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Myosin-Va Contributes to Manifestation of Malignant-Related Properties in Melanoma Cells

Authors:
Cleidson Padua Alves at Massachusetts General Hospital
Cleidson Padua Alves
Milene H. de Moraes at Federal University of Santa Catarina
Milene H. de Moraes
Josane de Freitas Sousa at Federal University of Pará
Josane de Freitas Sousa
Carmen Lucia S Pontes
Carmen Lucia S Pontes
Carmen Lucia S Pontes
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The Journal of Investigative Dermatology publishes basic and clinical research in cutaneous biology and skin disease.
Myosin-Va is highly expressed in melanoma cells, and its knockdown impairs cell adhesion and spreading on the fibronectin-coated surface. (a) Relative MYO5A mRNA expression detected by quantitative PCR in melanocytes (pMel1 to 4) versus melanoma cells, using β-actin for normalization and mean of all melanocytes as reference value. (b) Western blot of myosin-Va in a panel of melanoma cell lines of radial growth phase (RGP), vertical growth phase (VGP), and metastasis (M), including two genetic pairs (WM793 and 1205Lu; WM278 and WM1617). (c–e) Western blots for myosin-Va in (c, d) WM1617 and (e) UACC-257. WM1617 cells were lysed 3 days after transduction with lentiviral vectors carrying short hairpin RNAs (shRNAs) targeted to bacterial Lac-Z (shControl) or MYO5A (shMYO5A#1), or after stable selection with antibiotics for about 2–3 weeks in the case of shMYO5A#2–3 and respective shControl (Supplementary Figure S3). UACC-257 cells were lysed 3 days after transfection with small interfering RNA against myosin-Va (siMYO5A, obtained from Dharmacon SmartPool, LQ-019321-00-0002) or control (siControl, Dharmacon On-Target Non-Targeting Pool, D-001810-10). (f) Confocal images of F-actin-stained cells adhered to fibronectin-coated coverslips for the indicated times. Arrows indicate transduced cells visualized by green fluorescent protein expression (inserts). Bar = 20 μm. (g) Cells allowed to adhere on fibronectin-coated surface for the indicated times were counted and data were plotted as mean±SD from three independent experiments (*P<0.005). (h) Cell spreading. Imaged as in f, and the areas for 60 cells/time point were measured using ImageJ (*P<0.001).Download Power Point slide (356 KB)
Myosin-Va is highly expressed in melanoma cells, and its knockdown impairs cell adhesion and spreading on the fibronectin-coated surface. (a) Relative MYO5A mRNA expression detected by quantitative PCR in melanocytes (pMel1 to 4) versus melanoma cells, using β-actin for normalization and mean of all melanocytes as reference value. (b) Western blot of myosin-Va in a panel of melanoma cell lines of radial growth phase (RGP), vertical growth phase (VGP), and metastasis (M), including two genetic pairs (WM793 and 1205Lu; WM278 and WM1617). (c–e) Western blots for myosin-Va in (c, d) WM1617 and (e) UACC-257. WM1617 cells were lysed 3 days after transduction with lentiviral vectors carrying short hairpin RNAs (shRNAs) targeted to bacterial Lac-Z (shControl) or MYO5A (shMYO5A#1), or after stable selection with antibiotics for about 2–3 weeks in the case of shMYO5A#2–3 and respective shControl (Supplementary Figure S3). UACC-257 cells were lysed 3 days after transfection with small interfering RNA against myosin-Va (siMYO5A, obtained from Dharmacon SmartPool, LQ-019321-00-0002) or control (siControl, Dharmacon On-Target Non-Targeting Pool, D-001810-10). (f) Confocal images of F-actin-stained cells adhered to fibronectin-coated coverslips for the indicated times. Arrows indicate transduced cells visualized by green fluorescent protein expression (inserts). Bar = 20 μm. (g) Cells allowed to adhere on fibronectin-coated surface for the indicated times were counted and data were plotted as mean±SD from three independent experiments (*P<0.005). (h) Cell spreading. Imaged as in f, and the areas for 60 cells/time point were measured using ImageJ (*P<0.001).Download Power Point slide (356 KB)
… 
Ablation of myosin-Va inhibits colony formation, migration, and invasiveness of metastatic melanoma cells without affecting cell proliferation. (a–c) Lentiviral-transduced WM1617 cells, using three independent shMYO5A or shControls, were used to assess the following: (a) colony formation in soft agar after 30 days of growth, bar = 500 μm; (b) proliferation rates by absorbance measurement of crystal violet staining; (c) migration in transwell and invasion in the transwell-matrigel assay. Cells were kept in starvation conditions 24 hour before the assay and were then allowed to migrate/invade for 24 hours. Bar = 50 μm. (d) Migration in three-dimensional collagen. After 24 or 48 hours of incubation, distance from the spheroid edge to the invasive front was measured and the data from three independent experiments were plotted as a percentage of control. Bar = 100 μm. (e and f) Transwell invasion and proliferation rates of UACC-257 cells transiently transfected with duplex small interfering RNA (siRNA) targeted to MYO5A and irrelevant siRNA. Invasion assay was performed as described in c, and proliferation rates were estimated based on ATP measurements. Data were plotted as mean±SD from three independent experiments. Bar = 50 μm.Download Power Point slide (350 KB)
Ablation of myosin-Va inhibits colony formation, migration, and invasiveness of metastatic melanoma cells without affecting cell proliferation. (a–c) Lentiviral-transduced WM1617 cells, using three independent shMYO5A or shControls, were used to assess the following: (a) colony formation in soft agar after 30 days of growth, bar = 500 μm; (b) proliferation rates by absorbance measurement of crystal violet staining; (c) migration in transwell and invasion in the transwell-matrigel assay. Cells were kept in starvation conditions 24 hour before the assay and were then allowed to migrate/invade for 24 hours. Bar = 50 μm. (d) Migration in three-dimensional collagen. After 24 or 48 hours of incubation, distance from the spheroid edge to the invasive front was measured and the data from three independent experiments were plotted as a percentage of control. Bar = 100 μm. (e and f) Transwell invasion and proliferation rates of UACC-257 cells transiently transfected with duplex small interfering RNA (siRNA) targeted to MYO5A and irrelevant siRNA. Invasion assay was performed as described in c, and proliferation rates were estimated based on ATP measurements. Data were plotted as mean±SD from three independent experiments. Bar = 50 μm.Download Power Point slide (350 KB)
… 
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Myosin-Va Contributes to Manifestation of Malignant-Related
Properties in Melanoma Cells
Cleidson P. Alves1, Milene H. Moraes1, Josane F. Sousa1, Carmen Lucia S. Pontes1,
Anelisa Ramão1, Satoru Yokoyama3, Daniel M. Trindade1,4, David E. Fisher2, and Enilza M.
Espreafico1,*
1Department of Cellular and Molecular Biology and Pathogenic Bioagents, Faculty of Medicina of
Ribeirão Preto, University of São Paulo, 14049-900, Ribeirão Preto, São Paulo, Brazil.
2Department of Dermatology, Cutaneous Biology Research Center, Mass. General Hospital,
Harvard Medical School, MA 02115, USA
3Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama,
Toyama, Japan
TO THE EDITOR
Melanoma is a highly metastatic and therapeutically resistant cancer, whose incidence has
more than tripled in the last decades (Smalley
et al.
, 2010). Physiologically, melanocytes
produce and store melanin pigments in the melanosomes, which are transported to the cell
periphery and transferred to keratinocytes, a process that requires the tripartite complex
Rab27a/melanophilin/myosin-Va (Hume and Seabra, 2011). Myosin-Va is an actin-based
molecular motor that also serves a multitude of other functions, such as plasma membrane
receptor recycling, exocytosis, association with nuclear speckles and the centrosome (see
Woolner and Bement, 2010); interaction with PTEN, thereby modulating PI3K pathway
(van Diepen
et al
., 2009), interaction with Bcl-xL, proposed to promote invasion of islet-
tumor cells (Du
et al
., 2007); as biomarker of invasiveness for nonfunctioning pituitary
adenomas (Galland
et al
., 2010). Moreover, myosin-Va was shown to be up-regulated by
Snail to promote cancer cell invasion (Lan
et al.
, 2010), and was postulated to control
apoptosis by sequestering the pro-apoptotic protein Bmf, which is unleashed upon loss of
cell attachment (Puthalakath
et al
., 2001).
Up-regulation of
MYO5A
gene in melanoma and other cancer types was revealed in
different microarray studies compiled here (Table S1; Figure S1). However, these data did
not clarify whether
MYO5A
up-regulation was associated with melanocyte transformation
or simply reflected tissue specificity since comparison was against normal skin and
melanocytes are minor cells in the skin. Here, we extended this evidence by showing that
MYO5A
is up-regulated in a variety of melanoma cell lines in comparison with primary
melanocytes (Figure 1a), as well as in metastatic cells in comparison to paired vertical
growth phase cells (Figure 1b and S2), implicating myosin-Va in malignant transformation
and/or melanoma progression. Interestingly, in this WM panel, myosin-Va expression
correlated with that of the oncogenic transcription factor
MITF
(Sousa and Espreafico,
2008).
*Corresponding author: Av. Bandeirantes, 3900 14049-900 – Ribeirão Preto, SP, Brazil Tel: 55-16-3602-3348, Fax: 55-16-3633-1786
emesprea@fmrp.usp.br.
4Current address is Brazilian Biosciences National Laboratory (LNBio) at Brazilian Center for Research in Energy and Materials
(CNPEM), Campinas, São Paulo, Brazil
Conflict of interest The authors declare no conflict of interest.
NIH Public Access
Author Manuscript
J Invest Dermatol
. Author manuscript; available in PMC 2014 June 01.
Published in final edited form as:
J Invest Dermatol
. 2013 December ; 133(12): . doi:10.1038/jid.2013.218.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
To investigate the role of myosin-Va in melanoma cells, we knocked down this protein
using three different shRNAs (shMYO5A#1-3) carried by lentiviral vectors (Figure S3 and
Qin
et al.
, 2003) and an siRNA (siMYO5A). Once efficient knockdown was attained
(Figures 1c-e), functional studies were performed. Upon adhesion to fibronectin-coated
glass coverslips, MYO5A-depleted cells showed numerous small blebs on their surface and
reduced lamellipodia/filopodia formation (Figure 1f), besides deficient adhesion (Figure 1g)
and spreading (Figure 1h).
Next, we examined the role of myosin-Va in adhesion-independent growth. The ability to
form colony in soft agar, as analyzed after 25-30 days of incubation, was at least 50% lower
for MYO5A-depleted cells than controls, for the three different shRNAs used (Figure 2a).
Proliferation rates under adherent conditions were determined by crystal violet staining for
WM1617 (Figure 2b) or ATP measurements for UACC-257 (Figure 2f), and no differences
were observed, in the time courses analyzed, between MYO5A-knockdown and control
cells. Subsequently, we analyzed transwell migration and invasion and found rates 50 to
70% lower for shMYO5A#2/3-transfected WM1617 cells than controls (Figure 2c). Similar
decrease in transwell invasion was observed for siMYO5A-transfected UACC-257 cells
(Figure 2e). Next, we performed spheroid assays (as in Smalley and Herlyn, 2008) with
shMYO5A#1-transduced cells. Compact spheroids with intact appearance were added to a
tri-dimensional collagen gel and imaged after 24 and 48 hours of culture. Myosin-Va-
depleted cells exhibited migration distances from spheroid margin to invasion front 50 to
60% shorter than controls (Figure 2d). Also, knockdown cells that migrated out of the
spheroids looked smaller than controls after 48 hours, suggesting that myosin-Va-depleted
cells differ in the sensitivity to microenvironment factors during migration in collagen
matrix.
The multifunctional character of myosin-Va makes us believe that this molecular motor, in
addition to its role in cell adhesion/motility by promoting focal adhesion dynamics and
filopodia/lamillipodia growth (supported by work in progress from our group, Nader
et al
.),
may also perform a role in extracellular matrix proteolysis, mediating surface exposure and
positioning of matrix metalloproteinases. Indeed, the alignment of metalloproteinases along
the cytoskeleton seems to be a prerequisite for cell invasion in melanoma. Also, co-
localization of metalloproteinases with myosin-Va (Sbai
et. al
., 2011) in astrocytes, and a
role for RAB27A (Bobrie
et al.
, 2012) in the release of metalloproteinase-9 to promote
metastasis of mammary carcinoma cells have been shown. Moreover, evidence that
RAB27A (Akavia
et al
., 2010) functions as a driver of cancer supports the hypothesis that,
likewise, myosin-Va promotes malignancy by functioning in vesicular trafficking. Indeed,
endocytosis and recycling of plasma membrane receptors require Rab GTPases and
molecular motors with reflexes in adhesion dynamics, cell signaling and metabolism in
many instances shown to drive oncogenic transformation and invasion (Mosesson
et al.
,
2008). Furthermore, the relevance of our findings is supported by recent report
demonstrating that the formation of filopodia is a critical step in the metastasis cascade
(Shibue,
et. al.
, 2013).
Additionally, we cannot rule out the possibility that some of the effects observed could be
due to an increase in the rates of apoptosis in the MYO5A knockdown cells. Although we
have not observed alteration of viability after myosin-Va depletion in short term culturing
under regular conditions, increase of apoptosis rates under adhesion blockage and poor
recovery of frozen stocks were noted. In fact, recent independent findings reinforce
participation of myosin-Va in the control of apoptosis. Bmf sequestration to the actin
cytoskeleton, presumably in complex with myosin-Va/DLC2, promotes resistance to MEK-
inhibitors (Van Brocklin
et al.
, 2009). Accordingly, overexpression of myosin-Va tail
fragments harboring the binding site for DLC2 leads to apoptosis in melanoma cells likely
Alves et al. Page 2
J Invest Dermatol
. Author manuscript; available in PMC 2014 June 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
by disrupting Bmf and probably also Bim anchorage (Izidoro-Toledo and Borges
et. al
.,
2013). Finally, miR-145, which is a transcriptional target of p53 and known to act as a tumor
suppressor, was recently shown to target myosin-Va (Dynoodt
et. al.
, 2012). Therefore,
myosin-Va may integrate mechanisms that interconnect invasion/migration machinery and
resistance to apoptosis. Interdependencies between these processes are reviewed in
Alexander and Friedl (2012).
In summary, the data presented here show that myosin-Va promotes adhesion dynamics,
anchorage-independent survival, migration and invasion
in vitro
. Therefore, up-regulation of
myosin-Va during melanoma progression may be part of a general mechanism that promotes
malignant properties.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We are thankful to Silmara Reis Banzi and Benedita Oliveira Souza for their technical assistance, as well as to the
Laboratory of Confocal Microscopy of FMRP-USP. We are especially grateful to Dr Meenhard Herlyn (Wistar
Institute, Philadelphia, PE, USA) for providing the WM melanoma cell lines and Dr. David Baltimore (Caltech,
Pasadena, CA, USA) for providing lentiviral vectors used to make shMYO5A#1 and one of the control shRNAs.
This work was supported by grants to EME from Fundação de Amparo á Pesquisa do Estado de São Paulo
(FAPESP - #2009/50167-3) and CNPq (#401322/2005-0). CPA and MHM received fellowships from CAPES and
CNPq. JFS, DMT, AR and CLSP received FAPESP fellowships and EME was awarded with CNPq research
fellowship (311347/2011-8). DEF was supported by grants from NIH, the Adelson Medical Research Foundation,
the Melanoma Research Alliance, the Doris Duke Medical Foundation, and the US-Israel Binational Science
Foundation.
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Figure 1. Myosin-Va is highly expressed in melanoma cells and its knockdown impairs cell
adhesion and spreading on fibronectin-coated surface
(a) Relative
MYO5A
mRNA expression detected by qPCR in melanocytes (pMel1 to 4)
versus melanoma cells, using β-actin for normalization and mean of all melanocytes as
reference value. (b) Western-blot of myosin-Va in a panel of melanoma cell lines of radial
growth phase (RGP), vertical growth phase (VGP) and metastasis (M), including two
genetic pairs (WM793 and 1205Lu; WM278 and WM1617). (c-e) Western-blots for
myosin-Va in (c, d) WM1617 and (e) UACC-257. WM1617 cells were lysed 3 days after
transduction with lentiviral vectors carrying shRNAs targeted to bacterial Lac-Z (shControl)
or MYO5A (shMYO5A#1), or after stable selection with antibiotics for about 2-3 weeks in
the case of shMYO5A#2-3 and respective shControl (Figure S3). UACC-257 cells were
lysed 3 days after transfection with siRNA against myosin-Va or control. (f) Confocal
images of F-actin stained cells adhered to fibronectin-coated coverslips for the indicated
times. Arrows indicate transduced cells visualized by GFP expression (inserts). Scale bar =
20μm. (g) Cells allowed to adhere on fibronectin-coated surface for the indicated times were
counted and data were plotted as mean ± SD from 3 independent experiments. (h) Cell
spreading. Imaged as in (f) and the areas for 60 cells/time point were measured using
ImageJ.
Alves et al. Page 5
J Invest Dermatol
. Author manuscript; available in PMC 2014 June 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 2. Ablation of myosin-Va inhibits colony formation, migration and invasiveness of
metastatic melanoma cells without affecting cell proliferation
(a-c) Lentiviral transduced WM1617 cells, using three independent shMYO5A or
shControls, were used to assess: (a) Colony formation in soft-agar after 30 days of growth.
Scale bar = 500μm; (b) Proliferation rates by absorbance measurement of crystal violet
staining; (c) Migration in transwell and invasion in transwell-matrigel assay. Cells were kept
in starvation conditions 24 hour prior the assay and were then allowed to migrate/invade for
24 hours. Scale bar = 50μm. (d) Migration in 3D collagen. After 24 or 48 hours of
incubation - distance from spheroid edge to invasive front was measured and the data from
three independent experiments were plotted as a percentage of control. Scale bar = 100μm.
(e-f) Transwell invasion and proliferation rates of UACC257 cells transiently transfected
with duplex siRNA targeted to MYO5A and irrelevant siRNA. Invasion assay was done as
described in c, and proliferation rates were estimated based on ATP measurements. Data
were plotted as mean ± SD from 3 independent experiments. Scale bar = 50μm.
Alves et al. Page 6
J Invest Dermatol
. Author manuscript; available in PMC 2014 June 01.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Supplementary resource (1)

Supplementary Material
Data
May 2013
Cleidson Padua Alves · Milene H. de Moraes · Josane de Freitas Sousa · Carmen Lucia S. Pontes · Enilza M Espreafico
... Myosin-Va is also upregulated in metastatic melanoma cells and required for manifestation of malignant properties, such as anchorage independent survival, migration, and invasion 15 . In addition, by sequestering the pro-apoptotic factor Bmf 16 , myosin-Va modulates apoptosis triggered by mitochondrial outer membrane permeabilization (MOMP). ...
... The human metastatic melanoma cell line A375 harbors a BRAF V600E mutation and possesses predominantly fragmented mitochondria, which makes them a useful model for understanding mitochondrial dynamics in the context of cancer 5,40 . It is also worth underscoring that melanoma cells exhibit relatively high MYO5A gene expression 15 , thus provide a unique opportunity for studying MYO5A functions. Our data show that myosin-Va knockdown in A375 cells leads to higher rates of mitochondrial respiration, lower glucose consumption and lactate secretion, and higher ROS production. ...
... It is now widely known that the Warburg effect favors biomass building of proliferative tissues 45 . Therefore, the reduced clonogenic ability of myosin-Va knockdown cells is consistent with our metabolic data and corroborates previous findings 1, 15 . Exploring the possibility that myosin-Va functions as an effector molecule of the MAPK pathway to enhance mitochondrial cells showing the immunostaining of the mitochondrial network with anti-Hsp60 in cyan, the endogenous myosin-Va in yellow, and endogenous Drp1 in magenta, prior to ionomycin treatment (left panels), and after ionomycin treatment (right panels). ...
A novel role for Myosin-Va in mitochondrial fission
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Full-text available
  • May 2019
In cancer cells metabolic changes and mitochondrial morphology are coupled. It is known that the cytoskeleton and molecular motors are directly involved in regulating mitochondrial morphology. Here we show that myosin-Va, an actin-based molecular motor, is required for the malignant properties of melanoma cells and localizes to mitochondria in these cells. Knockdown of myosin-Va increases cellular respiration rates and ROS production and decreases glucose uptake and lactate secretion. In addition, knockdown of myosin-Va results in reduced mitochondrial fission and correspondingly elongated mitochondria. We show that myosin-Va interacts with the mitochondrial outer membrane protein Spire1C, an actin-regulatory protein implicated in mitochondrial fission, and that Spire1C recruits myosin-Va to mitochondria. Finally, we show that during mitochondrial fission myosin-Va localization to mitochondria increases, and that myosin-Va localizes to mitochondrial fission sites immediately adjacent to Drp1 punctae. We conclude that myosin-Va facilitates mitochondrial fission. These data implicate myosin-Va as a target for the Warburg effect in melanoma cells.
View
... Consistent with the oncogenic effects of PART1 in the TNBC cells, PART1 knockdown downregulated MYO5A, ZHX2 and BICC1, which have all been implicated in cancer progression [49][50][51][52]. In contrast, PART1 knockdown upregulated PPP2R3A, which is a suspected tumor suppressor [53]. ...
... It is elevated in metastatic colorectal cancer and promotes migration and metastasis of lung, breast, and colon cancer cell lines [68]. MYO5A also promotes anchorage-independent growth, invasion and migration in melanoma [50]. The corresponding reduction in motility/migration upon PART1 inhibition is consistent with the reduction MYO5A levels. ...
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Simple Summary Long non-coding RNAs (lncRNAs) play an important role in cancer progression. Herein we provide new information regarding the role of prostate androgen regulated transcript 1 (PART1). We show that the lncRNA PART1 is enriched in triple-negative breast cancers and cancer stem cell populations. We demonstrate its role in cancer cell and tumor growth and provide evidence for its association with worse survival in a subset of breast cancer patients. Importantly, our genome-wide analyses have revealed novel insights into the function of this lncRNA, demonstrating how it changes the microRNA (miRNA) landscape leading to genome-wide mRNA expression regulation. Our study suggests that PART1 represents an attractive target for the treatment of triple-negative breast cancers. Abstract Triple-negative breast cancers (TNBCs) are aggressive, lack targeted therapies and are enriched in cancer stem cells (CSCs). Novel therapies which target CSCs within these tumors would likely lead to improved outcomes for TNBC patients. Long non-coding RNAs (lncRNAs) are potential therapeutic targets for TNBC and CSCs. We demonstrate that lncRNA prostate androgen regulated transcript 1 (PART1) is enriched in TNBCs and in Aldefluorhigh CSCs, and is associated with worse outcomes among basal-like breast cancer patients. Although PART1 is androgen inducible in breast cancer cells, analysis of patient tumors indicates its androgen regulation has minimal clinical impact. Knockdown of PART1 in TNBC cell lines and a patient-derived xenograft decreased cell proliferation, migration, tumor growth, and mammosphere formation potential. Transcriptome analyses revealed that the lncRNA affects expression of hundreds of genes (e.g., myosin-Va, MYO5A; zinc fingers and homeoboxes protein 2, ZHX2). MiRNA 4.0 GeneChip and TaqMan assays identified multiple miRNAs that are regulated by cytoplasmic PART1, including miR-190a-3p, miR-937-5p, miR-22-5p, miR-30b-3p, and miR-6870-5p. We confirmed the novel interaction between PART1 and miR-937-5p. In general, miRNAs altered by PART1 were less abundant than PART1, potentially leading to cell line-specific effects in terms miRNA-PART1 interactions and gene regulation. Together, the altered miRNA landscape induced by PART1 explains most of the protein-coding gene regulation changes (e.g., MYO5A) induced by PART1 in TNBC.
View
... Initial research of MYO5A concentrated on its role in neurological diseases [35][36][37][38]. As research progressed, MYO5A was also found to perform a critical effect in malignant melanoma [39][40][41][42]. Subsequently, it was disclosed that MYO5A was associated with metastasis in a variety of cancers. ...
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  • Sep 2023
  • BMC CANCER
Background Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor worldwide. Circular RNA (circRNA) is of great value in tumorigenesis progression. However, the mechanism of circFNDC3B in ESCC remains to be clarified. Methods Firstly, the circular characteristics of circFNDC3B were evaluated by Actinomycin D and RNase R measurements. The functions of circFNDC3B in ESCC cells were examined by CCK-8, EdU and flow cytometry. Subsequently, the molecular mechanism of circFNDC3B was explained using luciferase reporter gene detection. Finally, we constructed xenograft model to prove the role of circFNDC3B in vivo. Results Our study revealed that circFNDC3B was more stable than its linear RNA and prominently upregulated in ESCC. Functional findings suggested that silencing of circFNDC3B reduced the proliferation and enhanced apoptosis of ESCC cells in vitro. Meanwhile, knockdown of circFNDC3B attenuated tumor progression in vivo. Next, miR-370-3p/miR-136-5p was discovered to bind circFNDC3B. miR-370-3p/miR-136-5p reversed the promotive effect on cell proliferation and the inhibitory effect on cell apoptosis of circFNDC3B. MYO5A was a downstream target of miR-370-3p/miR-136-5p. CircFNDC3B served as a sponge for miR-370-3p/miR-136-5p and alleviated the prohibitory effect of miR-370-3p/miR-136-5p on MYO5A, which accelerated ESCC progression. Conclusion circFNDC3B positively adjusted the MYO5A expression via spongy miR-370-3p/miR-136-5p, hence achieving the cancer-promoting effect on ESCC. circFNDC3B was a prospective diagnosis marker for ESCC.
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... Mutations in Myo5a lead to the pigmentation and neuronal defects seen in the rare disease Griscelli Syndrome (Mercer et al., 1991;Pastural et al., 1997;Van Gele et al., 2009). Myo5a has also been implicated in cancer progression (Lan et al., 2010;Alves et al., 2013). ...
Exploiting cryo-EM structures of actomyosin-5a to reveal the physical properties of its lever
Preprint
Full-text available
  • Mar 2023
Myosin 5a transports cellular cargos along actin filaments towards the cell periphery. Its long lever plays a key role in determining the large size of its powerstoke, stepping distance along F-actin, ability to bear load and its regulation by Ca2+. Despite this, little is known about the physical properties of the lever and how they contribute to the mechanics of walking. Using a combination of cryo-electron microscopy and molecular dynamics simulations, we resolved the first structure of myosin 5a comprising the motor domain and full-length lever (subfragment-1) bound to actin. From the flexibility captured in the cryo-electron microscopy data, we were able to characterise the stiffness of the lever. Here, we demonstrate how the structure and flexibility of the lever contribute to the regulation and walking behaviour of myosin 5a.
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... For instance, Rab1b is recruited to the Golgi apparatus in CM cells and enhances secretion of proinvasive and pro-angiogenic proteins in vitro and in vivo (174). The tripartite complex Rab27a-Melanophilin-MyosinVa was shown to regulate CM cell migration and invasion, and higher levels of these proteins are associated with advanced CM stages in patient-derived tumor samples (38,175). Rab27a also induces extracellular matrix (ECM) degradation by promoting the secretion of vesicles carrying the membrane type 1-MMP (39). ...
The Dark Side of Melanin Secretion in Cutaneous Melanoma Aggressiveness
Article
Full-text available
  • May 2022
Skin cancers are among the most common cancers worldwide and are increasingly prevalent. Cutaneous melanoma (CM) is characterized by the malignant transformation of melanocytes in the epidermis. Although CM shows lower incidence than other skin cancers, it is the most aggressive and responsible for the vast majority of skin cancer-related deaths. Indeed, 75% of patients present with invasive or metastatic tumors, even after surgical excision. In CM, the photoprotective pigment melanin, which is produced by melanocytes, plays a central role in the pathology of the disease. Melanin absorbs ultraviolet radiation and scavenges reactive oxygen/nitrogen species (ROS/RNS) resulting from the radiation exposure. However, the scavenged ROS/RNS modify melanin and lead to the induction of signature DNA damage in CM cells, namely cyclobutane pyrimidine dimers, which are known to promote CM immortalization and carcinogenesis. Despite triggering the malignant transformation of melanocytes and promoting initial tumor growth, the presence of melanin inside CM cells is described to negatively regulate their invasiveness by increasing cell stiffness and reducing elasticity. Emerging evidence also indicates that melanin secreted from CM cells is required for the immunomodulation of tumor microenvironment. Indeed, melanin transforms dermal fibroblasts in cancer-associated fibroblasts, suppresses the immune system and promotes tumor angiogenesis, thus sustaining CM progression and metastasis. Here, we review the current knowledge on the role of melanin secretion in CM aggressiveness and the molecular machinery involved, as well as the impact in tumor microenvironment and immune responses. A better understanding of this role and the molecular players involved could enable the modulation of melanin secretion to become a therapeutic strategy to impair CM invasion and metastasis and, hence, reduce the burden of CM-associated deaths.
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... Although we did not detect any aberrant expression of HOX genes, upregulation of HOXA9 as well as HOXB7 was reported by Göllner et al. in a resistant EZH2 negative AML cell line model. We furthermore identified upregulation of the direct EZH2 target genes CNN3 and AKAP13, that are involved in chemotherapy resistance in colon cancer and breast cancer, respectively 60,61 , and the genes MYO5A, AKT3 and SPECC1, which are implicated in the evasion of apoptosis [62][63][64] . Additionally, upregulation of TPD52, involved in proliferation, migration, invasion and apoptosis, was found in many cancer types including AML 65 . ...
Loss-of-function mutations in the histone methyltransferase EZH2 promote chemotherapy resistance in AML
Article
Full-text available
  • Mar 2021
Chemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine. We identify seven distinct EZH2 mutations leading to loss of H3K27 trimethylation via multiple mechanisms. Analysis of matched diagnosis and relapse samples reveal a heterogenous regulation of EZH2 and a loss of EZH2 in 50% of patients. We confirm that loss of EZH2 induces resistance against cytarabine in the cell lines HEK293T and K562 as well as in a patient-derived xenograft model. Proteomics and transcriptomics analysis reveal that resistance is conferred by upregulation of multiple direct and indirect EZH2 target genes that are involved in apoptosis evasion, augmentation of proliferation and alteration of transmembrane transporter function. Our data indicate that loss of EZH2 results in upregulation of its target genes, providing the cell with a selective growth advantage, which mediates chemotherapy resistance.
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... Functional studies demonstrated that MYO5A depletion impairs migration of different colon cancer cells in vitro and attenuates metastatic spread of these cells in a chicken chorioallantoic membrane assay in vivo [53]. While no subsequent studies addressed the mechanisms of MYO5A-driven CRC progression, depletion of this motor in HeLa cells and melanoma cells is also inhibits cell migration and invasion [125,126]. Cumulatively, this data strongly suggests that MYO5A acts as an important driver of metastatic dissemination of various tumors. ...
Myosin Motors: Novel Regulators and Therapeutic Targets in Colorectal Cancer
Article
Full-text available
  • Feb 2021
Simple Summary Colorectal cancer (CRC) is a deadly disease that may go undiagnosed until it presents at an advanced metastatic stage for which few interventions are available. The development and metastatic spread of CRC is driven by remodeling of the actin cytoskeleton in cancer cells. Myosins represent a large family of actin motor proteins that play key roles in regulating actin cytoskeleton architecture and dynamics. Different myosins can move and cross-link actin filaments, attach them to the membrane organelles and translocate vesicles along the actin filaments. These diverse activities determine the key roles of myosins in regulating cell proliferation, differentiation and motility. Either mutations or the altered expression of different myosins have been well-documented in CRC; however, the roles of these actin motors in colon cancer development remain poorly understood. The present review aims at summarizing the evidence that implicate myosin motors in regulating CRC growth and metastasis and discusses the mechanisms underlying the oncogenic and tumor-suppressing activities of myosins. Abstract Colorectal cancer (CRC) remains the third most common cause of cancer and the second most common cause of cancer deaths worldwide. Clinicians are largely faced with advanced and metastatic disease for which few interventions are available. One poorly understood aspect of CRC involves altered organization of the actin cytoskeleton, especially at the metastatic stage of the disease. Myosin motors are crucial regulators of actin cytoskeletal architecture and remodeling. They act as mechanosensors of the tumor environments and control key cellular processes linked to oncogenesis, including cell division, extracellular matrix adhesion and tissue invasion. Different myosins play either oncogenic or tumor suppressor roles in breast, lung and prostate cancer; however, little is known about their functions in CRC. This review focuses on the functional roles of myosins in colon cancer development. We discuss the most studied class of myosins, class II (conventional) myosins, as well as several classes (I, V, VI, X and XVIII) of unconventional myosins that have been linked to CRC development. Altered expression and mutations of these motors in clinical tumor samples and their roles in CRC growth and metastasis are described. We also evaluate the potential of using small molecular modulators of myosin activity to develop novel anticancer therapies.
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... MYO5A is an actin-dependent motor protein essential for the intracellular transport of organelles [15]. MYO5A plays an important role in malignant melanoma [16], and its expression was found to be elevated in a number of highly metastatic cancer cell lines and metastatic colorectal cancer tissues [17]. Moreover, overexpression of MYO5A is associated with neck lymph nodes metastasis of oral squamous cell carcinoma [18]. ...
Identification of three predictors of gastric cancer progression and prognosis
Article
Full-text available
  • Aug 2020
Abnormal gene expression is an established cause of gastric cancer (GC) initiation and progression. In this study, we aimed to identify several key genes that could be used to effectively predict progression and prognosis in patients with GC. The Cancer Genome Atlas and the Gene Expression Omnibus database were used to identify candidate genes. Fourteen genes were found to associate highly with progress, metastasis, and survival of GC. Five of these genes were overexpressed in tumor tissue compared to adjacent normal tissue. This was confirmed by RT‐PCR and western blotting for MYO5A, PLTP, and TPP1, while the CCK8 assay was used to show that these three genes promote GC cell proliferation. In summary, we demonstrate that MYO5A, PLTP, and TPP1 expression may be suitable markers for the progression and prognosis of GC.
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Genomic and transcriptomic profiling reveals key molecules in metastatic potentials and organ-tropisms of hepatocellular carcinoma
Article
  • Dec 2022
  • CELL SIGNAL
Metastasis is a landmark event for rapid postsurgical relapse and death of HCC patients. Although distinct genomic and transcriptomic profiling of HCC metastasis had been reported previously, the causal relationships of somatic mutants, mRNA levels and metastatic potentials were difficult to be established in clinic. Therefore, 11 human HCC cell lines and 7 monoclonal derivatives with definite metastatic potentials and tropisms were subjected to whole exome sequencing (WES) and whole transcriptome sequencing (WTS). TP53, MYO5A, ROS1 and ARID2 were the prominent mutants of metastatic drivers in HCC cells. During HCC clonal evaluation, TP53, MYO5A and ROS1 mutations occurred in the early stage, EXT2 and NIN in the late stage. NF1 mutant was unique in lung tropistic cell lines, RNF126 mutant in lymphatic tropistic ones. PER1, LMO2, GAS7, NR4A3 expression levels were positively associated with relapse-free survival (RFS) of HCC patients. The integrative analysis revealed 58 genes exhibited both somatic mutation and dysregulated mRNA levels in high metastatic cells. Altogether, metastatic drivers could accumulate gradually at different stages during HCC progression, some drivers might modulate HCC metastatic potentials and the others regulate metastatic tropisms.
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LINC01980 facilitates esophageal squamous cell carcinoma progression via regulation of miR-190a-5p/MYO5A pathway
Article
  • Apr 2020
Understanding the role of Long non-coding RNAs (lncRNAs) in tumorigenesis in diverse human malignancies would helpful for targeted therapies, containing esophageal squamous cell carcinoma (ESCC). However, the specific role and molecular mechanisms of LINC01980 in ESCC remain unclarified. In this study, we investigated the expression level, function role, and molecular mechanisms of LINC01980 in esophageal cancer cells and ESCC tissues. The high expression of LINC01980 was detected in ESCC tissues and cells, and predicted poor prognosis. LINC01980 promoted the cell proliferation, migration, invasion ability and epithelial-mesenchymal transition (EMT) progress in ESCC cells. In addition, a negative correlation between LINC01980 and miR-190a-5p or miR-190a-5p and MYO5A was observed in ESCC. We found that miR-190a-5p could directly bind with the mRNA of LINC01980 and MYO5A, and it was detected low expression in ESCC. We further demonstrated that the downregulation of MYO5A caused by overexpressing miR-190a-5p was released via upregulation of LINC01980. Functionally, LINC01980 acted as a competitively endogenous RNA (ceRNA) to impact the expression of MYO5A by sponging miR-190a-5p in ESCC. Therefore, these findings suggest that LINC01980 may act as an oncogenic lncRNA in ESCC and LINC01980/miR-190a-5p/MYO5A pathway contributes to the development of ESCC.
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A myosin-Va tail fragment sequesters dynein light chains leading to apoptosis in melanoma cells
Article
Full-text available
  • Mar 2013
Previous studies proposed that myosin-Va regulates apoptosis by sequestering pro-apoptotic Bmf to the actin cytoskeleton through dynein light chain-2 (DLC2). Adhesion loss or other cytoskeletal perturbations would unleash Bmf, allowing it to bind and inhibit pro-survival Bcl2 proteins. Here, we demonstrated that overexpression of a myosin-Va medial tail fragment (MVaf) harboring the binding site for DLC2 dramatically decreased melanoma cell viability. Morphological and molecular changes, including surface blebbing, mitochondrial outer membrane permeabilization, cytochrome-c and Smac release, as well as caspase-9/-3 activation and DNA fragmentation indicated that melanoma cells died of apoptosis. Immobilized MVaf interacted directly with DLCs, but complexed MVaf/DLCs did not interact with Bmf. Overexpression of DLC2 attenuated MVaf-induced apoptosis. Thus, we suggest that, MVaf induces apoptosis by sequestering DLC2 and DLC1, thereby unleashing the pair of sensitizer and activator BH3-only proteins Bmf and Bim. Murine embryonic fibroblasts (MEFs) lacking Bim and Bmf or Bax and Bak were less sensitive to apoptosis caused by MVaf expression than wild-type MEFs, strengthening the putative role of the intrinsic apoptotic pathway in this response. Finally, MVaf expression attenuated B16-F10 solid tumor growth in mice, suggesting that this peptide may be useful as an apoptosis-inducing tool for basic and translational studies.
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In vitro three-dimensional tumor microenvironment models for anticancer drug discovery
Article
Full-text available
Anticancer drug discovery has long been hampered by the poor predictivity of the preclinical models. There is a growing realization that the tumor microenvironment is a critical determinant of the response of cancer cells to therapeutic agents. The past 5 years have seen a great deal of progress in our understanding of how the three-dimensional microenvironment modulates the signaling behavior of tumor cells. The present review discusses how three-dimensional in vitro cell culture models can benefit cancer drug discovery through an accurate modeling of the tumor microenvironment, leading to more physiologically relevant experimental outcomes.
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Identification of miR-145 as a Key Regulator of the Pigmentary Process
Article
Full-text available
  • Aug 2012
  • J INVEST DERMATOL
The current treatments for hyperpigmentation are often associated with a lack of efficacy and adverse side effects. We hypothesized that microRNA (miRNA)-based treatments may offer an attractive alternative by specifically targeting key genes in melanogenesis. The aim of this study was to identify miRNAs interfering with the pigmentary process and to assess their functional role. miRNA profiling was performed on mouse melanocytes after three consecutive treatments involving forskolin and solar-simulated UV (ssUV) irradiation. Sixteen miRNAs were identified as differentially expressed in treated melan-a cells versus untreated cells. Remarkably, a 15-fold downregulation of miR-145 was detected. Overexpression or downregulation of miR-145 in melan-a cells revealed reduced or increased expression of Sox9, Mitf, Tyr, Trp1, Myo5a, Rab27a, and Fscn1, respectively. Moreover, a luciferase reporter assay demonstrated direct targeting of Myo5a by miR-145 in mouse and human melanocytes. Immunofluorescence tagging of melanosomes in miR-145-transfected human melanocytes displayed perinuclear accumulation of melanosomes with additional hypopigmentation of harvested cell pellets. In conclusion, this study has established an miRNA signature associated with forskolin and ssUV treatment. The significant down- or upregulation of major pigmentation genes, after modulating miR-145 expression, suggests a key role for miR-145 in regulating melanogenesis.Journal of Investigative Dermatology advance online publication, 16 August 2012; doi:10.1038/jid.2012.266.
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Rab27a Supports Exosome-Dependent and -Independent Mechanisms That Modify the Tumor Microenvironment and Can Promote Tumor Progression
Article
Full-text available
  • Aug 2012
  • CANCER RES
During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that Rab27a blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by Rab27a-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer in vivo validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context. Cancer Res; 72(19); 4920-30. ©2012 AACR.
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The Outgrowth of Micrometastases Is Enabled by the Formation of Filopodium-like Protrusions
Article
Full-text available
  • May 2012
Unlabelled: Disseminated cancer cells that have extravasated into the tissue parenchyma must interact productively with its extracellular matrix components to survive, proliferate, and form macroscopic metastases. The biochemical and cell biologic mechanisms enabling this interaction remain poorly understood. We find that the formation of elongated integrin β(1)-containing adhesion plaques by cancer cells that have extravasated into the lung parenchyma enables the proliferation of these cells via activation of focal adhesion kinase. These plaques originate in and appear only after the formation of filopodium-like protrusions (FLP) that harbor integrin β(1) along their shafts. The cytoskeleton-regulating proteins Rif and mDia2 contribute critically to the formation of these protrusions and thereby enable the proliferation of extravasated cancer cells. Hence, the formation of FLPs represents a critical rate-limiting step for the subsequent development of macroscopic metastases. Significance: Although the mechanisms of metastatic dissemination have begun to be uncovered, those involved in the establishment of extravasated cancer cells in foreign tissue microenvironments remained largely obscure. We have studied the behavior of recently extravasated cancer cells in the lungs and identified a series of cell biologic processes involving the formation of filopodium-like protrusions and the subsequent development of elongated, mature adhesion plaques, which contribute critically to the rapid proliferation of the micrometastatic cells and thus are prerequisites to the eventual lung colonization by these cells.
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Melanosomes on the move: A model to understand organelle dynamics
Article
Full-text available
  • Oct 2011
  • BIOCHEM SOC T
Advances in live-cell microscopy have revealed the extraordinarily dynamic nature of intracellular organelles. Moreover, movement appears to be critical in establishing and maintaining intracellular organization and organellar and cellular function. Motility is regulated by the activity of organelle-associated motor proteins, kinesins, dyneins and myosins, which move cargo along polar MT (microtubule) and actin tracks. However, in most instances, the motors that move specific organelles remain mysterious. Over recent years, pigment granules, or melanosomes, within pigment cells have provided an excellent model for understanding the molecular mechanisms by which motor proteins associate with and move intracellular organelles. In the present paper, we discuss recent discoveries that shed light on the mechanisms of melanosome transport and highlight future prospects for the use of pigment cells in unravelling general molecular mechanisms of intracellular transport.
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Cancer invasion and resistance: Interconnected processes of disease progression and therapy failure
Article
  • Dec 2011
Cancer progression and outcome depend upon two key functions executed by tumor cells: the growth and survival capability leading to resistance to therapy and the invasion into host tissues resulting in local and metastatic dissemination. Although both processes are widely studied separately, the underlying cell-intrinsic and microenvironmentally controlled signaling pathways reveal substantial overlap in mechanism. Candidate signaling hubs that serve both tumor invasion and resistance include growth factor and chemokine signaling, integrin engagement, and components of the Ras/MAPKs, PI3K, and mTOR signaling pathways. In this review, we summarize these and other mechanisms controlled by the microenvironment that jointly support cancer cell survival and resistance, as well as the invasion machinery. We also discuss their interdependencies and the implications for therapeutic dual- or multi-pathway targeting.
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An Integrated Approach to Uncover Drivers of Cancer
Article
  • Dec 2010
Systematic characterization of cancer genomes has revealed a staggering number of diverse aberrations that differ among individuals, such that the functional importance and physiological impact of most tumor genetic alterations remain poorly defined. We developed a computational framework that integrates chromosomal copy number and gene expression data for detecting aberrations that promote cancer progression. We demonstrate the utility of this framework using a melanoma data set. Our analysis correctly identified known drivers of melanoma and predicted multiple tumor dependencies. Two dependencies, TBC1D16 and RAB27A, confirmed empirically, suggest that abnormal regulation of protein trafficking contributes to proliferation in melanoma. Together, these results demonstrate the ability of integrative Bayesian approaches to identify candidate drivers with biological, and possibly therapeutic, importance in cancer.
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Differential gene expression profiles of invasive and non-invasive non-functioning pituitary adenomas based on microarray analysis
Article
  • Mar 2010
  • ENDOCR-RELAT CANCER
Non-functioning pituitary adenomas (NFPAs) may be locally invasive. Markers of invasiveness are needed to guide patient management and particularly the use of adjuvant radiotherapy. To examine whether invasive NFPAs display a specific gene expression profile relative to non-invasive tumors, we selected 40 NFPAs (38 of the gonadotroph type) and classified them as invasive (n=22) or non-invasive (n=18) on the basis of magnetic resonance imaging and surgical findings. We then performed pangenomic analysis with the 44k Agilent human whole genome expression oligonucleotide microarray in order to identify genes with differential expression between invasive and non-invasive NFPAs. Candidate genes were then tested in qRT-PCR. Prediction class analysis showed that the expression of 346 genes differed between invasive and non-invasive NFPAs (P<0.001), of which 233 genes were up-regulated and 113 genes were down-regulated in invasive tumors. On the basis of Ingenuity networks and the degree of up- or down-regulation in invasive versus non-invasive tumors, 35 genes were selected for expression quantification by qRT-PCR. Overexpression of only four genes was confirmed, namely IGFBP5 (P=0.02), MYO5A (P=0.04), FLT3 (P=0.01), and NFE2L1 (P=0.02). At the protein level, only myosin 5A (MYO5A) immunostaining was stronger in invasive than in non-invasive NFPAs. Molecular signature allows to differentiate 'grossly' invasive from non-invasive NFPAs. The product of one of these genes, MYO5A, may be a useful marker of tumor invasiveness.
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