in the inner well for 2 h. MDA-MB468 cells exposed to rWNT-5A migrated less than the vehicle-treated control (Figure 2A). The same phenomenon was observed in MDA-MB468-5A, as compared to control MDA-MB468-EV cells ( Figure 2B). We found similar effects of WNT-5A on MDA-MB468 cells invasion, as the penetration of the cells through the matrigel was significantly inhibited in cells treated with rWNT-5A(0.4 mg/mL for 24 h) when compared to vehicle-treated control cells ( Figure 2C) and in MDA-MB468-5A cells in comparison with MDA-MB468-EV control cells ( Figure 2D). We also observed that the invasion of MDA-MB231 cells was significantly reduced in the presence of rWNT-5A (0.4 mg/mL) for 24 h when compared to vehicle-treated (0.1% BSA in PBS) controls ( Figure 2E). These results indicate that WNT-5A restricts the migration and invasion of MDA-MB468 and MDA-MB231 cells.
Figure 2 e WNT-5A significantly decreases the migration and invasion of breast cancer cells. The Trans-well migration assay was performed using inserts coated with Collagen-I and the invasion assay was carried out using BD Matrigelä invasion chambers (as described in Materials and methods). (A) MDA-MB468 cells were either untreated or treated with rWNT-5A (0.4 mg/mL) in the upper chamber of the Trans-well and allowed to migrate for 24 h. Cells that had migrated to the bottom of the membrane were counted manually after staining with DAPI. The migration of rWNT-5A exposed cells was normalized against migration of vehicle exposed control cells. (B) MDA-MB468-EV and MDA-MB468-5A cells were placed in the upper chamber of the Trans-well and allowed to migrate for 24 h. Cells that had migrated to the bottom of the membrane were counted manually after staining with DAPI. The migration of MDA-MB468-5A cells was normalized against migration of MDA-MB468-EV control cells. (C) MDA-MB468 cells were either untreated or treated with rWNT-5A (0.4 mg/mL) in the upper Matrigel invasion chamber and allowed to migrate for 24 h. Cells that had invaded to the bottom of the membrane were counted manually after staining with DAPI. The invasion of rWNT-5A exposed cells was normalized against invasion of vehicle-exposed control cells. (D) MDA-MB468-EV and MDA-MB468-5A cells were placed in the upper Matrigel invasion chamber and allowed to migrate for 24 h. Cells that had invaded to the bottom of the membrane were counted manually after staining with DAPI. The invasion of MDA-MB468-5A cells was normalized against invasion of MDA-MB468-EV control cells. (E) MDA-MB231 cells were treated or not with rWNT-5A (0.4 mg/mL) in the upper Matrigel invasion chamber and allowed to migrate for 24 h. Cells that had invaded to the bottom of the membrane were counted manually after staining with DAPI. The invasion of rWNT-5A-exposed cells was normalized against invasion of vehicle-exposed control cells. The control used for all rWNT-5A stimulations was PBS supplemented with 0.1% BSA. Statistical comparisons between means were made with Student's t-test (AeE). All error bars represent standard error of the mean (n [ 4). *p < 0.05; **p < 0.01; ***p < 0.001.

WNT-5A modulates the activation of pERK1/2 in breast cancer cells
The ERK pathway is one of the most intensively studied mammalian MAPK pathways. It is deregulated in approximately one-third of all human cancer cases (Reddy et al., 2003), which attests to its importance. There were two reasons for studying ERK1/2 signaling in the current context. First, it drives breast cancer cell migration and invasion (Chen et al., 2009;von Thun et al., 2012) and second, it can be regulated by Cdc42 (Deroanne et al., 2005;Zuo et al., 2011). Breast cancer cells (MDA-MB468 and MDA-MB231) were stimulated with rWNT-5A (0.4 mg/mL) in a time-dependent manner. The phosphorylation of ERK1/2 were analyzed after 1 h, 6 h, 12 h, and 24 h by Western blotting using a polyclonal antibody specific to phosphorylated sites at Thr202/Tyr204. Phosphorylation of these sites is coupled to the activity of ERK1/2 and thus a reliable and indirect method for estimating ERK1/2 activity (Osabe and Negishi, 2011;Procaccia et al., 2010). Based on this, we have from here onwards referred to our determinations of phosphor-ERK1/2 levels as ERK1/2 activities.Both breast cancer cell lines had the same activation kinetics of ERK1/2 when stimulated with rWNT-5A. WNT-5A initially induced activation of ERK1/2 followed by a phase in which the activity fell below the basal level ( Figure 3A and B; Supplementary Figure 3A). These results are validated by statistical analysis of pERK/ERK integrated-density values of MDA-MB468 cells treated with or without rWNT-5A. The results demonstrated statistical decreases after 6 h and 12 h of rWNT-5A stimulation ( Figure 3A). Complementary experiments reveals that this pERK1/2 activation in MDA-MB468 cells occurred already after 5 m (Supplementary Figure 3B). We conclude that the initial increase was Ca 2þ dependent, because when BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N 0 ,N 0 -tetraacetic acid)-loaded MDA-MB468 cells were stimulated with rWNT-5A, the initial increase in ERK1/2 activity was diminished (Supplementary Figure 3C).
We also used confocal microscopy of MDA-MB468 cells to confirm the WNT-5A changes in ERK1/2 activation. rWNT-5A induced a marked increase in pERK1/2 staining within 1 h. Semi-quantitative estimation of the fluorescent intensity revealed a significant increase in pERK1/2 staining at the membrane as well as in the cytoplasm, as compared to untreated cells. However, at 6 h we observed a significant decrease in the staining intensity of pERK1/2 both at the membrane and in the cytoplasm ( Figure 4A and B). So, these  (n [ 4). *p < 0.05; ***p < 0.001. data from confocal microscopy confirm the results from the Western blotting ( Figure 3). Also, the MDA-MB468 cells transfected and expressing the WNT-5A protein (MDA-MB468-5A) showed a marked reduction in pERK1/2 staining, supporting the Western blot findings ( Figure 4C and D).
These data suggest that prolonged exposure of cells to WNT-5A also impaired ERK1/2 activity. The experiments with the MEK1/2 inhibitor (U0126) showed that inadequate ERK1/2 signaling impaired migration of MDA-MB468 cells (Supplementary Figure 3D), suggesting its direct role in migration of MDA-MB468 cells. Together these data indicate that apart from an initial increase, WNT-5A confine ERK1/2 activity in breast cancer cells and that impaired ERK1/2 activity renders breast cancer cells less migratory and invasive.

3.4.
Silencing of Cdc42 upregulates ERK1/2 activity in MDA-MB468-5A cells MDA-MB468 cells transfected to express the WNT-5A protein had elevated levels of the active GTP-bound form of Cdc42 and reduced levels of active ERK1/2 ( Figure 5A). These changes occurred simultaneously with a reduction of basal cell migraton and invasion in MDA-MB468-5A cells compared to the control MDA-MB468-EV cells. These results led us to ask whether WNT-5A-induced suppression of ERK1/2 activity is mediated via an increased Cdc42 activity in these cells. To answer this question, we employed a siRNA knockdown approach. The transfection method was optimized on MDA-MB468-5A cells (data not shown) after which the cells were transfected with 75 nM siRNA specific for Cdc42 mRNA. MDA-MB468-5A cells transfected with siRNA against Cdc42 mRNA had significant increased activity of ERK1/2, as compared to control MDA-MB468-5A cells treated with scramble siRNA ( Figure 5B). The results suggest that in MDA-MB468-5A cells, the WNT-5Ainduced inhibition of ERK1/2 activity is mediated via Cdc42.

Transfection of Cdc42 mutant forms interfere with migration and invasion of MDA-MB468 cells
To further confirm that Cdc42 activity inhibits migration and invasion of MDA-MB468 cells, we took a complementary approach in which we transiently transfected parental MDA-  Figure 4A and B). MDA-MB468 cells transfected with the constitutively active Cdc42 mutant (Cdc42L61) exhibited significantly less migration and invasion than the control cells ( Figure 6A and B). In addition, the inhibition of migration and invasion of Cdc42L61 transfected MDA-MB468 cells occurred in parallel with a reduced ERK1/2 activity ( Figure 6C). In another approach, we transiently transfected MDA-MB468 cells with the dominant negative Cdc42 mutant (Cdc42N17) (Edlund et al., 2002) and studied their response to a 24 h incubation with rWNT-5A. In comparison with control cells, cells transfected with Cdc42N17 had increased activity of ERK1/2 in response to rWNT-5A stimulation ( Figure 6D). These results support our conclusion that increased Cdc42 activity reduces the activity of ERK1/2 as well as breast cancer cell migration and invasion.

3.6.
WNT-5A inhibits the invasive migration of MDA-MB468 cells by inhibiting Gelatinase-B (MMP9) Our next goal was to locate possible downstream targets of Cdc42-ERK1/2 signaling that could be linked to breast cancer cell migration and invasion. A possible candidate for this linkage is gelatinase-B (MMP9), as it is activated by ERK1/2 (Beliveau et al., 2010;Moshal et al., 2006) and can modulate cell migration (Chen et al., 2009;Rolli et al., 2003). We first investigated the activity of MMP9 in MDA-MB468-5A cells through Gelatin-Zymography. The activity of MMP9 was significantly reduced in the media from MDA-MB468-5A cells compared to the media from MDA-MB468-EV control cells ( Figure 7A). The quantitative analysis confirmed that the activity of MMP9 was significantly reduced in WNT-5Aexpressing MDA-MB468 cells ( Figure 7B). In order to further verify these data, MMP9 protein expression was analyzed in MDA-MB468-5A and MDA-MB468-EV cells by Immunofluorescence Microscopy. We observed an overall decrease in MMP9 protein expression in MDA-MB468-5A cells, as compared to control cells (Supplementary Figure 5). The decrease in both expression level and activity of MMP9 in MDA MB468-5A cells compared to control cells suggests that this gelatinase is involved in WNT-5A-modulated breast cancer cell migration and invasion. We analyzed the media collected from parental MDA-MB468 cells transiently transfected with the constitutively active mutant of Cdc42 (Cdc42L61) for MMP9 activity to determine if Cdc42 signaling suppressed gelatinase-B activity. The activity of MMP9 in Cdc42L61-transfected MDA-MB468 cells was significantly less than in control cells ( Figure 7C and D). With an alternative approach, we demonstrated a statistically significant rWNT-5A-induced increase in MMP9 activity in media from parental MDA-MB468 cells transiently transfected with the dominant negative Cdc42 mutant (Cdc42N17; Figure 7E). Further evidence that MMP9 is an important downstream mediator of WNT-5A-regulated breast cancer cells migration comes from experiments in which we utilized active human rMMP9. rMMP9 (20 ng/mL) was added to MDA-MB468-5A cells to counteract the demonstrated ability of After this treatment, the cells were lysed in PLB buffer and analyzed for their content of Cdc42, pERK1/2, ERK1/2, and WNT-5A proteins by Western blotting. Quantifications of pERK1/2 in MDA-MB468-5A cells treated with Cdc42 siRNA or scrambled siRNA were carried out by calculating Integrated Density Values of the pERK1/2 bands and normalizing it against that of total ERK. Statistical comparisons between means were made with Student's t-test. All error bars represent standard error of the mean (n [ 6). **p £ 0.01.
WNT-5A to impair MMP9 activity ( Figure 7B). In accordance with our previous results, addition of rMMP9 increased the migration of MDA-MB468-5A cells in comparison to vehicletreated control cells ( Figure 7F).

Discussion
In the present study, WNT-5A activated Cdc42 in MDA-MB468 and MDA-MB231 breast cancer cell lines (both ATCC-certified and -characterized). Treatment with rWNT-5A for 4 h, indicate that Cdc42 is one of the signaling proteins activated by WNT-5A in these cells. This increase in Cdc42 activity persisted in this study, as shown by elevated Cdc42 activity in resting MDA-MB468-5A cells that are continuously expressing and releasing the WNT-5A protein. The specificity of the WNT-5A-induced Cdc42 activation was confirmed by using recombinant human sFRP1, which significantly inhibited the rWNT-5A induced Cdc42 activation when given in a 1:10 ratio.
In the next set of experiments, we performed Trans-well migration and invasion assays to test the effect of increased activity of Cdc42 in breast cancer cells as Cdc42 is known to positively regulate cell proliferation, migration, and invasion of tumor cells ( There is a recent report of an antimigratory function of Cdc42 in aggressive breast cancer cells (Zuo et al., 2011), a result that supports our finding. In this report, the authors used a siRNA approach to demonstrate that knocking down of Cdc42 led to an increase in the basal migration and invasion of MDA-MB231 and C3L5 cells. Consequently, we highlighted Cdc42 as a downstream mediator of non-canonical WNT-5A signaling in breast cancer cells and suggest an essential role of Cdc42 activation in reducing breast cancer cell migration and invasion. Cdc42 has been shown to control the activation of ERK1/2 in a cell-type specific manner (Deroanne et al., 2005;Szczur et al., 2006;Zugasti et al., 2001;Zuo et al., 2011), so we explored the possibility that WNT-5A regulates ERK1/2 activity through Cdc42. It has also been shown that MAPKs play an important role in cell migration (Huang et al., 2004) and, in particular, ERK1/2 is an integral signal in breast cancer cell migration (Chen et al., 2009). In our current experiments, we stimulated Quantification of MMP9 activity in media from MDA-MB468 cells transfected with pRK5myc-Cdc42L61 was performed by calculating the integrated densitometric value of its inverted band and normalizing it against that obtained from pRK5myc-EV transfected MDA-MB468 cells. (E) Quantification of MMP9 activity in the media from MDA-MB468 cells transiently transfected with pRK5myc-Cdc42N17 (dominant negative mutant of Cdc42) followed by rWNT-5A stimulation for 24 h. rWNT-5A stimulated MDA-MB468 cells (transfected with pRK5myc vector) served as control. (F) MDA-MB468-5A cells were either untreated or treated with active human rMMP9 (from Calbiochem, 20 ng/mL) by its addition to the upper chamber of the Trans-well after which the cells were allowed to migrate for 24 h. Cells that had migrated to the bottom of the membrane were counted manually after staining with DAPI. The migration of rMMP9 exposed cells was normalized against the migration of vehicle-exposed control cells. Statistical comparisons between means were made with Student's t-test. All error bars represent standard error of the mean (n [ 7). **p < 0.01.
both MDA-MB468 and MDA-MB231 cells with rWNT-5A and followed the activation kinetics of ERK1/2 for 24 h, using a phospho-specific pERK1/2 antibody to monitor the activity of this kinase (Osabe and Negishi, 2011). We observed identical ERK1/2 activation kinetics in both cell lines: upon rWNT-5A stimulation there was an initial and transient increase in the activity of ERK1/2, peaking at 1 h, and after that, the activity declined and remained below the basal level for at least 24 h. The initial transient increase was Ca 2þ dependent as the WNT-5A-induced increase in ERK1/2 activity was neutralized when the cells were loaded with BAPTA, a Ca 2þ chelator. These results agree with previous findings in other cell types that Ca 2þ positively regulates activation of ERK1/2 (Chao et al., 1992;Estrada et al., 2003;Moshal et al., 2006). rWNT-5A triggers a Ca 2þ -dependent ERK activation with rapid onset and transient duration, prior to WNT-5A-induced statistically significant increase in Cdc42 activity. The significant decrease in ERK1/2 activity after 6 h and 12 h in rWNT-5A-stimulated MDA-MB468 cells fits well with the decrease in ERK1/2 activity level in MDA-MB468-5A cells. The immunofluorescence and semi-quantitative data confirmed our findings from the Western blot experiments. Together, these data point to the ability of WNT-5A signaling to restrict the activity of ERK1/2 via a Cdc42-dependent mechanism that results in impaired migration and invasion of breast cancer cells. This proposal is in agreement with the observed increase in ERK1/2 activity and cell migration in Cdc42-depleted MDA-MB231 cells (Zuo et al., 2011). However, a recent study demonstrated increased migration of breast cancer cells in response to rWNT-5A treatment after a very short stimulation period (Zhu et al., 2012). It is possible that short-term stimulation with rWNT-5A can elicit an early and transient increase in cell migration as suggested by our finding of an early and transient increase in ERK1/2 activity. However in a breast cancer context the effects of prolonged WNT-5A exposure on cell migration would be more relevant for how WNT-5A affects breast cancer metastasis. In the present study, we tried to mimic the in vivo situation of prolonged WNT-5A exposure by transfecting MDA-MB468 cells with a WNT-5A plasmid. Interestingly enough, these transfected cells exhibit increased Cdc42 activity, decreased ERK1/ 2 activity, and decreased migration and invasion.
The effects of WNT-5A on Cdc42 and ERK1/2 suggest an inverse relationship between Cdc42 and ERK1/2 in MDA-MB468 cells, a suggestion that is further supported by the finding that inhibition of Cdc42 resulted in increased ERK1/2 activity (Zugasti et al., 2001;Zuo et al., 2011). In MDA-MB468-5A cells, which have high endogenous Cdc42 activity, silencing of Cdc42 induced a significant increase in ERK1/2 activity, suggesting that WNT-5A-mediated Cdc42 signaling suppresses the activity of ERK1/2 in these cells. As previously discussed, transient transfection of MDA-MB468 cells with the constitutively active Cdc42 mutant (Cdc42L61) impaired their migration and invasion and was accompanied by a significant decrease in ERK1/2 activity. In a reverse approach, we transfected these cells with a dominant negative mutant of Cdc42 (Cdc42N17) and noted that rWNT-5A stimulation caused increased ERK1/2 activity. On the basis of these results, we conclude that WNT-5A impairs breast cancer cell migration and invasion by increasing the activity of Cdc42, thereby decreasing ERK1/2 activity. MMP9 activity has been implicated in breast cancer cell migration (Rolli et al., 2003) and ERK1/2 activity has been positively associated with increased MMP activity (Yao et al., 2004). This information led us to next investigate the role of MMP9 in WNT-5A-impaired breast cancer cell migration and invasion. Cell-culture medium collected from MDA-MB468-5A cells revealed a significant reduction in MMP9 activity compared to empty-vector transfected control cells. Similarly, immunofluorescence staining clearly revealed downregulation of MMP9 expression in MDA-MB468-5A cells compared to empty vector control cells and there was decreased MMP9 activity in the culture medium from parental MDA-MB468 cells transfected with constitutive active Cdc42 mutant (Cdc42L61). In an alternate approach, we observed a WNT-5A-triggered increase in MMP9 activity when parental MDA-MB468 cells were transiently transfected with dominant negative Cdc42 mutant (Cdc42N17). These data indicate that WNT-5A impairs MMP9 activity via Cdc42 signaling. Finally, to show that the WNT-5A-induced inhibition of MDA-MB468 breast cancer cell migration is dependent on reduced MMP9 activity, we studied MDA-MB468-5A cell migration in the absence and presence of active human recombinant MMP9. The addition of rMMP9 to MDA-MB468-5A cells led to increased migration compared to vehicle-treated control cells. Taken together, these results lead us to conclude that WNT-5A-mediated impaired breast cancer cell migration and invasion is at least in part explained by a reduced release of MMP9. Interestingly, it has recently been demonstrated that WNT-5A induced MMP9 mRNA expression through a ERK1/2 signaling mechanism in microglia (Halleskog et al., 2012), which appears contrary to our present findings on breast cancer cells. If the WNT-5A-induced MMP9 mRNA expression relates to an increase in MMP9 activity this is another good example of how WNT-5A can have different and opposing effect depending on the cellular context.
In human breast cancer tissue it has been shown that WNT-5A protein expression accompanied prolonged disease-free survival and overall survival. In agreement, we here provide evidence that although WNT-5A activates Cdc42, it causes a downstream decrease in ERK1/2 and MMP9 activities those results in impaired breast cancer cell migration and invasion. We believe that WNT-5A-modulated Cdc42-ERK1/2 signaling plays an integral part in restricting breast cancer progression and metastasis. Future studies are warranted to identify potential additional downstream targets complementing the observed effect on MMP9 activity.
Tao (Chinese University of Hong Kong, Hong Kong) for providing WNT-5A plasmid. This work was supported by the Swedish Cancer Foundation, the Swedish Research Council, the S€ oderberg Foundations, Sk ane University Hospital Research Foundations, the Gunner Nilsson's Cancer Foundation (all to T.A). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.