A Truncated Form of the Polarity Protein SCRIB Induces Apoptosis in MDA-MB231 Cells by
Regulating Components of Several Signalling Pathways
Maria Makkou1,2, Takis Makatounakis2 and Joseph Papamathekis1,2*
1Department of Biology, University of Crete, Greece
2Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas (FORTH), Greece
Submission: July 15, 2019; Published: July 26, 2019
*Corresponding Address: J Papamatheakis, Department of Biology, University of Crete, N Plastira 100, 70013 Heraklion, Crete, Greece
How to cite this article:Maria Makkou, Androniki Kretsovali, Takis Makatounakis, Joseph Papamathekis. A Truncated Form of the Polarity Protein SCRIB
Induces Apoptosis in MDA-MB231 Cells by Regulating Components of Several Signalling Pathways. Canc Therapy & Oncol Int J. 2019; 14(4): 555894.
Polarity proteins are fundamental to the establishment and maintenance of breast tissue integrity, whereas alterations of their amino acid sequence and protein structure can drive breast cancer formation and progression. Scrib is a multi-functional scaffold protein whose proper localization within the cell is pivotal for its correct function. We report here that the ectopic expression of a truncated form of the polarity protein Scrib, mainly composed of the LRR domain, drives MDA-MB231 breast cancer cells to caspase 3-mediated apoptotic cell death. Furthermore, we identify the truncated Scrib as a regulator of JNK/c-Jun, Notch and Wnt/β-catenin signalling pathways. Our results indicate that the ectopic expression of the truncated Scrib in a breast cancer cell line with a specific background of genetic mutations (KRAS, AFRGAP1, BRAF, MYCL) activates a program of intracellular molecular alterations and promotes apoptosis.
Keywords: Cell polarity; Scrib; Breast cancer; Apoptosis
Cell polarity is essential for the structure and function of all body tissues. Cell polarity determines crucial cellular functions such as the establishment of the apico-basal cell polarity, asymmetric cell division and cell migration. Epithelial cells are a typical example of polarized cells (apico-basal polarity) with the apical surface exposed to the lumen and the basal surface attached to the basement membrane. Three main protein complexes are responsible for the establishment of cell polarity, the Crumbs (Crumbs3-PALS1-PATJ) and Par (Par3-Par6-aPKC) complexes identify the apical domain of the cell, while the Scribble complex [lethal giant larvae (Lgl) - Scribble (Scrib) - Disc large (Dlg)] defines the basal domain Wen & Zhang .
The polarity protein Scrib is known to interact with structural and signalling proteins, regulating cell-cell adhesion, cytoskeleton remodeling and the activation state of several signalling pathways Feigin & Muthuswamy , McCaffrey LM & Macara . Scrib consists of an N-terminal region with 16-leucine-rich repeats (LRR) that is responsible for its membrane targeting, following 4 PDZ domains that interact with proteins such as ZO-2, APC, β-PIX, zyxin family and a C-terminal region that contains three spectrin binding motifs Stephens et al. . Alterations in the regulation or sequence of scrib gene leads to abnormal protein levels and subcellular localization Lin et al. , Wan et al. . Various point mutations or gene amplifications have been reported in several cases of cancer, including breast cancer (https://www.cbioportal.org/, Lin et al. .
Scrib mutations that lead to truncated Scrib versions missing the LRR domain result in a phenotype that mimics Scrib loss Zeitler et al. . In Drosophila such mutations lead to complete deregulation of cell polarity and growth control, highlighting the key role of the LRR domain in Scrib-mediated functions Zeitler et al. . In comparative terms, Drosophila mutants missing the PDZ domains exhibit a phenotype similar to low level expression if Scrib Zeitler et al. . According to genomic data from the Cancer Genome Atlas (TCGA), several human cancers (including breast cancer) harbor nonsense mutations in scrib, leading to the expression of truncated forms of the protein. Several studies support the involvement of Scrib in human cancers. Loss of Scrib in cooperation with myc oncogene has been correlated with blocking apoptosis, cell transformation and breast cancer formation Zhan et al. . Furthermore, mutations that lead to
loss of Scrib act complementarily with mutations leading to Ras
and Notch activation, which promote tumorigenesis and tumor
progression Brumby et al. . Importantly, although Scrib is
known to interfere with various signaling pathways to control
crucial cellular functions and to promote tumorigenesis, whether
truncated versions of Scrib play a role in similar processes is not
known. In this study, we use a triple-negative breast cancer cell
model to investigate how overexpression of a truncated version
of Scrib protein affects components of signaling pathways that
are involved in apoptosis. Our data suggest that the truncated
Scrib protein encompassing amino-acids 94-494 is a regulator
of multiple signalling events and indicate a key role in apoptotic
cell death in this breast cancer model.
The MDA-MB231 cell line was purchased from ATCC. Cells
were cultured in high glucose Dulbecco’s modified Eagle’s
medium (DMEM) (Life Technologies #41965‐062) supplemented
with 10% heat-inactivated fetal bovine serum (FBS) (PAA Gold,
GE Healthcare #A15151) and 1% penicillin‐streptomycin (Life
Technologies #15140122). Cells were cultured in a humified
incubator at 37 °C under 5% CO2. To induce expression of
Scrib94-494, cells were treated with 1μg/ml doxycycline
(dissolved in distilled water). For the generation of doxycycline
(dox) inducible cell lines, the pLenti CMV rtTA3 Blast (w756-1)
plasmid (Addgene #26429) was introduced into MDA-MB 231
cells and transfected cells were selected using 5μg/ml blasticidin
(Sigma Aldrich). Subsequently, a dox inducible Scrib94-494-
RFP expression vector was introduced into MDA-MB231 cells
followed by puromycin (5μg/ml) (Sigma Aldrich) selection.
For the Scrib 94-494 RFP tagged expression vector, Scrib 94-
494 was obtained from the pLK01 plasmid (Addgene, #37252)
by SalI/StuI restriction enzyme digestion and subcloned into the
SmaI/SalI sites of the JRed-C2 vector (Addgene, #54788). Finally,
the JRed-C2_Scrib 94-494 plasmid was digested with BamHI
and NheI restriction enzymes and the Scrib-RFP fragment was
subcloned into XbaI and BamHI sites of the pMA2780 vector
(Addgene, #25438). The integrity of the final construct was
confirmed by sequencing.
Antibodies against cleaved caspase-3 (#9661S), phosphoc-
Jun (#3270), Notch1 (#3608), Notch2 (#5732) and Notch3
(#5276) were purchased from Cell Signaling Technology.
Antibodies against Scrib (C-20) (sc-11049), phospho-JNK (G-7)
(sc-6254), JNK (D-2) (sc-7345) and β-catenin (E-5) (sc-7963)
were purchased from Santa Cruz. The anti-Killer Red (AB961)
and α-tubulin (#T9026) antibodies were purchased from
Evrogen and Sigma, respectively. All antibodies were used in 1:1000 dilution for western blotting
For lentivirus production, the lentiviral constructs and
second-generation packaging plasmids pMD2.G (Addgene,
#12259) and psPAX2 (Addgene, #12260) were co-transfected
into HEK293T cells. Lentivirus-containing supernatant was
collected 48 and 72 h after transfection and filtered through a
0.45 mm filter before use. Transfections were performed using
the calcium phosphate DNA precipitation method Chen .
Whole cell protein extracts were prepared by lysing the
cells in-well with ice-cold RIPA lysis buffer (50mM Tris-HCl
pH 8.0, 150mM NaCl, 2mM MgCl2, 2mM CaCl2, 0,5% sodium
deoxycholate, 1% NP40, 0,1% SDS, 10% Glycerol, 1x PhosSTOP
phosphatase inhibitors and 1x Complete protease inhibitors),
for 10 min on ice and then for 20min on a rotating wheel at 40C.
The lysates were clarified by centrifugation (10min, 16000rpm,
40C) and total protein concentration was estimated using a
Bradford reagent according to the manufacturer’s instructions.
The supernatant was transferred to a new tube and SDS loading
buffer was added before boiling for 10 min at 95 oC. 30μg of
protein were separated on SDS-PAGE gels and transferred to
PVDF membranes (Johnson 0,45μm). The presence of phosphoand
total proteins was detected using the appropriate antibodies.
Blots were developed with the ECL system (Thermo Scientific).
For cell cycle analysis, 100.000 cells from each sample were
trypsinized, washed with PBS, treated with 200 μg/ml RNAse
A (Qiagen) for 30 min at 37OC and stained with propidium
iodide (PI -Sigma) according to the manufacturer’s instructions.
The analysis was conducted by flow cytometry (FACS Calibur
analyzer). The cell cycle profile was further analyzed using the
ModFit LT software.
The cells were fixed with 4% paraformaldehyde for 15 min
at room temperature. The cells were washed 3 times with 0.05%
Tween-PBS and permeabilized in 0.5% NP40-PBS (Roche) for 5
min at room temperature followed by three rinses. Thereafter,
samples were blocked with PBS 1% BSA for 1h. Then, the
samples were incubated with primary antibodies diluted in PBS
with 3% bovine serum albumin (BSA, Sigma) for 1 h at room
temperature. After washing 3 times with PBS, the samples were
incubated with fluorophore-conjugated secondary antibodies
diluted in PBS with 3% BSA at room temperature for 1h. Then
the cells were washed for another 3 times. Finally, cells were
mounted with ProLong Diamond Antifade Mountant with DAPI
(ThermoFisher, #P36962). Confocal microscopy was carried out
on a Laser-scanning confocal Leica SP8-X.
Dysregulation of oncogenic signaling pathways, such as Ras,
has been indicated to correlate with Scrib protein mutations,
leading to tumor formation and progression Dow et al. .
Data from the Cancer Genome Atlas (TCGA) database indicate
that several cancers including breast cancer, harbor mutations in
the sequence of scrib that lead to truncated forms of the protein
(Figure 1). For instance, mutations that result to premature stop
codons and lead to scrib alleles that encode the N-terminal LRR
domain and lack the C-terminal part of the protein are found
in breast invasive ductal carcinoma, in melanoma, as well as
in uterine endometroid carcinoma (Figure 1b) (https://www.
cbioportal.org/). Furthermore, Metodieva et al.  showed
that some breast cancers apply distinct scrib exon usage pattern,
which results in overexpression of conserved exons encoding
the N-terminal LRR domain and loss of the exons encoding the
C-terminus, therefore lacking the cell cycle checkpoint function
of the C-terminal part of Scrib.
To examine the role of a truncated scrib mutant mainly
consisting of the LRR domain of Scrib (Figure 1a), we used MDAMB231,
a highly aggressive triple negative human breast cancerderived
cell line. In these cells which scrib gene is amplified
in one chromosome while it is deleted in its chromosomal
pair. We employed a tetracycline inducible system to control
the ectopic expression of Scrib94-494 – RFP in these cells.
Following dox treatment, Scrib 94-494 over-expressing cells
demonstrated a rounder morphology and a decrease in cellcell
contacts as compared to un-induced controls (Figure 1c).
Immunofluorescence analysis using a Scrib polyclonal antibody,
which recognizes the C-terminal part of the protein, revealed
that endogenous Scrib is localized both at the cell surface and
the cytoplasm of the MDA-MB231 Scrib 94-494 un-induced
cells. Upon dox treatment we observed diffuse cytoplasmic
localization of endogenous Scrib which colocalized with the
ectopically expressed truncated Scrib (Figure 1c).
We observed that the induction of Scrib 94-494 was
accompanied by reduction in the total cell number. To examine if
this reduction was associated with cell cycle arrest, the Scrib 94-
494 expressing cells and the un-induced controls were stained
with PI and analyzed by flow cytometry. We found that Scrib 94-
494 expressing cells underwent a G0/G1 cell cycle arrest (Figure
2a). The role of Scrib 94-494 in apoptosis was investigated by
Western blotting analysis of cleaved caspase-3. Analysis of
whole cell lysates indicated the presence of cleaved caspase-3 in
Scrib 94-494 expressing cells, suggesting the involvement of the
truncated Scrib in caspase-mediated apoptosis (Figure 2b). Our
combined results indicate that the LRR domain of Scrib might
have an important role in cell cycle arrest as well as cell death
It is well established that scrib is a key regulator of several
signal transduction pathways Stephens et al. . It was
previously shown in Drosophila that JNK normally promotes
apoptosis in scrib mutant cells, whereas scrib mutants lead to
tumour overgrowth and invasion by acting synergistically with
the activated Ras or Notch signaling pathways Brumby et al.  &
Leong et al. . To examine the role of Scrib 94-494 expression
in JNK (c-Jun N-terminal kinase)/c-Jun and Notch signaling
pathways, we carried out western blot analysis of whole-cell
lysates of Scrib 94-494 expressing cells. Western blot analysis
revealed activation of the (JNK)/c-Jun signaling axis in Scrib
94-494 expressing cells, as indicated by phosphorylation status
of JNK and c-Jun signaling proteins (Figure 3). To investigate
the impact of ectopic expression of Scrib 94-494 on Notch
signaling, we used Notch isoform-specific antibodies that detect
both full-length and the NTM region of Notch, consisting of a
short extracellular juxtamembrane peptide, a transmembrane
sequence and the intracellular domain (NICD). Our results
indicated that the protein levels of both full-length and the
NTM region of Notch1, Notch2 as well as Notch3 were reduced
in Scrib 94-494 expressing cells (Figure 3). Overall, these data
suggest that Scrib 94-494 activates JNK/c-Jun signalling axis by
post-translational phosphorylation of its components, whereas
influences Notch signalling regulating Notch protein levels.
Several studies support the crosstalk between MAPK
signalling and the canonical Wnt signalling pathway Bikavilli
et al. . In the absence of the Wnt ligand, β-catenin
phosphorylation results in its proteasomal degradation
MacDonald et al. . To assess β-catenin protein levels in Scrib
94-494 expressing cells, we carried out western blot analysis
of whole cell lysates. Significant reduction of β-catenin protein
levels was observed in Scrib 94-494 expressing cells that could
be driven by the β-catenin destabilization. Collectively, our data
indicate that a Scrib 94-494-mediated molecular mechanism
positively regulates the JNK/c-Jun signalling axis, while at the
same time reduces Notch and β-catenin total protein levels in
MDA-MB231 cells [16-20].
Our findings suggest that a truncated form of Scrib possesses
a key regulatory role in signal transduction by affecting
components of the JNK/c-Jun, Notch and Wnt/β-catenin
signaling pathways in a breast cancer cell model that harbor
multiple oncogenic mutations. It was previously shown that
JNK activation normally promotes apoptosis of scrib mutant
cells in Drosophila, whereas scrib mutations can lead to tumour
overgrowth and invasion in cells with constitutively activated
Ras or Notch signaling Brumby et al.  & Leong et al. .
Furthermore, Scrib activates JNK/c-Jun signalling pathway and
promotes apoptosis in a normal mammary epithelia cell line
Zhan et al. . In our in vitro model, we have shown that a
truncated scrib mutant mainly composed of the LRR domain,
which is crucial for its proper localization, activates JNK/c-Jun
signalling pathway while reduces Notch and β-catenin protein
levels. Additionally, the truncated Scrib induces cell cycle arrest
at G1/G0 phase and promotes apoptosis in MDA-MB231 human
breast cancer cells. Based on our findings we speculate that
overexpression of Scrib 94-494 might drive apoptosis of MDAMB231
cells via regulation of components of several signalling
In conclusion, our results demonstrate that a truncated
form of Scrib protein, mainly consisting of the LRR domain, is
able to regulate JNK/c-Jun, Notch and Wnt/β-catenin signalling
pathways, providing hints for a crosstalk among them that leads
cells to apoptotic cell death. The detection of truncated forms
of scrib in human breast cancer specimen could find application
as a prognostic marker of the disease and could provide a mean
to assess easily the activation status of JNK, Notch and Wnt/β-
catenin signalling pathways; something that could be of utmost
importance in clinical practice.