https://www.pnas.org/doi/10.1073/pnas.1321510111
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RETRACTED: Hypoxia-inducible factors mediate coordinated RhoA-ROCK1 expression and signaling in breast cancer cells
Contributed by Gregg L. Semenza, November 19, 2013 (sent for review October 7, 2013)
December 9, 2013
111 (3) E384-E393
Significance
Breast cancers often contain regions of reduced O2
availability, leading to increased activity of hypoxia-inducible
factors (HIFs). Here, we demonstrate that HIFs activate transcription of
the Rho family member RHOA and Rho kinase 1 (ROCK1)
genes, leading to cytoskeletal changes that underlie the invasive cancer
cell phenotype. ROCK1 is a kinase that regulates myosin light-chain
activity, leading to actin-myosin contraction, which is the basis for
cell movement. Coordinately increased levels of RhoA and ROCK1 mRNA in
human breast cancers predicted patient mortality. These results
demonstrate that a microenvironmental stimulus, hypoxia, can activate a
critical signal transduction pathway, independent of genomic
alterations, to drive cancer progression.
Abstract
Overexpression
of Rho kinase 1 (ROCK1) and the G protein RhoA is implicated in breast
cancer progression, but oncogenic mutations are rare, and the molecular
mechanisms that underlie increased ROCK1 and RhoA expression have not
been determined. RhoA-bound ROCK1 phosphorylates myosin light chain
(MLC), which is required for actin-myosin contractility. RhoA also
activates focal adhesion kinase (FAK) signaling. Together, these
pathways are critical determinants of the motile and invasive phenotype
of cancer cells. We report that hypoxia-inducible factors coordinately
activate RhoA and ROCK1 expression and signaling in breast cancer cells,
leading to cell and matrix contraction, focal adhesion formation, and
motility through phosphorylation of MLC and FAK. Thus, intratumoral
hypoxia acts as an oncogenic stimulus by triggering hypoxia-inducible
factor → RhoA → ROCK1 → MLC → FAK signaling in breast cancer cells.
Invasion and metastasis are
complex processes leading to dissemination of cancer cells from the
primary tumor to distant organs. A critical step is cytoskeletal
reprogramming, which transforms rigid, immobile epithelial cells to
motile, invasive cancer cells. Members of the Rho family of GTPases play
a central role in this process by functioning as molecular switches
that control morphogenesis and movement (1).
Rho proteins mediate both polymerization of actin (F-actin formation)
to create stress fibers, which are antiparallel actin filaments that are
crosslinked by myosin, and activation of myosin to trigger
contractility (2, 3). Active (GTP-loaded) Rho binds to Rho-associated coiled-coil–forming kinase (ROCK), resulting in activation of the kinase (4).
This activation mediates the phosphorylation of myosin light chain
(MLC) directly as well as indirectly by inhibiting myosin phosphatase
(MYPT), leading to actin-myosin contraction (5, 6). ROCK also phosphorylates LIM kinase, which inhibits actin depolymerization (7).
For
cells to move, force generated by actin-myosin contractility is used to
pull on the extracellular matrix (ECM) at focal adhesions, and ECM
stiffness promotes the formation of focal adhesions (8).
Conversely, substrate stiffness is induced by cell contraction and
leads to the activation of focal adhesion kinase (FAK), which is
required for mechanosensing and cell motility (9–11).
A positive regulatory loop exists between Rho family member A (RhoA)
and FAK signaling. In mouse models, FAK plays a critical role in breast
cancer progression (12, 13).
ROCK1 and RHOA gene expression are coordinately up-regulated in motile cells isolated from metastatic breast cancers (14).
Clinical and experimental data indicate that increased expression of
RhoA or ROCK1 is associated with breast cancer progression (15–19).
Somatic mutations do not account for RhoA or ROCK1 overexpression in
the majority of breast cancers, and the underlying molecular mechanisms
remain undefined.
The presence of intratumoral hypoxia, i.e., reduced O2 availability within cancer as compared with normal tissue, is associated with an increased risk of invasion and metastasis (20–23).
Cancer cells respond to the hypoxic microenvironment through the
activity of hypoxia-inducible factors 1 (HIF-1) and 2 (HIF-2). HIFs are
transcription factors that are composed of an O2-regulated HIF-1α or HIF-2α subunit and a constitutively expressed HIF-1β subunit (24).
We used genetic and pharmacologic loss-of-function studies in mouse
models to demonstrate that HIF-1, HIF-2, or both activate the
transcription of a battery of genes whose protein products are required
for discrete steps in the process of breast cancer invasion and
metastasis via lymphatic and blood vessels (25–29).
In primary tumor biopsies, elevated HIF-1α protein levels are
associated with an increased risk of metastasis and mortality that is
independent of breast cancer grade or stage (30–33). Increased HIF-2α levels also are associated with cancer progression (34).
Given
the essential role of HIFs and the RhoA–ROCK1 pathway in breast cancer
invasion, we hypothesized that the motility of breast cancer cells may
be enhanced under hypoxic conditions by a molecular mechanism involving
interplay between these two pathways. Our studies revealed that HIFs
regulate RhoA and ROCK1 expression and activity directly, as determined
by MYPT and MLC phosphorylation in vitro and in vivo. HIF-dependent
RhoA–ROCK1 signaling resulted in cell contraction, cell-induced matrix
contraction, formation of focal adhesions, FAK activation, and increased
cell motility. The coordinate activation of RHOA and ROCK1
expression by HIFs was associated with decreased survival of breast
cancer patients. Taken together, these results provide a molecular
mechanism by which intratumoral hypoxia activates a critical
signal-transduction pathway that is required for breast cancer motility,
invasion, and metastasis.
Results
HIFs Mediate Increased Cell Motility, Formation of Stress Fibers, and Matrix Contraction in Hypoxic Breast Cancer Cells.
Cell motility is a necessary prerequisite for tissue invasion (35).
Previous studies have examined the influence of hypoxia on cell
motility using Boyden chamber assays, which do not permit dynamic or
single-cell resolution and are confounded by the influence of
gravitational force and pore size. Other studies have used video
microscopy to examine breast cancer cells that were exposed to hypoxia,
replated, and analyzed for short (20-min) periods of time (36).
We previously generated MDA-MB-231 subclones, which were stably
transfected with an empty vector (shEV) or expression vectors encoding
shRNA targeting both HIF-1α and HIF-2α (sh1/2α), and found that the
sh1/2α subclone showed impaired lymphatic and vascular metastasis after
injection into the mammary fat pad (29, 37). There was no difference in the viability of the two MDA-MB-231 subclones under either 20% or 1% O2 (29). We dynamically monitored the random motility of shEV and sh1/2α cells exposed to 20% or 1% O2
on collagen-coated surfaces for 22 h. Mean cell velocity determined at
4-h intervals revealed increased velocity starting at 14 h of exposure
to 1% O2, whereas cells exposed to 20% O2 retained a constant velocity throughout the experiment (Fig. 1A and Movies S1–S4).
Hypoxia-induced increases in cell velocity were HIF dependent and led
to an increase in the maximum displacement of cells from their origin (Fig. 1 A and B).
The time-lapse movies revealed that the morphology of the MDA-MB-231
sh1/2α subclone was rounded and lacked the protrusions indicative of a
motile cell phenotype that were noted in the shEV subclone.
...
Increased formation of actin
stress fibers and enhanced contractility are common features of motile
cells in 2D culture conditions (38).
Immunofluorescent staining of polymerized actin (F-actin) using
FITC-conjugated phalloidin revealed an HIF-dependent increase in the
formation of stress fibers following exposure of cells to hypoxic
conditions for 24 h (Fig. 1C).
Cells
make attachments to the ECM at focal adhesion sites and transmit
contractile forces to the substratum via actin stress fibers. MLC
phosphorylation on serine-19 (pMLCS19) is required to
coordinate the formation of stress fibers. To determine the individual
and joint contribution of HIF-1α and HIF-2α to the regulation of MLC
phosphorylation, MDA-MB-231 subclones transduced with expression vectors
encoding shRNA targeting either HIF-1α (sh1α) or HIF-2α (sh2α) were
also established (29). The levels of pMLCS19 were increased significantly in the shEV subclone following 24-h exposure to 1% O2 (Fig. 1D). Knockdown of HIF-1α completely abrogated and HIF-2α knockdown significantly reduced pMLCS19
induction by hypoxia. The impaired hypoxia-induced MLC phosphorylation
was associated with absent or reduced ability, respectively, of sh1α and
sh2α subclones to induce matrix contraction when embedded in type I
collagen (Fig. 1E). This finding was recapitulated in fibroblasts transduced with the same shRNA vectors (Fig. S1),
suggesting that intratumoral hypoxia also may induce HIF-dependent
cytoskeletal changes in stromal cells and demonstrating that these
responses are not dependent on somatic mutations present in breast
cancer cells. Taken together, the data presented in Fig. 1
demonstrate that inhibition of HIFs decreases cell motility, the
formation of stress fibers, MLC phosphorylation, and cell-induced ECM
contraction in response to hypoxia.
Increased RhoA and ROCK1 Expression and Activity in Hypoxic Breast Cancer Cells.
RhoA-bound
ROCK1 phosphorylates and inhibits MYPT; it also directly phosphorylates
and activates MLC, resulting in increased levels of pMLCS19.
To determine whether RhoA or ROCK1 plays a role in hypoxia-induced MLC
activation, RhoA and ROCK1 mRNA and protein levels were analyzed in
cells exposed to 20% or 1% O2 (Fig. 2 A and B).
Quantitative real-time RT-PCR (qRT-PCR) revealed that RhoA and ROCK1
(but not ROCK2) mRNA levels increased under hypoxic conditions in a
panel of nontumorigenic (MCF10A), tumorigenic but nonmetastatic (MCF-7
and T47D), and metastatic (MDA-MB-231 and MDA-MB-435) breast cell lines (Fig. 2A and Fig. S2). RhoA and ROCK1 protein levels also increased modestly after 48 h at 1% O2
and were associated with MYPT phosphorylation at threonine-853, which
is a ROCK1-specific phosphorylation site that inhibits MYPT activity.
pMLCS19 was enhanced in T47D, MDA-MB-231, and MDA-MB-435
cells under hypoxic conditions but was not detectable in MCF10A or MCF-7
cells cultured under either 20% or 1% O2 (Fig. 2B).
Overall, the magnitude of RhoA and ROCK1 expression correlated with the
metastatic potential of the cell lines, as is consistent with studies
in mouse models showing that RhoA and ROCK1 are required for breast
cancer metastasis (16, 18, 39).
To determine the clinical relevance of RhoA and ROCK1 overexpression in
human breast cancer, survival data from two independent cohorts (40, 41)
were analyzed by stratifying patients according to RhoA, ROCK1, ROCK2,
or combined RhoA and ROCK1 mRNA levels in the primary tumor (Fig. 2 C and D). ROCK2 mRNA expression, which was not induced by hypoxia (Fig. S2A),
did not correlate with patient survival in either dataset. ROCK1 mRNA
levels also were not significantly associated with survival. In
contrast, RhoA mRNA levels above the median were associated with
decreased patient survival. The most significant difference in survival
was observed when patients who expressed high levels (above the median
level of expression) of both RhoA and ROCK1 mRNA in their primary tumor
were compared with patients who expressed low levels of both mRNAs.
Taken together, the data presented in Fig. 2
show that RhoA and ROCK1 mRNA and protein expression are coordinately
induced by hypoxia and that combined overexpression predicts mortality
of breast cancer patients.
Hypoxia-Induced RhoA and ROCK1 Expression Is HIF Dependent.
Gene-expression data from 597 breast cancers (42)
was used to compare levels of RhoA and ROCK1 mRNA with expression of
prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), P4HA2, VEGF, LOX,
PLOD1, and ANGPTL4 mRNA, which are HIF regulated in breast cancer cells.
RhoA and ROCK1 mRNA levels were significantly correlated with five of
the six HIF target genes analyzed, unlike ROCK2 mRNA (expression of
which is not regulated by hypoxia or HIFs) (Fig. S3 A and B). As a positive control, the expression of L1CAM (another known HIF target gene) was analyzed; its expression also was correlated with five of the six HIF target genes (Fig. S3A). These data provide evidence for HIF-dependent RhoA and ROCK1 expression in human breast cancers.
In
MDA-MB-231 cells, an increase in RhoA and ROCK1 protein levels occurred
following 12 h of hypoxic exposure and continued for at least 48 h (Fig. S3C).
The hypoxic induction of RhoA and ROCK1 mRNA and protein was inhibited
significantly in the sh1α, sh2α, and sh1/2α subclones (Fig. 3 A and B and Fig. S3D).
The knockdown of HIF-1α or HIF-2α blocked ROCK1-dependent
phosphorylation of MYPT (at T853 and T696, which are also ROCK-dependent
phosphorylation sites) and MLC (at S19) under hypoxic conditions in
MDA-MB-231 cells (Fig. 3B) and fibroblasts (Fig. S3E).
Furthermore, exposure of breast cancer cells to hypoxia was sufficient
to induce HIF-dependent activation of RhoA, as measured by a specific
Rho-GTP binding assay, without the addition of growth factors (Fig. 3C).
To assess RhoA and ROCK1
expression in vivo, MDA-MB-231 shEV and sh1/2α subclones were injected
orthotopically into the mammary fat pad of immunodeficient mice, and
tumors were harvested on day 52. We have reported previously that
primary tumor growth and metastasis of the sh1/2α subclone to lymph
nodes and lungs was reduced significantly relative to the shEV subclone (29, 37).
RhoA and ROCK1 mRNA levels were decreased significantly in tumors
derived from sh1/2α as compared with shEV subclones; this decrease was
comparable to the reduced expression of HIF-1α and P4HA1 mRNA (Fig. 3D). Immunohistochemistry revealed intense nuclear HIF-1α staining in perinecrotic (hypoxic) regions of shEV tumors (Fig. 3E) but not in sh1/2α tumors (Fig. S3F).
Analysis of adjacent shEV tumor sections revealed increased RhoA and
ROCK1 expression that colocalized with HIF-1α in perinecrotic regions (Fig. 3E, Bottom Row). Taken together, the data presented in Fig. 3
demonstrate that HIFs mediate the coordinate expression of RhoA and
ROCK1 in hypoxic breast cancer cells both in vitro and in vivo.
RHOA and ROCK1 Are Direct HIF Target Genes....(sivuteksti)
Three HIF binding sites also were identified in the ROCK1 gene (Fig. 4 C and D).
Site 1, located in intron 1, showed hypoxia-inducible binding of
HIF-1α, HIF-2α, and HIF-1β. Site 2, located in intron 27, bound HIF-1α
and HIF-1β but not HIF-2α. Site 3, located in intron 32, bound HIF-1α,
HIF-2α, and HIF-1β (Fig. 4D). Taken together, the data presented in Fig. 4 demonstrate that HIF-1 and HIF-2 bind directly to multiple sites in the RHOA and ROCK1 genes in hypoxic breast cancer cells, as is consistent with the coordinate regulation of RHOA and ROCK1 expression observed in vitro and in vivo (Fig. 2) and the inhibitory effects of HIF-1α and HIF-2α knockdown on RHOA and ROCK1 expression (Fig. 3A).
HIFs Mediate the Formation of Stress Fibers and Focal Adhesions.
To
determine if enhanced RhoA signaling under hypoxic conditions is
sufficient to promote the formation of focal adhesions, MDA-MB-231 cells
exposed to 20% or 1% O2 for 24 h were stained with
FITC-conjugated phalloidin to detect polymerized actin (F-actin; green)
and with anti-vinculin primary antibody with rhodamine-conjugated
secondary antibody to detect focal adhesions (red) by fluorescence
microscopy (Fig. 5A). Both actin polymerization/stress fiber formation and focal adhesion formation were enhanced under hypoxic conditions (Fig. 5B).
Fig. 5.
FAK activation, as measured by phosphorylation at T397 (pFAKT397), also was increased under hypoxic conditions, regardless of the ECM protein used as substratum (Fig. 5C and Fig. S5A). Analysis of pFAKT397 in a panel of breast cell lines revealed that, although total FAK protein levels were not related to disease progression, pFAKT397 levels were correlated with metastatic potential and pMLC status (compare Fig. 5D with Fig. 2B).
Total FAK protein levels were not altered by hypoxia or HIF knockdown,
but both HIF-1α and HIF-2α were required for enhanced pFAKT397 levels under hypoxic conditions in MDA-MB-231 cells (Fig. 5 E and F). Enhanced pFAKT397 was associated with an increase in focal adhesion density and size in a HIF-dependent manner under hypoxic conditions (Fig. 5G and Fig. S5B). Treatment of MDA-MB-231 cells with the ROCK1 inhibitor Y-27632 blocked hypoxia-induced FAKT397 phosphorylation (Fig. 5H). Based on the data presented in Fig. 5, we conclude that HIF-dependent induction of RHOA and ROCK1 expression caused increased formation of focal adhesions leading to FAK activation in hypoxic breast cancer cells.
Focal Adhesions Are Required for Enhanced Cell Motility Under Hypoxic Conditions.
Cell
motility requires the transmission of force through focal adhesions,
and focal adhesion size has been shown to correlate directly with cell
velocity (44).
Therefore, the effect of focal adhesions on cell velocity induced by
hypoxia was investigated by generating MDA-MB-231 subclones stably
transfected with shEV vector or a vector encoding either of two
independent shRNAs targeting FAK (Fig. 6A). The formation of focal adhesions was disrupted by FAK knockdown, as indicated by anti-vinculin immunofluorescence (Fig. 6B).
The velocity of hypoxic cells during random migration on slides coated
with type I collagen or fibronectin was reduced by FAK knockdown to
levels similar to those of sh1/2α cells (Fig. 6C and Fig. S6A). The maximum displacement of FAK-knockdown cells also was reduced dramatically, to levels similar to those in sh1/2α cells (Fig. 6D and Fig. S6B).
Fig. 6.
To
inhibit the formation of focal adhesions by a method independent of
genetic or pharmacologic approaches, substrate stiffness was modulated
using functionalized polyacrylamide gels. As in glass and tissue culture
dishes, incubating cells under hypoxia for 24 h on stiff polyacrylamide
surfaces (>2,000 Pa) led to an increase in the formation of focal
adhesions in shEV but not in sh1/2α cells (Fig. 6E).
Plating cells on a soft substratum (<200 Pa) blocked hypoxia-induced
formation of focal adhesions, an effect similar to that seen with FAK
or HIF knockdown. Similarly, cell velocity was enhanced by hypoxia on
stiff but not on soft surfaces and required HIF activity (Fig. 6F). Taken together, the data presented in Fig. 6
demonstrate that the increased cell velocity that is induced by hypoxia
in a HIF-dependent manner requires the formation of focal adhesions.
Discussion
The results of this study show that hypoxia-induced, HIF-dependent coordinate transcriptional activation of the RHOA and ROCK1
genes leads to pathway activation that is manifested by actin
polymerization, MLC phosphorylation, actin-myosin cell contractility,
cell-induced matrix contraction, and enhanced breast cancer cell
motility (Fig. 7).
Increased formation of focal adhesions and FAK phosphorylation, which
also resulted from RhoA-ROCK1 activation, were required for enhanced
cell motility under hypoxic conditions. Enhanced motility was abrogated
by the knockdown of HIF-1α and HIF-2α or FAK or by inhibiting the
formation of focal adhesions by plating cells on a soft substratum.
Taken together, these results delineate a molecular mechanism by which
hypoxia, independent of genomic alterations, induces a
signal-transduction pathway that triggers cytoskeletal reorganization,
which underlies the motility of cancer cells and is an essential
attribute of the invasive-metastatic phenotype.
HIFs Mediate Enhanced Motility of Hypoxic Breast Cancer Cells.
In
this study we dynamically evaluated the role of HIFs and chronic
hypoxia in breast cancer cell motility. No change in cell motility was
observed during the first 14 h of incubation at 1% O2, but cell motility increased during the last 8 h of observation. HIFs are induced rapidly upon exposure to 1% O2,
and the delayed effect of hypoxia reflects the time required to induce
signaling to the FAK pathway via increased RhoA and ROCK1 mRNA and
protein expression. Immunoblot assays of MDA-MB-231 lysates confirmed
that RhoA and ROCK1 induction occurs at 12 h and is maintained through
48 h of hypoxic exposure. Our data indicate that HIF → RhoA → ROCK1
signaling is required to induce cell motility under conditions of
chronic hypoxia similar to those experienced in vivo. Clinical studies
have demonstrated that the median pO2 in advanced breast cancers is 10 mmHg (∼1.5% O2), compared with 65 mmHg (∼9.5% O2) in normal breast tissue, and that breast cancers with pO2 <10 mmHg are associated with increased risk of metastasis and patient mortality (45).
Our immunohistochemical analysis revealed striking coexpression of
HIF-1α, RhoA, and ROCK1 protein within the perinecrotic region of
orthotopic breast tumors, as is consistent with coordinate
transcriptional regulation of RHOA and ROCK1 by HIFs. The
significant correlation of RhoA and ROCK1 mRNA levels with expression of
other HIF target genes in >500 human breast cancers indicates that
this observation is clinically relevant.
Increased Stiffness Primes Tumors for Hypoxic Induction of FAK Phosphorylation.
Women with dense breast tissue have an increased risk of breast cancer (46, 47). Tumor fibrosis promotes tumor stiffness, which in turn promotes cancer progression via FAK activation (48, 49).
This study compared cells plated on a soft substratum mimicking normal
breast tissue and cells plated on a stiff substratum that more closely
resembled breast cancer tissue (49).
The formation of focal adhesions, FAK phosphorylation, and cell
motility were enhanced on the stiff substratum as compared with soft
substratum, and this effect of substrate stiffness was dependent upon
HIF activity. Thus, ECM stiffness and hypoxia synergistically activate
RhoA → ROCK1 → FAK signaling that is required for breast cancer cell
motility and invasion.
Levels of HIF-1α, RhoA, and ROCK1 Correlate with Metastatic Status.
Our
results show that HIF-1α, RhoA, and ROCK1 are coexpressed in breast
cell lines and cancer tissue. Expression was lowest in the
nontransformed mammary epithelial cell line (MCF-10A) and highest in the
metastatic breast cancer cell lines (MDA-MB-231 and MDA-MB-435). HIF-1α
expression in breast cancer is associated with decreased survival in
multiple studies (30–33).
In this study, decreased metastasis-free survival was predicted by RhoA
and ROCK1 co-overexpression, with remarkably consistent results between
two independent patient cohorts. The finding that ROCK1 mRNA
overexpression significantly impacted mortality only in the context of
RhoA mRNA overexpression is consistent with the role of RhoA as an
obligate activator of ROCK1. Inhibition of HIF, RhoA, or ROCK1
expression using RNA interference in MDA-MB-231 cells has been shown to
impair metastasis significantly (16, 18, 29). Likewise, targeting either HIF or ROCK1 pharmacologically abrogates metastasis in animal models (18, 29, 37, 39, 50).
FAK Activity, but Not FAK Expression, Is Increased in Metastatic Breast Cancer Cells.
Our
results demonstrate that FAK is a major effector of RhoA/ROCK1 in
hypoxic breast cancer cells. Interestingly, total levels of FAK or MLC
protein did not correlate with metastatic potential in the mammary cell
lines investigated. This result is supported by studies showing that a
large fraction of breast cancers, as well as preinvasive ductal
carcinomas in situ, express elevated FAK protein levels and that the FAK
activation state is more informative than total protein levels (51). These findings in breast cancer are in contrast to those in melanoma, in which HIF-dependent FAK gene transcription promotes invasion (52).
Given that MLC and FAK protein levels are not limiting in breast
cancer, our study suggests that FAK may be poised for activation by an
hypoxic tumor microenvironment. In contrast, RhoA mRNA and protein
levels varied with the metastatic potential of the mammary cell lines,
and increased RhoA levels under hypoxic conditions were associated with
activation of ROCK1; increased expression of ROCK1 also was induced by
hypoxia in an HIF-dependent manner.
Therapeutic Implications.
Both
of the metastatic cell lines studied were derived from triple-negative
breast cancers, which do not express estrogen, progesterone, or HER2
receptors, respond poorly to chemotherapy (53), and are characterized by increased expression of the HIF transcriptome (42).
Because Rho/Rock/FAK signaling is potently activated by HIFs under
hypoxia, treatment protocols that use FAK or HIF inhibitors (54, 55) may be especially beneficial for breast cancer patients with high HIF levels in their primary tumors.
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