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<p class="art-type" id="articleinfo">Research Article</p>
<p class="art-title">A Provocative Molecular Link between Mammographic
Density and BRCA1-loss associated TNBC</p>
<p class="art-author"><?php $authors="Jingyao Xu<sup>1</sup>, Gbinigie Olusola<sup>1</sup>, Alexus Footman<sup>1</sup>, Nora Hansen<sup>1</sup>, Aswathy Miriam Cheriyan<sup>1</sup>, Krishna Koganti<sup>1</sup>, Vaishali Reddy<sup>1</sup>, Samir Yezdani<sup>1</sup>, Vikram Eddy<sup>1</sup>, Henry De&#700;smond<sup>1</sup>, Nicolas Bakinde<sup>2</sup>, Joel Okoli<sup>3</sup>, Gabriela Oprea<sup>4</sup>, Kathleen Gundry<sup>4</sup>, E Shyam P Reddy<sup>1</sup> and Veena N Rao<sup>1*</sup>"; echo (stristr($authors,$coauthor))?str_replace($coauthor,"<a href='".$extpath."authors/".$courl."' target='_blank'>".$coauthor."</a>",$authors):$authors; ?></p>
<p class="art-affl"><sup>1</sup>Cancer Biology Program, Department of OB/GYN, Morehouse School of Medicine, Georgia Cancer Center for Excellence, Grady Health
System, Atlanta, USA<br/><sup>2</sup>Depatment of Internal Medicine, Morehouse School of Medicine, Georgia Cancer Center for Excellence, Grady Health System, Atlanta, USA<br/><sup>3</sup>Department of Surgery, Morehouse School of Medicine, Georgia Cancer Center for Excellence, Grady Health System, Atlanta, USA<br/><sup>4</sup>Department of Pathology, Emory University Hospital, Atlanta, USA</p>
<p class="art-aff"><b>*Corresponding author: <?php $corresponding_author="Veena N Rao"; echo ($coauthor!="" && $coauthor==$corresponding_author)?"<a href='".$extpath."authors/".$courl."' target='_blank'>".$coauthor."</a>":$corresponding_author;?></b>, Professor and Co-Director Cancer Biology
Program, GCC Distinguished Cancer Scholar
Department of OB/GYN, Morehouse School of Medicine, Georgia Cancer Center for Excellence, Rm 10C011, Grady Memorial Hospital, 80 Jesse Hill Jr. Drive, Atlanta, Georgia 30303-3031, USA, Tel: 404-489-9993, Fax: 404-489-9220, E-mail: <a href="mailto:vrao@msm.edu">vrao@msm.edu</a></p>
<p class="art-aff"><b>Received:</b> May 13, 2019 <b>Accepted:</b> June 13, 2019 <b>Published:</b> June 21, 2019</p>
<p class="art-aff"><b>Citation:</b> Xu J, Olusola G, Footman A, et al. A Provocative Molecular Link between
Mammographic Density and BRCA1-loss
associated TNBC. <i>Int J Human Genet Genet
Disord</i>. 2019; 1(1): 1-8. doi: <a href="https://doi.org/10.18689/ijhg-1000101">10.18689/ijhg-1000101</a></p>
<p class="art-aff"><b>Copyright:</b> &copy; 2019 The Author(s). This work
is licensed under a Creative Commons
Attribution 4.0 International License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the
original work is properly cited.</p>
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<p class="art-subhead" id="abstract">Abstract</p>
<p class="art-para">Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer
that has a high mortality rate and disproportionately affects young African American (AA) women who carry mutations in the BRCA1 gene. Approximately 80% of breast
cancers which develop in BRCA1-mutant carriers will have TNBC and the molecular
mechanism facilitating tumor development is unclear. Our earlier work suggested Ubc9
to play a critical role in BRCA1 loss mediated TNBC cell migration and metastasis. Collagen is one of the major components of the stromal extracellular matrix (ECM) network that influences tissue density. Its re-organization act as a scaffold aiding cancer
cells to migrate causing metastasis. Ubc9 is known to increase the production of
collagen, a key component of fibroglandular breast tissue, as well as tumorigenesis. Our
work is based on the hypothesis that loss of BRCA1 in women with high breast density
causes abnormal Ubc9 levels which upregulates collagen, fibronectin and inhibits SIRT1, &#946;-catenin expression facilitating TNBC. We tested this hypothesis by studying the
expression of total collagen, fibronectin, Ubc9, SIRT1, &#946;-catenin in BRCA1 mutant TNBC
cells and tumor sample derived from patient with dense breasts using immunofluorescence, immunohistochemistry, and collagen assay. Our results suggest for the first time that
mutation or loss of BRCA1 function in women with fibrocystic breasts can lead to over
expression of Ubc9, induction of collagen and; fibronectin, inhibition of SIRT1 and
nuclear accumulation of &#946;-catenin which could contribute to TNBC development. This
network will aid not only in the identification of potential mechanism-based biomarkers
that could detect disease early, but also enforce preventive measures that could reduce
the risk for TNBC in women with high MD thus reducing the mortality associated with
these cancers to achieve health equity.</p>
<p class="art-para"><b>Keywords:</b> BRCA1; Mammographic density; Ubc9; TNBC; Collagen; Fibronectin; SIRT1; ER-&#945;, &#946;-Catenin; BAT; WAT.</p>
<p class="art-para"><b>Abbreviations:</b> TNBC: Triple Negative Breast Cancers; ER: Estrogen Receptor; AA: African American; MD: Mammographic density; BAT: Brown Adipose Tissue; WAT: White
Adipose Tissue; EMT: Epithelial Mesenchymal Transition; ECM: Extracellular Matrix; SIRT1: Sirtuin1.</p>
<p class="art-subhead" id="intro">Introduction</p>
<p class="art-para">Breast cancers are represented by a heterogeneous group
of tumors. They are characterized by a wide spectrum of
clinical, pathological and molecular features as well as by their
responses to therapy <a href="#1" id="ref1">[1]</a>. Among women in the US, breast
cancer is the most common cancer diagnosis (excluding skin
cancer), and it is the leading cause of cancer death second to
lung cancer <a href="#2" id="ref2">[2]</a>. According to the American Cancer Society, an
estimated 268,600 new cases of female breast cancer will be
diagnosed in women in the U.S in 2018 and an estimated
41,760 will die from it <a href="#2" id="ref2">[2]</a>. Recently, more attention has been
devoted to the classification of primary breast cancer into
molecular subtypes using markers like estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth
factor receptor (HER) <a href="#3" id="ref3">[3</a>,<a href="#4" id="ref4">4]</a>. Luminal subtypes make up the
hormone receptor&#8211;expressing tumors and generally carry a
favorable prognosis. Human epidermal growth factor receptor
2 (HER2) subtypes refer to predominantly hormone receptor&#8211; negative tumors with a specific gene expression pattern <a href="#4" id="ref4">[4</a>-<a href="#7" id="ref7">7]</a>. TNBC - defined by the lack of ER, PR and HER-2 receptors, - is
a highly aggressive subtype of breast cancer and accounts for
15-20% of all breast cancer cases <a href="#5" id="ref5">[5</a>,<a href="#8" id="ref8">8</a>-<a href="#11" id="ref11">11]</a>. Interestingly, about
70% of TNBC is associated with basal like subtype and 76% of
basal like cancers are triple negative. Histologically, TNBC is
high grade with a high mitotic index and can be either invasive
ductal carcinoma or metaplastic carcinoma <a href="#3" id="ref3">[3</a>,<a href="#12" id="ref12">12]</a>. TNBC is a
pressing area of research for both researchers and clinicians
alike because (1) TNBC is a poor prognostic factor for diseasefree and overall survival, (2) no targeted therapy is readily
available for TNBC, (3) there is a clustering of TNBC cases
specifically in premenopausal women and in women of
African descent, and (4) the overlap of BRCA1-associated
breast cancers with the TNBC phenotype is significant
<a href="#1" id="ref1">[1</a>,<a href="#3" id="ref3">3</a>,<a href="#10" id="ref10">10</a>,<a href="#13" id="ref13">13</a>-<a href="#15" id="ref15">15]</a>.</p>
<p class="art-para">Although the incidence rates of breast cancer between
Non-Hispanic white women and AA women are getting
closer, the mortality rates in AA women are higher <a href="#16" id="ref16">[16]</a>. TNBC
is a heterogeneous disease group divided into six different
subtypes based on gene expression analysis/profiles: BL1, BL2, IM, M, MSL, and LAR <a href="#17" id="ref17">[17]</a>. The classification of TNBC into
molecular subtypes is essential for developing personalized
treatments as each subtype responds uniquely to specific
treatments. For example, BL1 and BL2 are both of the &#8220;basallike&#8221; type and initially are sensitive to platinum drugs, such as
cisplatin. M and MSL are both of the &#8220;mesenchymal&#8221; type and
are initially sensitive to kinase inhibitors, such as dasatinib. IM
is of the &#8220;immunomodulatory&#8221; type, and LAR is of the &#8220;luminal&#8221; type and is initially sensitive to non-steroidal androgen
receptor hormone therapy, such as bicalutamide <a href="#17" id="ref17">[17]</a>.</p>
<p class="art-para">Although these are a few of the current treatments used, there is still no targeted therapy for TNBC as there exists for
other types of breast cancers. In addition to the lack of effective
treatment for it, the basal-like subtype is the most aggressive
and, considering the statistics above, is unsurprisingly most
prevalent in AA women <a href="#18" id="ref18">[18</a>,<a href="#19" id="ref19">19]</a>. Notably, tumors with BRCA1
mutation are also of the basal-like subtype <a href="#20" id="ref20">[20]</a>.</p>
<p class="art-para">Women who are germline BRCA1 mutation carriers have
a 15% increased risk of developing breast cancer and, if
diagnosed, a 40-60% risk of a TNBC diagnosis. In fact, over
75% of breast tumors found in women with a BRCA1 mutation
are of the TNBC subtype <a href="#21" id="ref21">[21]</a>. As with breast cancer mortality
and prevalence of the basal-like subtype, the frequency of
BRCA1 mutations is higher in AA women. A recent study, in
which 289 self-identified AA patients, all of whom had been
diagnosed with primary invasive breast cancer, were evaluated
for a variety of germline mutations, including BRCA1
mutations, found that 80% of these patients carried mutations
in their BRCA1 and BRCA2 genes. Other studies have also
shown that there are higher frequencies of BRCA1 mutations
in breast cancer patients of African descent, especially
descendants with an origin of ancestry from Nigeria and the
Bahamas <a href="#18" id="ref18">[18</a>,<a href="#22" id="ref22">22]</a>.</p>
<p class="art-para">SUMOylation has been shown to regulate transcription
factor activities that are involved in several cellular signaling
pathways, such as: BRCA1, cell cycle, and steroid hormones in
breast cancer pathogenesis <a href="#23" id="ref23">[23</a>-<a href="#26" id="ref26">26]</a>. We have shown that wild
type BRCA1 proteins, unlike the disease-associated mutant
BRCA1 proteins, bind the sole SUMO E2-conjugating enzyme
Ubc9 and function as a tumor suppressor <a href="#25" id="ref25">[25]</a>. BRCA1 is a
multifunctional protein that activates ER-&#945; transcriptional
activation and tumor suppression <a href="#21" id="ref21">[21]</a>. BRCA1 mutation, deficiency or abnormal subcellular localization causes elevated
Ubc9 levels and ER-&#945; repression leading to TNBC <a href="#24" id="ref24">[24</a>,<a href="#25" id="ref25">25]</a>. Using patient-derived cell lines with known BRCA1 mutations, we reported the in-vivo association of BRCA1 and Ubc9 in
normal mammary epithelial cells, but not in BRCA1 mutant
TNBC cells. We observed an increased expression of Ubc9 in
BRCA1 mutant TNBC cells while knockdown of Ubc9 expression
in these cells decreased their proliferation and migration <a href="#27" id="ref27">[27]</a>. For the first time, we were able to show that high Ubc9
expression due to BRCA1 mutation may trigger an early
growth and transformation advantage to normal breast and
ovarian epithelial cells resulting in aggressive cancers <a href="#27" id="ref27">[27]</a>. In
addition to TNBC, it has also been reported that Ubc9 is either
upregulated or over-expressed in other tumor cells as well, such as: ovarian, lung, head, neck, and melanoma etc., <a href="#28" id="ref28">[28]</a>.</p>
<p class="art-para">Breast tissue architecture has a central role in breast
biology and function along with other tissue biophysical
parameters like X-ray density and mechanical stiffness. Mammography is the most common imaging technique used
for the screening of breast cancer and it can help in
differentiating certain biophysical markers of human breast, such as mammographic density (MD) <a href="#29" id="ref29">[29</a>,<a href="#30" id="ref30">30]</a>. The classification
of breast density can be identified as &#8220;low&#8221; density or &#8220;high&#8221; density. MD is defined by the ratio of fibroglandular breast
tissue (epithelial cells, fibroblasts, and connective tissue) to
the amount of adipose tissue in the breast. The higher the
percentage of fibroglandular tissue, the denser the breast
<a href="#31" id="ref31">[31</a>,<a href="#32" id="ref32">32]</a>. Collagen is produced by fibroblasts and is a key
component in fibroglandular breast tissue. Dense breast
tissue gives a light appearance on the mammogram and is
composed of stromal and epithelial cells, whereas non-dense
breast appears darker and is composed of adipose tissue <a href="#13" id="ref13">[13]</a>. Recently, there have been studies showing a positive
correlation between mammographic breast density and risk
for the development of breast cancer. This association has
caused breast density to emerge as a critical phenotypic
marker of increased breast cancer risk, resulting in
mammographic breast density to be one of the strongest
known risk factors for breast cancer. In fact, the only other
two risk factors that are stronger are BRCA1 mutations and
age <a href="#33" id="ref33">[33]</a>. Women with over 75% dense tissue have four to six
times the risk of breast cancer due to the increased
fibroglandular tissue content, compared to those with very
little to no dense tissue <a href="#12" id="ref12">[12</a>,<a href="#21" id="ref21">21</a>,<a href="#31" id="ref31">31</a>,<a href="#32" id="ref32">32]</a>. Furthermore, high
mammographic breast density has also been positively
associated with breast tumor characteristics that are predictive
of worse prognosis including larger tumor size, positive lymph
nodes, and advanced stage. It seems logical then that tumors
in high MD breasts may also progress more rapidly <a href="#34" id="ref34">[34]</a>.</p>
<p class="art-para">Recently, it was found that breast density has a strong
association with ER-negative diseases, such as those common
in the TNBC subtypes. Additionally, it was predicted that
tumors in dense breast have increased interaction with
stromal and epithelial cells, resulting in more aggressive
tumors <a href="#35" id="ref35">[35]</a>. This theory suggests a mechanism by which
fibroglandular breast tissue may play a role in tumorigenesis. To that effect, it has also been shown that a positive correlation
exists between TNBC and COL4A2 in a study conducted by
Jing Song et al. <a href="#36" id="ref36">[36]</a>. It was found that collagen type IV alpha
2 (COL4A2) was highly expressed in TNBC. Additionally, when
cells were treated with collagen siRNA1 and siRNA2, the
number of cells undergoing early apoptosis increased
significantly. When the collagen was inhibited, a higher
percentage of cells were found in the G2 phase while a lower
percentage was found in the S phase <a href="#36" id="ref36">[36]</a>. Ubc9 plays a critical
role in the production of collagen, a key component of
fibroglandular breast tissue, as well as tumorigenesis. In a
rheumatoid arthritis (RA) study conducted by Li et al. <a href="#37" id="ref37">[37]</a>, the
role of Ubc9 in the progression of RA was shown using a
collagen-induced arthritis (CIA) model. The expression levels
of Ubc9 were first observed in the joints of the CIA model and
compared to the levels of Ubc9 in the normal joints model. The study found that the CIA joints had a significantly higher
Ubc9 expression. Once Ubc9 was inhibited by treatment with
Ubc9 siRNA, there was a significant reduction in the arthritis
score and joint destruction <a href="#37" id="ref37">[37]</a>. This study provides evidence
that Ubc9 plays a critical role in collagen production. Recently, De Filipis et al. have shown high MD to be negatively
associated with CD36, an integral membrane protein that
regulates many cellular processes such as differentiation of
adipocytes <a href="#38" id="ref38">[38]</a>. In a separate study of brown adipose fat in
Type 2 Diabetes patients, Ubc9 knockdown increased the
expression of CD36, suggesting a mechanism by which Ubc9
down regulates brown adipose tissue deposition <a href="#39" id="ref39">[39]</a>. They
further showed that reducing the expression of CD36 resulted
in a decrease in the amount of fat cells and an increase in the
amount of collagen in the ECM, both of which would lead to
a high MD.</p>
<p class="art-para">Fibronectin (FN) is a glycoprotein of the extracellular
matrix that plays a major role in cell adhesion, migration, and
oncogenic transformation <a href="#40" id="ref40">[40]</a>. SIRT1, a nicotinamide adenine
dinucleotide (NAD)-dependent histone-deacetylase, is linked
to longevity, metabolism, stress response, genomic stability
and energy homeostasis <a href="#41" id="ref41">[41]</a>. Our recent work suggests for
the first time a novel molecular mechanism by which BRCA1
by tethering Ubc9 induces SIRT1, caveolin-1, and ER-a
expression inhibiting TNBC metastasis to the lung <a href="#42" id="ref42">[42]</a>. In fact, SIRT1 expression was found to be significantly reduced in
TNBC subtype <a href="#43" id="ref43">[43]</a> which correlates with our observation. Wild-type BRCA1, but not the mutated BRCA1 regulates the
expression of the nuclear, active non-phosphorylated form of
&#946;-catenin in vitro <a href="#44" id="ref44">[44]</a>. Sumoylation of &#946;-catenin was shown to
be involved in the deregulated proliferation of myeloma cells
<a href="#45" id="ref45">[45]</a>. Our data suggests for the first time how BRCA1
dysfunction results in deregulated expression of Ubc9, induction of collagen, fibronectin, inhibition of SIRT1 and
nuclear active form of &#946;-catenin contributing to EMT in
women with high MD leading to TNBC.</p>
<p class="art-subhead" id="methods">Materials and Methods</p>
<p class="art-para"><b>Cell culture</b></p>
<p class="art-para">HCC1937; and HCC1937 BRCA1 cells were obtained from
American Type Culture Collection (Rockville, MD, USA), CAL51
cells were obtained from CD Biosciences Inc. and cultivated as
described previously <a href="#25" id="ref25">[25]</a>. Stable cell line HCC1937 BRCA1a, CAL51 BRCA1a Mut#1 has been described previously <a href="#42" id="ref42">[42]</a>. CAL51 cells were grown in DMEM medium with 10% FBS, 0.6 &#181;g/ml Insulin, 5 &#215; 10<sup>3</sup> &#181;g/ml transferrin and 1% PS. HCC1937
cells were grown in RPMI 1640 medium with 10% FBS and 1% PS.</p>
<p class="art-para"><b>Expression plasmids</b></p>
<p class="art-para">The BRCA1/1a expression plasmids BRCA1a, BRCA1a
Mut#1 (K109R), BRCA1 C7-C51(1-182 amino acids (aa), BRCA1
C7-C51 Mut#1(K109R) were cloned as described previously
<a href="#25" id="ref25">[25]</a>.</p>
<p class="art-para"><b>Antibodies and reagents</b></p>
<p class="art-para">The antibodies used in this study were primary polyclonal
Rabbit anti-Collagen antibody (Santa Cruz biotechnology), primary polyclonal Rabbit anti-Fibronectin antibody (Santa
Cruz Biotechnology), primary polyclonal Rabbit anti-&#946;Catenin antibody (Santa Cruz Biotechnology), primary
polyclonal Goat anti-Ubc9 N-15 antibodies (Santa Cruz
Biotechnology).</p>
<p class="art-para"><b>Immunofluorescence analysis</b></p>
<p class="art-para">HCC1937, HCC1937 BRCA1, and CAL51 cells were cultured
alone or transfected with pCDNA3, BRCA1a, BRCA1a Mut#1, for 24 hours in six-well plates onto glass cover slips overnight. The cells were washed and fixed in icy methanol for 5 minutes, and blocked using 10% BSA for 60 minutes, followed by
primary polyclonal Rabbit anti-collagen antibody 1:250, primary polyclonal anti-fibronectin and &#946;-catenin antibody (Santa Cruz), 1:500 diluted in 1.5% BSA made in PBS at 25&#176;C
for 1 hour and Alexa 488 goat anti-Rabbit/Alexa 568 goat anti-mouse (Molecular Probes) diluted in 1.5% BSA/PBS for
50 minutes and stained (Hoechst 33258, Pentahydrate, Life
technologies). The cover slips were mounted with Vectashield
mounting medium for fluorescence (H-1000 from Vector). The stained cells were examined by LSM 700 Confocal
Microscope, equipped with 63X oil Ph immersion objectives. Composite filter cubes were used for the 488-405 as described
previously <a href="#25" id="ref25">[25]</a>.</p>
<p class="art-para"><b>
Collagen assay</b></p>
<p class="art-para">HCC1937 and HCC1937 pCDNA3 cells were homogenized
in distilled H<sub>2</sub>O and an equal amount of 12M HCl was added so
the samples were hydrolyzed at 120&#176;C for 3 hrs. Samples were
clarified with active charcoal be evaporated to dryness under a
vacuum in a 96-well plate. Samples were measured at 560 nm
after 90 minutes incubation at 60&#176;C with Chloramine T reagent
and compared to collagen standard.</p>
<p class="art-para"><b>
Patient selection and data collection</b></p>
<p class="art-para">The surgical pathology files at Grady Memorial Hospital
and Emory University Hospital were searched for breast
carcinomas that did not immunohistochemically (IHC) stain
for ER, PR, and HER2 neu. When HER2 was equivocally
expressed on IHC, the amplification status was further studied
by fluorescence in situ hybridization (FISH) for HER2. These
comprised the TNBC&#700;s. In addition to clinical pathology data
and genetic mutation, whole slides were examined for the
presence of tumor and for the appearance of benign tissue
adjacent to the tumor. Cases were selected based on whether
there was enough tumor available for staining. Individual
patient data was de-identified, and there was no patient
contact. This study did not pose any risk to patients and was
approved by the Institutional Review Board.</p>
<p class="art-para"><b>
Hormone receptor and HER studies</b></p>
<p class="art-para">At the time of diagnosis, the predictive and prognostic
breast carcinoma markers ER (clone 6F11 Novocastra), PR (clone 16 Novocastra) and HER2 neu (clone CB11 Novocastra) were performed on formalin fixed, paraffin embedded (FFPE) tissue and evaluated following College of American
Pathologists (CAP) guidelines. When HER2 was equivocally
expressed on IHC, the amplification status was further studied
by fluorescence <i>in situ</i> hybridization (FISH) for HER2. The
scoring of the IHC results was semi quantitative, using 0-3 for
intensity and a percentage of tumor cells staining, following
the current American Society of Clinical Oncology and the
College of American Pathologists guidelines <a href="#46" id="ref46">[46]</a>. In tumors
with a HER grade of IHC 2+, FISH was used to further
determine the overexpression of HER.</p>
<p class="art-para"><b>
Tissue Microarrays (TMAs)</b></p>
<p class="art-para">All microscopic slides from TNBC were reviewed, and the
most representative tumor block was selected as the donor
block for the TMA. Two 1-mm cores from each tumor were
included in the TMA recipient block.</p>
<p class="art-para"><b>
Immunohistochemistry</b></p>
<p class="art-para">Slides (4 microns) were cut from the TMAs and were
evaluated for Ubc9 IHC staining. Goat monoclonal antibody (ab21193, Abcam) (1:200 dilution) was used as primary
antibody. Goat IgG was added as negative control. The tissue
was pretreated at 65&#176;C for 2 hours, followed by
deparaffinization using standard procedures. Antigen retrieval
was carried out in antigen retrieval solution (10 mmol/l Tris, 1
mmol/l EDTA, pH 9.0) before sheep serum was added. Then
slides were incubated with Ubc9 antibody for more than 16
hours at 4&#176;C, overnight. After 3 washes with PBST and further
incubation with horseradish peroxidase (HRP)-conjugated
secondary antibody for 1 hour at 37&#176;C, positive signals were
detected with the chromogen 3,3&#8242;-diamiobenzidine (DAB). Positive expression of Ubc9 was mainly detected in the
cytoplasm and/or nucleus. For the staining, Ubc9 clone
Ep298Y, Novus biological was used in dilution of 1:100. For
Ubc9 antigen retrieval was performed by microwaving slides
at 800 W for 10 minutes followed by 560 W for 10 minutes in
citrate buffer. (1M sodium citrate at pH of 6.0) and cooling in
running water immediately. The primary antibody for the
biomarker was incubated for 60 minutes at room temperature. Diaminobenzidine tetra hydrochloride (DAB) solution was
incubated for 10 minutes after which copper-sulphate
solution (0.5% copper sulfate in 0.8% sodium chloride) was
applied to the slides and incubated for 10 minutes and
counterstained with hematoxylin for 2-3 minutes, followed by
rinsing in tap water. Slides were de-hydrated by immersing in
three alcohol baths for 10 seconds and cleared in two xylene
baths followed by application of cover slip. Negative and
positive controls were performed by omitting the primary
antibody and including control tissue as specified by the
manufacturer, respectively. The scoring of the IHC results was
semi quantitative, using the percentage of positive cells to
produce a final score in the range 0 to100. The cases were
scored without knowledge of the clinical pathology
parameters or patient outcome. For Ubc9 the TMA were
scored twice. The staining of the two core samples from each
tumor was evaluated separately. The average of the individual
cores was used for the final score.</p>
<p class="art-subhead" id="results">Results and Discussion</p>
<p class="art-para"><b>
BRCA1a inhibits the expression of collagen in BRCA1-
deficient HCC1937 and CAL51 TNBC cells</b></p>
<p class="art-para">To study whether BRCA1a inhibits collagen expression in
BRCA1-mutant HCC1937 and CAL51 TNBC cells, we have
studied the expression of collagen in these cells by using
immunofluorescence and collagen assay kits. Our results
show higher levels of expression of collagen in HCC1937 cells
compared to HCC1937 cells stably transfected with BRCA1a
by immunofluorescence analysis using collagen antibodies (<a href="#f001">Figure 1A</a>). Similarly, collagen content was found to be higher
in HCC1937 pCDNA3 and CAL51 pCDNA3 cells compared to
BRCA1a transfected HCC1937 and CAL51 cells using the total
collagen assay kit (<a href="#f001">Figures 1B</a> and <a href="#f001">1C</a>). These results support
our hypothesis that loss of binding of mutant/defective
BRCA1 to Ubc9 in TNBC could cause increased collagen
expression which may contribute to the pathogenesis of
TNBC (<a href="#f002">Figure 2</a>).</p>
<div class="art-img" id="f001">
<img src="<?php echo $imgpath;?>images/ijhg-101-f001.gif" class="img-responsive center-block"/></div><br/>
<div class="art-img" id="f001">
<img src="<?php echo $imgpath;?>images/ijhg-101-f002.gif" class="img-responsive center-block"/></div>
<p class="art-para"><b>Ubc9 is expressed at elevated levels in BRCA1 mutant TNBC
tumor tissue obtained from an AA woman with high breast
density</b></p>
<p class="art-para">Our group has previously found BRCA1 proteins, unlike
the pathogenic mutant BRCA1/1a proteins to bind Ubc9 <a href="#25" id="ref25">[25]</a>
and function as a tumor suppressor in TNBC cells <a href="#25" id="ref25">[25</a>,<a href="#27" id="ref27">27]</a>. Furthermore, positive Ubc9 expression was found to correlate
with poor clinical outcome in Nigerian women with TNBC
<a href="#23" id="ref23">[23]</a>. To study whether Ubc9 is expressed at elevated levels in
TNBC tumor tissue obtained from an AA woman with high
MD and BRCA1 mutation, we studied Ubc9 expression in this
sample by IHC staining using Ubc9 antibody. We observed
increased expression of Ubc9 in TNBC tissues obtained from
AA women with high MD (<a href="#f003">Figure 3</a>) compared to match
benign tissue using IHC analysis suggesting a potential role
for Ubc9 in triggering TNBC development. A finding once
again reinforces a role for Ubc9 in the development of BRCA1-
linked TNBC.</p>
<div class="art-img" id="f001">
<img src="<?php echo $imgpath;?>images/ijhg-101-f003.gif" class="img-responsive center-block"/></div>
<p class="art-para"><b>BRCA1 unlike the Ubc9 binding BRCA1 mutant inhibits
fibronectin protein expression in TNBC cells</b></p>
<p class="art-para">Fibronectin is an adhesive glycoprotein of the extracellular
matrix that is known is known to play a role in cell adhesion, migration in various human cancers and is expressed at
elevated levels in TNBC <a href="#40" id="ref40">[40]</a>. To investigate whether wild type
BRCA1a downregulates the expression of fibronectin in TNBC
cells, we studied the expression of fibronectin in CAL51 TNBC
cells in the absence and presence of wild type BRCA1a and
BRCA1a Mut#1(K109R) by immunofluorescence analysis
using fibronectin antibodies. Our results show inhibition of
fibronectin levels in BRCA1a unlike BRCA1a Mut#1 (<a href="#f004">Figure 4A</a>). Similarly, we repeated the experiments using a carboxy
terminal truncated BRCA1 C7-C51(aa1-182) and BRCA1 C7-
C51 Mut#1 (K109R). We observed inhibition of fibronectin
levels in BRCA1 C7-C51 unlike BRCA1 Mut#1 in CAL51 TNBC
cells (<a href="#f004">Figure 4B</a>). Since the BRCA1 Mut#1 does not bind to
Ubc9 it suggests that binding of BRCA1 to Ubc9 may be
required for downregulation of fibronectin levels in TNBC
cells. It also suggests that the NH2 terminal BRCA1-182 aa is
enough for inhibition of fibronectin levels in TNBC cells.</p>
<div class="art-img" id="f001">
<img src="<?php echo $imgpath;?>images/ijhg-101-f004a.gif" class="img-responsive center-block"/>
<img src="<?php echo $imgpath;?>images/ijhg-101-f004b.gif" class="img-responsive center-block"/>
</div>
<p class="art-para"><b>BRCA1a increases the nuclear &#946;-catenin protein expression
in TNBC cells</b></p>
<p class="art-para">Both &#946;-catenin and BRCA1 proteins were shown to be
required for normal mammary gland development. BRCA1, but
not mutated BRCA1, was shown to interact and increase the
levels of non-phosphorylated and active nuclear form of
&#946;-catenin in TNBC cells <a href="#44" id="ref44">[44]</a>. This group provided the first
evidence to suggest that loss of BRCA1 in TNBC cells leads to
impaired expression of nuclear form of &#946;-catenin which can
lead to breast cancer <a href="#44" id="ref44">[44]</a>. To examine whether BRCA1a, the
isoform of BRCA1 induces &#946;-catenin expression in TNBC, we
studied the expression of &#946;-catenin in HCC1937 pCDNA3, HCC1937 BRCA1a, CAL51pCDNA3, and CAL51 BRCA1a stable
cells lines using &#946;-catenin antibodies by immunofluorescence
analysis (<a href="#f005">Figures 5A</a> and <a href="#f005">5B</a>). Our results show nuclear &#946;-catenin
to be induced both in HCC1937 BRCA1a and CAL51 BRCA1a
cells compared to vector transfected HCC1937 and CAL51
TNBC cell lines. The results suggest BRCA1a to be like BRCA1 in
regulating &#946;-catenin protein expression in TNBC cells <a href="#45" id="ref45">[45]</a>.</p>
<div class="art-img" id="f001">
<img src="<?php echo $imgpath;?>images/ijhg-101-f005.gif" class="img-responsive center-block"/></div>
<p class="art-subhead">Conclusions</p>
<p class="art-para">TNBC is the most invasive and aggressive breast cancer
that has a high mortality rate and disproportionately affects
young AA women who have a much higher frequency of
BRCA1 mutations <a href="#18" id="ref18">[18</a>,<a href="#19" id="ref19">19</a>,<a href="#22" id="ref22">22]</a>. There have been studies showing a positive correlation between high MD and the risk for the
development of TNBC but the molecular pathway facilitating
tumor development is not understood <a href="#12" id="ref12">[12</a>,<a href="#13" id="ref13">13</a>,<a href="#31" id="ref31">31</a>,<a href="#35" id="ref35">35]</a>. Collagen
one of the major components of the stromal ECM network
influences tissue density <a href="#36" id="ref36">[36</a>,<a href="#38" id="ref38">38]</a>, and its reorganization acts as
a scaffold aiding cancer cells to migrate causing metastasis
<a href="#36" id="ref36">[36]</a>. Our previous work suggested Ubc9 to play a critical role
in BRCA1 loss mediated TNBC metastasis <a href="#42" id="ref42">[42]</a>. Ubc9 is shown
to increase the production of collagen, a key component of
the fibroglandular breast tissue, as well as, tumorigenesis <a href="#37" id="ref37">[37]</a>. Our results suggest for the first time that mutations or loss of
BRCA1 function in women with high MD can lead to
overexpression of Ubc9, induction of collagen, fibronectin, and inhibition of SIRT1 and; cytoplasmic localization of
&#946;-catenin which could rapidly progress to TNBC development. This study highlights the need for not only frequent breast
screening and, use of digital mammography but also
screening for potential mechanism-based biomarkers (like
Ubc9, SIRT-1, &#946;-catenin, etc.) that can predict TNBC early in
women with high mammography density and BRCA1
mutation thus reducing the mortality associated with these
aggressive cancers leading to health equity.</p>
<p class="art-subhead">
Conflict of Interest</p>
<p class="art-para">Authors have no conflict of interest to disclose.</p>
<p class="art-subhead">
Acknowledgements</p>
<p class="art-para">We thank all the members of Drs. Rao and Reddy labs for
their help. We thank RCMI core facilities at Morehouse School
of Medicine, for their assistance. This work was supported in
part by Georgia Cancer Coalition Distinguished Cancer
Scholar award, NIH-NCRR-RCMI grant G-12-RR003034, U54
RR02613, 5P20RR11104 and NIHMD research endowment
grant 2S21MD000101, U54 CA118638 and It&#700;s the Journey
Inc. Georgia 2-Day walk for breast cancer and Georgia core to
V.N.R. V.N.R&#700;s lab was also supported in part by private funds
from the VOYA foundation.</p>
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