Weinberg Lab Research Summary
Research in our laboratory is increasingly
focusing on three major areas: First, what molecular
and biochemical mechanisms are responsible for triggering
cell senescence? Second, how does the stroma of a tumor,
such as a carcinoma, influence the biology of the tumor
as a whole? Third, how do cancer cells within a primary
tumor acquire the ability to invade and metastasize?
Molecular and Biochemical Mechanisms of Cell
Senescence. Senescence is a cell phenotype
that has been defined in the context of in vitro
culture of cells. Thus, after a certain number of cell
divisions, many types of cultured cells will halt proliferation
and enter into a non-growing state that is often termed
replicative senescence. The precise molecular mechanisms
that are responsible for triggering entrance into this
state are complex and confounding.
Much evidence implicates suboptimal conditions of culture
as a key element that is responsible for triggering
entrance into senescence. Thus, if cells are cultured
at a level of ambient oxygen that more closely approximates
that within living tissues, in vitro proliferation is
extended. Moreover, certain types of epithelial cells
can be coaxed to proliferate longer if they are provided
with stromal support, i.e., support by mesenchymal cells
such as fibroblasts and the extracellular matrix that
the latter construct.
One key source of senescence is clearly the reactive
oxygen species (ROS) that are generated as a consequence
of normal metabolism and may conspire with ambient oxygen
to induce oxidized DNA bases. The most commonly observed
of these bases is 8-oxo-dG. Thus, the accumulation of
these oxidized bases in the DNA may overwhelm the ability
of the DNA repair apparatus to restore normal DNA structure,
resulting in turn in the accumulation of these bases
in the DNA. This in turn might trigger a halt to cell
proliferation that is induced by proteins such as p53
and p16INK4A.
In fact, this oxidation may alter the guanosine before
it is incorporated into the DNA. This oxidation is normally
countered by an enzyme, 8-oxo-dGTPase, normally termed
MTH1, which hydrolyzes this oxidized nucleoside triphosphate
in order to avoid its inadvertent incorporation into
the DNA. We have found that knockdown of MTH1 expression
induces rapid senescence and the accumulation of significant
damage in the genomic DNA of cells. This provides us
with a powerful tool to measure the influence of DNA
oxidation on the entrance into cell senescence.
It remains unclear precisely how a halt to cell proliferation
is imposed, once a cell has sensed extensive physiologic
stress and incurred damage. A prominent effector of
this halt is p16INK4A, which blocks the advance
of cells through the G1 phase of the cell cycle. We
have recently been exploring the possibility that its
mechanism of activation, which has been elusive until
now, depends on the activation of stress-activate protein
kinases of the p38 family and are currently exploring
how inhibition of these enzymes will affect the expression
of the important p16INK4A cyclin-dependent
kinase inhibitor.
Tumor Stoma and the Growth of Carcinomas.
Carcinomas arise in epithelial tissues that are composed
of both epithelial and stromal cells. The latter encompass
a variety of mesenchymal cell types, including fibroblasts,
myofibroblasts, adipocytes, macrophages, mast cells,
endothelial cells, pericytes, and lymphocytes. Normal
epithelial cells depend on various types of cell physiologic
support in order to sustain their survival and proliferation.
While the process of tumor progression yields cells
that acquire increasing independence from stromal support,
the great majority of carcinomas are formed from neoplastic
cells that continue to be dependent on nearby stroma.
We have been interested in the possibility that the
stroma of a tumor changes as tumor progression advances.
Thus, we have undertaken a series of experiments in
which weakly growing carcinoma cells are mixed with
the stromal cells extracted from a number of human breast
carcinomas. The growth the resulting mixed tumors has
then been followed and has revealed that the stromal
cells from the majority of breast cancers are more competent
to drive tumor progression than are the stromal cells
from the normal human breast. This change in biological
make-up is reflected by the change in the types of fibroblasts
that are present in the tumor-associated stroma: myofibroblasts
increasingly replace fibroblasts. Tumors arising through
the admixture of myofibroblasts show a greatly increased
vascularization, indicating that angiogenesis is a key
factor in limiting tumor growth, and that the latter
can be accelerated by providing myofibroblasts with
potent angiogenesis-inducing powers.
The mechanism(s) used by myofibroblasts to accelerate
angiogenesis are quite interesting. These cells release
a cytokine, call variously stroma-derived factor-1 (SDF-1)
or CXCL12. The latter encourages the recruitment of
endothelial precursor cells (EPCs) from the circulation
into the tumor-associated stroma, whereupon the EPCs
are induced to differentiate into the endothelial cells
that proceed to construct the capillaries forming the
tumor-associated neovasculature. Because angiogenesis
is a rate-limiting step in tumor formation, the presence
of these myofibroblasts in the tumor-associated stroma
greatly accelerates the growth of the tumor as a whole.
A topic of great interest is the origins of the tumor-associated
stromal cells. While many may originate through proliferation
of stromal cells in the normal, adjacent host stroma,
others may originate in the circulation and thus in
the bone marrow of the host. Indeed, we have found that
primary tumors are able to encourage the mobilization
and recruitment of stromal fibroblasts from the bone
marrow, indicating that such tumors extend their reach
to distant corners of the body in order to expedite
their own agenda of proliferation. Our current research
is focused on identifying the nature of the signals
released by primary tumors in order to stimulate this
stromal recruitment and on the identities of the bone
marrow cell populations that serve as precursors to
tumor-associated stromal cells.
Mechanisms of Tumor Cell Invasiveness and Metastasis.
Carcinomas constitute ~80% of the tumors encountered
in the oncology clinic, and metastases are responsible
for ~90% of all cancer-associated deaths. These figures
have focused our attentions on the mechanisms that enable
carcinoma cells to invade and metastasize. In fact,
the ability to invade and metastasize is a complex,
multistep process that involves a number of distinct
changes in cell phenotype. Thus, the invasion-metastasis
cascade has been proposed to be constituted of the following
discrete steps: local invasiveness, intravasation (invasion
into blood and lymphatic vessels), transport through
the circulation, extravasation (escape from blood vessels
into the surrounding tissue parenchyma), formation of
a micrometastasis, and finally, colonization (growth
of a micrometastasis into a macroscopic metastasis).
The biological complexity of this cascade rivals that
of the initial steps of tumorigenesis, raising the question
of whether a number of distinct mutations must occur
within tumor cells in order to enable them to execute
this series of complex biological processes. At the
same time, it provokes the question of how cancer cells
are able to acquire these multiple abilities in a relatively
short period of time.
We have been working over the past several years with
a series of transcription factors that are normally
active during early embryogenesis and during wound healing.
These transcription factors all are capable of inducing
epithelial cells (the progenitors of carcinomas) to
undergo the epithelial-mesenchymal transition (EMT),
a transdifferentiation process that allows epithelial
cells to acquire many of the attributes of motile, invasive
stromal cells such as fibroblasts. Each of these transcription
factors is capable of acting pleiotropically to induce
the multiple cellular changes that are associated with
the EMT. These include the acquisition of fibroblastic
morphology, the downregulation of E-cadherin and cytokeratins,
the induction of N-cadherin and vimentin, (often) the
secretion of matrix metalloproteinases (MMPs), and the
acquisition of invasive behavior.
To date, we have studied in some detail the actions
of four of these transcription factors, which play key
roles in specific steps of embryogenesis involving EMTs,
such as gastrulation and the emigration of cells from
the neural crest. The Twist, FOXC2, Goosecoid, and Slug
transcription factors all seem capable of programming
much, if not all, of the EMT program when expressed
ectopically in epithelial cells. We have arrived at
these transcription factors through a variety of experimental
routes. Twist and FOXC2 were uncovered through an expression
array screen of genes that were expressed in highly
metastatic mouse breast cancer cells but not in their
non-invasive, non-metastatic counterparts. Goosecoid
was identified because of its known role in specifying
the Spemann organizer, which helps to program gastrulation.
And Slug was identified because of its known role in
enabling neural crest cells to become motile and invasive.
The role of several of these transcription factors
in facilitating metastasis has been demonstrated by
inhibiting their expression in otherwise metastatic
cells. Thus, shutdown of Twist and Slug expression in
metastatic mouse breast cancer cells and human melanoma
cells respectively results in suppression of their metastatic
behavior. Unproven by these experiments is whether ectopic
expression of one or another of these transcription
factors in normally non-invasive cancer cells enables
the latter to acquire all of the capabilities needed
to execute the entire invasion-metastasis cascade.
Significantly, the expression of several of these transcription
factors is associated with specific subtypes of human
malignancies. For example, Twist is expressed preferentially
in invasive lobular carcinomas of the breast, which
are known to be highly invasive and carry a poor clinical
prognosis, while rarely being expressed in invasive
ductal carcinomas, which have a far more favorable clinical
course. Similarly, FOXC2 is expressed in almost half
of the basaloid subclass of human breast cancers, which
carry a poor prognosis, while being rarely expressed
in the epithelioid tumors that generally have a good
prognosis for the breast cancer patient.
Observations like these suggest that various types
of human cancers opportunistically upregulate specific
early embryonic transcription factors in order to gain
many of the attributes of the cells associated with
high-grade malignancy. By activating these normally
latent transcription factors, cancer cells gain access
to their pleiotropic activities and thus are able to
acquire the multiple traits associated with the EMT
and invasiveness in a single step.
Research does not yet reveal how expression of these
various transcription factors is induced. Nonetheless
there is clear evidence that carcinoma cells in the
epithelial compartment of a tumor receive various signals
from the nearby reactive stroma that causes some of
the transcription factors to be expressed in the carcinoma
cells, enabling them to activate the EMT and acquire
invasive and metastatic powers. Our current research
is focused on uncovering the signals that are involved
in inducing expression of these transcription factors
and the mechanisms that enable them to communicate with
one another.
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