dual role of tgfβ in crc : tgfβ/smad4, crc, emt ......2019/05/03 · worldwide. in saudi arabia,...

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TGFβ Induced SMAD4 Dependent Apoptosis Proceeded by EMT in CRC

Abdul K. Siraj1*, Poyil Pratheeshkumar1*, Sasidharan Padmaja Divya1, Sandeep Kumar

Parvathareddy1, Rong Bu1, Tariq Masoodi1, Yan Kong1, Saravanan Thangavel1, Nasser

Al-Sanea2, Luai H Ashari2, Alaa Abduljabbar2, Samar Alhomoud2, Fouad Al-Dayel3 and

Khawla S. Al-Kuraya1#

1Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. 2Department of Surgery, Colorectal Unit, Riyadh, Saudi Arabia. 3Department of Pathology, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh, 11211, Saudi Arabia. *These authors have contributed equally

Running Title: Dual role of TGFβ in CRC

Keywords: TGFβ/Smad4, CRC, EMT, Apoptosis, KLF5, Sox4

Additional information.

Financial Support: Authors declare that there is no funding information to be disclosed

for this manuscript.

Conflicts of interest: The authors declare no potential conflicts of interest.

Word Count: Abstract: 208, Total word count (introduction to discussion): 3900

Total number of Tables: 1

Total number of Figures: 5

#Corresponding author and to whom reprints should be addressed:

Khawla S. Al-Kuraya, MD, FCAP Human Cancer Genomic Research, Research center King Faisal Specialist Hospital and Research Cancer MBC#98-16, P.O. Box 3354, Riyadh 11211 Saudi Arabia Tel; (966)-1-205-5167; Fax (966)-1-205-5170 Email: [email protected]

on September 30, 2020. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 3, 2019; DOI: 10.1158/1535-7163.MCT-18-1378

mailto:[email protected]

http://mct.aacrjournals.org/

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ABSTRACT

Colorectal cancer (CRC) is one of the leading cause of cancer-related deaths

Worldwide. In Saudi Arabia, CRC is more aggressive and presents at younger age,

warranting new treatment strategies. Role of TGFβ/Smad4 signaling pathway in

initiation and progression of CRC is well documented. Current study examined the role

of TGFβ/Smad4 signaling pathway in a large cohort of Saudi CRC, followed by in vitro

analysis to dissect the dual role of TGFβ on inducing epithelial to mesenchymal

transition (EMT) and apoptosis. Our study demonstrated high frequency of Smad4

alterations with low expression of Smad4 protein identifying a sub-group of aggressive

CRC to be an independent marker for poor prognosis. Functional studies using CRC

cells show that TGFβ induces Smad4 dependent EMT followed by apoptosis. Induction

of mesenchymal transcriptional factors, Snail1 and Zeb1 was essential for TGFβ-

induced apoptosis. Our results indicate that KLF5 acts as an oncogene in CRC cells

regardless of Smad4 expression and inhibition of KLF5 is requisite for TGFβ-induced

apoptosis. Furthermore, TGFβ/Smad4 signal inhibits the transcription of KLF5 that in

turn switches Sox4 from tumor promoter to suppressor. A high incidence of Smad4

alterations were found in the Saudi CRC patients. Functional study results indicate that

TGFβ induces Smad4 dependent EMT followed by apoptosis in CRC cells.




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INTRODUCTION

Colorectal cancer (CRC) is one of the most common malignancy and the second

leading cause of death by cancer in Western populations (1). However, there have been

remarkable changes in the rate of CRC incidence and mortality over the past few

decades in other ethnic population (2). In Saudi Arabia, CRC presents the number one

cancer affecting Saudi males and the third most common among females (3). Several

studies also have shown that CRC affecting Saudi population tend to be more

aggressive and present with high stage (4,5). CRC has a complex pathogenesis

involving multiple genetic and epigenetic alterations caused by somatic mutations that

lead to uncontrolled tumor cell proliferation, invasiveness and distant metastatic

potential (6,7).

Epithelial to mesenchymal transition (EMT) is a bidirectional process by which epithelial

cells lose their cell polarity and cell-cell adhesion, and gain a motile mesenchymal cell

phenotype that is believed to cause metastasis (8). Furthermore, EMT has been shown

to be attributed to chemotherapy resistance and enabling cancer cell survival (9). One

of the well-known inducer of EMT is transforming growth factor β (TGFβ) (10,11).

TGFβ/Smad4 signaling pathway control the signal transduction from cell membrane to

nucleus and is responsible for many important cellular processes such as tumor

initiation, progression, apoptosis and migration (12).

Although TGFβ is one of the potent inducer of EMT, it has paradoxical affect as tumor

growth suppressor by inducing cell cycle arrest and apoptosis in early stages of tumor




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formation (13). As the main mediator of canonical TGFβ signaling pathway, Smad4

plays pivotal role in the switch of TGFβ function on tumorigenesis (14). Smad4 is a key

mediator of the TGFβ signaling and act as tumor suppressor in CRC. Inactivation of

Smad4 at the gene or protein level is associated with progression of several types of

tumors including CRC (15-17).

Loss of Smad4 protein expression is found in 20% to 40% of CRC cases and strongly

correlated with poor prognosis of CRC patients (17-21). Studies also showed that

Smad4 has been mutated in about 15% of CRC tumors (22). However, data of Smad4

alteration in Middle Eastern CRC is limited (23,24). Therefore, we sought to explore the

role of Smad4 inactivation in a large cohort of CRC patients from Saudi Arabia and its

prognostic value in this ethnic group. Furthermore, a recent study has shed light on the

dichotomous effect of TGFβ/Smad4 signaling in pancreatic ductal adenocarcinoma

(PDA) (25) and its role in EMT and apoptosis. They revealed interesting results that

TGFβ induces PDA cells to undergo Smad4-dependent EMT followed by apoptosis.

In this study, we identified high incidence of Smad4 alterations (mutation, deletion and

low expression) in CRC in Saudi Arabia, a country with a particularly high incidence of

CRC developing at younger age, suggesting a potential role in CRC pathogenesis.

Interestingly, low expression of Smad4 was an independent poor prognostic marker.

Furthermore, functional analysis of relevant CRC cell lines support previously reported

role of TGFβ/Smad4 on inducing EMT and expression of mesenchymal markers Snail,

Twist and Zeb and highlighted the role of Smad4 deficiency in mediating drug

resistance (26-28). Similar to the observed role of TGFβ/Smad4 in PDA, this study also




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revealed that TGFβ /Smad4 signal inhibit the transcription of KLF5. Taken together, our

study demonstrate that TGFβ induces Smad4 dependent EMT followed by apoptosis in

CRC cells. This study further highlights the potential therapeutic role of KLF5 inhibition

in CRC treatment.

MATERIALS AND METHODS

Clinical Samples

A total of 1050 archival tissue samples from patients diagnosed with CRC at the King

Faisal Specialist Hospital and Research Centre were collected from Department of

Pathology. Detailed clinicopathological data were noted from case records and

summarized in Supplementary Table S1. Of these 1050 cases, 426 cases were

subjected to targeted capture sequencing. All samples were obtained from patients with

approval from Institutional Review Board of the hospital. For the study, waiver of

consent was obtained for archived paraffin tissue blocks from Research Advisory

Council (RAC) under project RAC#2170 025.

Tissue microarray and immunohistochemical evaluation

All samples were analyzed in a tissue microarray (TMA) format. TMA construction was

performed as described earlier (29). Briefly, tissue cylinders with a diameter of 0.6 mm

were punched from representative tumor regions of each donor tissue block and

brought into recipient paraffin block using a modified semiautomatic robotic precision

instrument (Beecher Instruments, Woodland, WI). Two cores of CRC were arrayed from




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each case. Standard protocol was followed for IHC staining, details of which are

provided in Supplementary Materials and Methods.

Smad4 immunohistochemical expression was seen predominantly in the nuclear

compartment and nuclear expression was quantified using the intensity score as

described previously (17). Briefly, the intensity of nuclear staining in the tumor cells

were scored from 0 to 4, with score 0 indicating absent Smad4 staining and score 4

indicating highest intensity of staining. Cases showing ≥3+ intensity score were

considered as normal for Smad4 expression. Score 0-2 were considered as low

expression of Smad4.

Tissue Culture Experiments

All human CRC cell lines; CX-1, CACO-2, HCT116, COLO-320, HCT15, DLD1, HT29,

SW480 and LOVO were purchased from ATCC and cultured in RPMI-1640 media

containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 U/ml streptomycin at

370 C in a humidified atmosphere containing 5% CO2. Authentication of all cell lines

were performed in house using short tandem repeats (STR) PCR. The results were

similar to the published data. All treatment experiments were performed in reduced FBS

(2%) condition. Apoptosis analysis was performed using annexinV/PI dual staining and

measured by flow cytometry as previously described (30). Information of antibodies

used for western blotting are listed in Supplementary Materials and Methods.




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Plasmid and Transfection

Plasmid DNA encoding human KLF5 and shRNA’s targeting human Snail1, Zeb1 and

KLF5 were purchased from Origene (Rockville, MD). The overexpression of KLF5 and

knockdown of Snail1, Zeb1 and KLF5 in CRC cells were performed using

Lipofectamine™2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's

protocol. Briefly, CRC cells were seeded in 6-well culture plates; when approximately

50% confluent, cells were transfected with 4 μg plasmid. Stable overexpression clones

resistant to G418 and stable knockdown clones resistant to puromycin were isolated;

overexpression of KLF5 and knockdown of Snail1, Zeb1 and KLF5 protein production

were confirmed by immunoblotting.

Immunofluorescence analysis

Immunofluorescence assay was performed as described previously (31). Briefly, CRC

cells grown on glass coverslips in 6-well plates and fixed with ice-cold methanol. After

permeabilization with 0.2% Triton X-100 and blocking with 5% horse serum in PBS

solution, cells were stained with antibodies to Smad4 (1:100), or E-cadherin (1:200) for

1 h at 37 °C. After washing, cells were incubated with fluorescent-conjugated secondary

antibodies and mounted using DAPI. The cells were visualized using Olympus BX63

fluorescence microscope.




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Sphere forming assay

Cells (500/well) were plated on Corning 24-well ultra-low attachment plates (Sigma-

Aldrich) grown in serum free DMEM-F12 (ATCC) supplemented with B27 (Thermo

Fisher), 20 ng/ml epidermal growth factor (Sigma-Aldrich), 0.4% bovine serum albumin

(Sigma-Aldrich) and 4 μg/ml insulin (Sigma-Aldrich). Fresh medium was supplemented

every 2 days. The spheroids were counted and photographed at day 14. For secondary

spheroid formation, the primary spheroids were dissociated into single cells, and

cultured on 24-well ultra-low attachment plates using spheroid culture medium for

another 10 days.

Statistical analysis

Contingency table analysis and 2 tests were used to study the relationship between

clinico-pathological variables and Smad4 mutation/expression. Survival curves were

generated using the Kaplan-Meier method, with significance evaluated using the

Mantel-Cox log-rank test. The limit of significance for all analyses was defined as a P-

value of < 0.05; two-sided tests were used in the calculations. The JMP11.0 (SAS

Institute, Inc., Cary, NC) software package was used for data analyses.

For all functional studies, data are expressed as the mean ± SD of triplicate

samples and was repeated for at least two separate experiments. Statistical analysis

was performed using one-way analysis of variance (ANOVA), and p<0.05 was

considered statistically significant.




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RESULTS

Clinico-pathological data

The characteristics of the 1050 CRC patients are summarized in Supplementary Table

S1. The median age at the time of surgery was 56 years (inter quartile range [IQR], 47.0

-68.0 years) with a male: female ratio of 1.1. Most of the tumors were located in the left

colon (79.7%). 77.9% of patients had a moderately differentiated tumor and 51% were

either stage III or stage IV.

Smad4 mutation and their clinico-pathological correlation

We first performed high depth capture sequencing of 426 CRC cases, and found 63

mutations in 13.6% of cases. Sanger Sequencing confirmed 54 somatic mutations

present in 12.2% (52/426) CRC cases (Supplementary Fig. S1A). Out of these, 68.5%,

18.5%, 11% and 2% were missense, stop-gained, frame-shift and inframe mutations

respectively (Supplementary Table S2). The Smad4 mutation was significantly

associated with low Smad4 protein expression examined by IHC (p=0.0032). However,

no association was found between Smad4 mutation and 5 years overall survival

(Supplementary Table S3).

ACCESSION NUMBER

The target capture sequencing data of 426 tumors have been deposited to the

European Nucleotide Archive (ENA) with accession number PRJEB32337.




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Smad4 deletion on FISH and its correlation with clinico-pathological parameters

We also performed the FISH assay to examine the Smad4 deletion in the primary tumor

tissues of CRC samples (Supplementary Fig. S1B). In our series of 1050 CRC cases,

FISH analysis was interpretable in 991 cases. TMA for 59 cases were non-

representative due to lack of tumor cells. Of the cases analyzed, only Smad4 gene

deletion was detected in 4.2% (44) cases of CRC. Interestingly, chromosome 18q

monosomy (only one Smad4 signal and one CEN18q signal) was detected in 559

(53.2%) cases (Supplementary Table S4a). Altogether, Smad4 deletion was identified in

603 (60.8%) of our cohort, and was significantly associated with distant metastasis (p =

0.0247) and low Smad4 protein expression (p < 0.0001). Smad4 deletion was also

found to be significantly associated with MSS tumor (p < 0.0001), which is in agreement

with previous reports (32,33) (Supplementary Table S4b)

Low expression of Smad4 protein is associated with poor prognosis

Of the 1050 colorectal cancer cases investigated by immunohistochemistry, low

expression of Smad4 was noted in 67.1% (680/1013) of cases by IHC (Supplementary

Fig. S1C). Thirty-seven cases were non-interpretable due to loss of tissue cores or

absent tumor cells in core. The staining pattern ranged from absent staining to intense

4+ nuclear staining. Low expression of Smad4 was found to be significantly associated

with poor prognostic markers such as larger tumors (T3, p = 0.0008), lymph node

involvement (p=0.0006), TNM stage IV tumors (p = 0.0012), Dukes’ stage C (p =

0.0006) and MSS tumors (p < 0.0001) (Table 1).




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Of particular importance was the association of low Smad4 protein expression with poor

5-year overall survival on univariate analysis using Kaplan-Meier curve (71.6% vs

78.9%, X2 = 7.33, p = 0.0068, Supplementary Fig. S1D). On multivariate analysis using

Cox proportional hazards regression model after adjusting for possible confounders of

survival, low expression of Smad4 was indeed found to be an independent poor

prognostic marker (HR = 1.77; 95% CI = 1.26–2.55; p = 0.0009).

Smad4 alterations and their associations

Smad4 alterations (mutation or low expression or deletion by FISH) were noted in

82.2% (342/416) of our cases. A significant association was noted between Smad4

alteration and lymph node involvement (p = 0.0439) as well as with MSS tumors (p <

0.0001) (Supplementary Table S5).

TGFβ induces Smad4 dependent apoptosis in CRC cells

To explore the role of Smad4 in TGFβ induced apoptosis in CRC cells, we analyzed the

expression of Smad4 in a panel of nine CRC cell lines by immunoblotting (Fig. 1A).

Based on Smad4 expression, we selected two CRC cells lines, HCT116 and DLD1 that

had over-expression of Smad4 as well as two other cell lines, HT29 and SW480 that

had negligible Smad4 expression for further experimentation. Smad4 abundance in

these cells was also verified by immunofluorescence analysis (Fig. 1B). We investigated

the effect of TGFβ on inhibiting cell viability and inducing apoptosis in these cells. There

was inhibition of cell viability and induction of apoptosis in both CRC cell lines that had

overexpression of Smad4; however, TGFβ has no effect on cell viability and apoptosis

in Smad4 low expressing cells (Fig. 1C-F).




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To verify the role of Smad4 in TGFβ induced cell death, we knockout Smad4 in Smad4

proficient cells (HCT116 and DLD1) (Supplementary Fig. S2A) followed by treatment

with TGFβ for 48 hours and analyzed cell proliferation and apoptosis. Silencing of

Smad4 significantly suppressed TGFβ induced inhibition of cell proliferation as

confirmed by clonogenic assay (Supplementary Fig. S2B-C). As shown in

supplementary Fig. S2D, Smad4 knockout cells became resistant to TGFβ treatment as

compared to Smad4 proficient control. Furthermore, we stably overexpressed Smad4 in

Smad4 deficient cells (HT29 and SW480) (Supplementary Fig. S2E). Forced expression

of Smad4 significantly decreased cell proliferation after treatment with TGFβ

(Supplementary Fig. S2F-G). Overexpression of Smad4 in HT29 and SW480 cells

became sensitive to TGFβ treatment as compared with cells transfected with empty

vector (Supplementary Fig. S2H).

TGFβ induces EMT followed by apoptosis in CRC cells

TGFβ has been known to induce both EMT and apoptosis in vitro (34,35). To

understand these events in detail, we treated Smad4 proficient and Smad4 deficient

CRC cells with TGFβ at different time points. As shown in Fig. 2A, Smad4 proficient

CRC cells lost cellular polarity and started forming elongated spindle-like protrusions

after 12 hours of TGFβ treatment. From 24-48 hours of TGFβ treatment, most spindle

like Smad4 proficient cells began to shrink and started dying. In contrast, Smad4

deficient cells retained its original morphology with negligible cell death throughout this

period (Fig. 2A). Immunofluorescence analysis showed a downregulation of E-cadherin




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expression after TGFβ treatment in Smad4 proficient cells, whereas the Smad4

deficient cells retained E-cadherin expression (Fig. 2B).

As shown in Fig. 2C, there was a time course increase in apoptosis following treatment

with TGFβ after 24 hours in Smad4 proficient cells confirming that this cell death was

due to apoptosis. However, TGFβ had no effect on Smad4 deficient cells (Fig. 2C). We

also investigated the expression of E-cadherin and apoptotic protein markers after

TGFβ treatment at different time points (24 and 48 hours) on these cells by immuno-

blotting. As shown in Fig. 2D, treatment of TGFβ successfully down-regulated E-

cadherin expression and induced the cleavage of caspase-3 in Smad4 proficient CRC

cells, whereas the expression of E-cadherin and caspase-3 remained unchanged in

Smad4 deficient cells (Fig. 2D). Together, these findings suggest that TGFβ induces

Smad4-dependent EMT followed by apoptosis in CRC cells.

Snail1 and Zeb1 promote EMT and apoptosis

Transcription factors, Snail1 and Zeb1 plays a key role in initiating TGFβ induced EMT

process (36,37). As shown in Fig. 3A-B, TGFβ treatment prominently induced the

expression of Snail1 and Zeb1 after 48 hours in Smad4 proficient CRC cells but not in

Smad4 deficient cells. Silencing of Snail1 and Zeb1 in Smad4 proficient cells markedly

inhibited the TGFβ-induced E-cadherin downregulation (Fig. 3C). Besides, knockdown

of Snail1 and Zeb1 suppressed the induction of EMT-like morphological changes by

TGFβ in Smad4 proficient cells (Fig. 3D). Next, we sought to determine the role of

Snail1 and Zeb1 in TGFβ-induced apoptosis. As shown in Fig. 3E-F, silencing of Snail1




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and Zeb1 significantly inhibited the TGFβ-induced apoptosis in Smad4 proficient cells,

showing the essential role of these transcriptional factors on TGFβ-induced apoptosis.

Sox4 is required for TGFβ driven apoptosis

Sox4 expression has shown to be elevated in a wide variety of human cancers (38-40).

To understand the role of Sox4 in TGFβ driven apoptosis, we silenced Sox4 in Smad4

proficient cells using two different shRNA’s and treated with TGFβ for 48 hours and

analyzed apoptosis. As shown in Fig. 4A, knockdown of Sox4 significantly suppressed

TGFβ-induced apoptosis in Smad4 proficient CRC cells. There was no further

significant increase in apoptosis even after addition of 5-FU in Sox4 depleted cells (Fig.

4A). This data clearly demonstrate that Sox4 is essential for TGFβ driven apoptosis in

Smad4 proficient cells. Next, we tested the role of Sox4 in TGFβ-induced EMT.

Knockdown of Sox4 had no effect on EMT morphology (Fig. 4B) or E-cadherin

expression (Fig. 4C). There was an increase in Sox4 expression in scramble control

cells after TGFβ treatment (Fig. 4C). Moreover, silencing of Snail1 and Zeb1 had no

effect on Sox4 expression (Fig. 4D). These results show that Sox4 has no role in TGFβ-

induced EMT. To test the role of Sox4 in spheroid growth, we generated spheroids from

Smad4 proficient cells and stemness of the spheroids were confirmed using stem cell

markers (Fig. 4E). Next, we determined the expression of Sox4 in adherent and

spheroid cells with and without TGFβ treatment. Cells grown as spheroids showed

higher Sox4 expression than cells grown in adherent conditions (Fig. 4F). Moreover,

there was an increase in Sox4 expression in both cells after TGFβ treatment (Fig. 4F).

To verify the role of Sox4 in spheroid growth, we silenced Sox4 in Smad4 proficient




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cells and grown in spheroid medium. As shown in Fig. 4G-H, silencing of Sox4

significantly decreased the spheroid growth. This data was further verified using

immunoblotting, where knockdown of Sox4 markedly decreased the stem cell marker

proteins expression in spheroids (Fig. 4I). Above results indicate that Sox4 acts as a

tumor initiator in Smad4 proficient cells. Taken together, these data suggest that during

TGFβ-induced EMT, Sox4 switches its function from pro-tumorigenic to pro-apoptotic.

Silencing of KLF5 sensitizes Smad4 deficient cells to TGFβ -induced apoptosis

KLF5 has been shown to promote cancer cell proliferation in several cancers including

CRC (41,42). To study the role KLF5 in TGFβ-induced apoptosis, we treated Smad4

proficient and Smad4 deficient cells with TGFβ for 48 hours and analyzed the

expression of KLF5 by immunoblotting. As shown in Fig. 5A, there was a prominent

inhibition of KLF5 expression after TGFβ treatment in Smad4 proficient cells, but the

KLF5 expression remained unchanged in Smad4 deficient cells. The basal expression

of KLF5 was relatively higher in Smad4 deficient cells than Smad4 proficient cells (Fig.

5A). Next, we silenced KLF5 in Smad4 deficient cells using two different shRNA’s and

treated with TGFβ and 5-FU either alone or in combination for 48 hours and analyzed

apoptosis. As shown in Fig. 5B, silencing of KLF5 markedly increased TGFβ-induced

apoptosis in Smad4 deficient CRC cells. Furthermore, treatment of 5-FU significantly

sensitized the KLF5 depleted cells (Fig. 5B). This data clearly demonstrate that

inhibition of KLF5 is essential for TGFβ driven apoptosis. This finding was in

concordance with recent reports in pancreatic cancer cells (25). David et al., reported

that TGFβ induced EMT inhibits KLF5 in Smad4 proficient cells and induce apoptosis

(25). To test the role of KLF5 in spheroid growth, we generated spheroids from Smad4




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deficient cells and stemness of the spheroids were confirmed using stem cell markers

(Fig. 5C). To test the role of KLF5 in spheroid growth, we silenced KLF5 in Smad4

deficient cells and grown in spheroid medium. Silencing of KLF5 significantly decreased

the spheroid growth (Fig. 5D-E) and stemness properties (Fig. 5F). Above results,

clearly indicate that KLF5 acts as an oncogene in CRC cells regardless of Smad4

expression and inhibition of KLF5 is essential for TGFβ induced apoptosis.

To test the role KLF5 in TGFβ induced EMT, we stably overexpressed KLF5 in Smad4

proficient cells, treated with TGFβ, and analyzed the E-cadherin expression. As shown

in Fig. 5G, overexpression of KLF5 inhibited TGFβ-induced downregulation of E-

cadherin in Smad4 proficient cells. This data evidently demonstrate the association

between KLF5 and EMT. Next, we tested the effect of KLF5 overexpression in TGFβ

induced apoptosis. Forced expression of KLF5 significantly inhibited TGFβ-induced

apoptosis in Smad4 proficient cells (Fig. 5H). Above results, clearly exhibit the

oncogenic function of KLF5. Finally, we silenced both Sox4 and KLF5 in Smad4

deficient cells to confirm the link between these proteins in TGFβ-induced apoptosis. As

shown in Fig. 5I, depletion of KLF5 promotes apoptosis in Smad4 deficient cells after

TGFβ treatment, whereas there was a significant inhibition of apoptosis on Sox4

depletion. All these results indicate that Sox4 promote apoptosis upon KLF5 depletion.

DISCUSSION

In the current study, we used a large cohort of clinical samples to address the clinical

significance of Smad4 alterations in Saudi CRC. We evaluated the Smad4 protein

expression in more than 1000 Saudi CRC patients as well as genomic mutation and




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gene deletion. The frequency of Smad4 alteration was similar to what has been

reported in CRC from other ethnicities (17,23,43-45). Consistent with previous reports,

loss of Smad4 expression exhibited strong correlation to poor overall survival in CRC

patients (46-48). Importantly, we show that low levels of Smad4 protein can identify a

subset of patients with Dukes’ C CRC who have a high probability of recurrence

following potentially curative surgery and 5-FU based adjuvant chemotherapy.

The role of TGFβ signaling in CRC progression and prognosis is very complex and has

previously shown bilateral dependence based on Smad4 status (49). Smad4 is an

important mediator of TGFβ signaling pathway and function as a tumor suppressor gene

in CRC (50). Our study shows that Smad4 expression was reduced significantly with

advancing tumor stage (Table 1). Our in vitro results using CRC cell lines identified a

mechanistic basis for duality of TGFβ in CRC. We show that under conditions of TGFβ

stimulation, presence of the common transactivator protein Smad4 in CRC cells

undergo apoptosis, while in its absence cancer cells promote tumor growth.

Interestingly, in agreement with recent report (25), TGFβ-induced Smad4 dependent

apoptosis is preceded by EMT. Moreover, TGFβ-induced activation of Snail1 and Zeb1

were required for EMT-related apoptosis. TGFβ-induced EMT is generally considered

as a pro-tumorigenic event. However, our study demonstrates that TGFβ exerts tumor

suppression in CRC cells by inducing EMT signal, which inhibits the transcription of

KLF5 that in turn switches Sox4 from tumor promoter to suppressor.

Our study demonstrated the essential role of Sox4 on inducing TGFβ/Smad4-mediated

apoptosis. However, Sox4 showed no direct effect on inducing EMT. Sox4 act as a




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tumor suppressor in those cells that undergo a TGFβ/Smad4 induced EMT, showing

that EMT switches Sox4 function from pro-tumorigenic to pro-apoptotic in CRC cells.

Interestingly, knockdown of KLF5 was sufficient to revert pro-tumorigenic function of

TGFβ and induce apoptosis in Smad4 deficient cells. Furthermore, we show that TGFβ-

mediated EMT downregulates KLF5 expression in Smad4 proficient cells that convert

Sox4 from anti-apoptotic to pro-apoptotic. These data indicate that KLF5 may be a vital

determinant of Sox4 function in CRC. Moreover, forced expression of KLF5 in Smad4

proficient CRC cells prominently inhibited TGFβ-induced EMT and apoptosis, further

confirming the oncogenic function of KLF5.

EMT has been shown previously to contribute to chemoresistance in various epithelial

cancers (51,52). Our data show that Smad4 proficient CRC cells were sensitive to 5-FU

chemotherapy even under TGFβ stimulation, while Smad4 deficient cells were

resistance to 5-FU chemotherapy. However, targeted inhibition of KLF5 using shRNA in

Smad4 deficient CRC cells were able to revert the chemoresistance under TGF

stimulation. This is important from translational stand point as it might suggest the

possibility of therapeutic value of inhibiting KLF5 in Smad4 deficient CRC patients.

However, additional studies are needed to further validate these findings and move

KLF5 inhibitor to preclinical testing.

In conclusion, our results show that Smad4 alteration is frequent in CRC from Middle

Eastern ethnicity and can identify patients at advanced stage (Dukes’ C) and

significantly poor outcome. Our functional studies in CRC cells reinforce the role of




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Smad4 in TGFβ-induced EMT signaling cascade. Furthermore, our results revealed that

TGFβ/Smsd4 signal inhibits the transcription of KLF5 that in turn switches Sox4 from

tumor promoter to suppressor. Therefore, we conclude that TGFβ induces Smad4

dependent lethal EMT in CRC cells.

ACKNOWLEDGMENTS

We thank Dr. Maqbool Ahmed, Roxanne Melosantos, Rafia Begum, Wael Haqawi,

Zeeshan Qadri, Dionne Rae Rala, Ingrid Francesca Victoria, Maha Al Rasheed, for

technical assistance.




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Table 1. Correlation of Smad4 IHC expression with clinico-pathological parameters in

colorectal carcinoma

Total Low Normal p value

N % N % N %

Total Number of Cases 1013 680 67.1 333 32.9

Age ≤ 50 years 332 33.0 220 66.3 112 33.7 0.7646 > 50 years 674 67.0 453 67.2 221 32.8

Sex Male 535 52.8 358 66.9 177 33.1 0.8795 Female 478 47.2 322 67.4 156 32.6

Tumour Site Left colon 800 80.8 548 68.5 252 31.5 0.0520 Right colon 190 19.2 116 61.0 74 39.0

Histological Type Adenocarcinoma 899 89.4 615 68.4 284 31.6 0.0039 Mucinous Carcinoma 107 10.6 58 54.2 49 45.8

pT T1 32 3.4 13 40.6 19 59.4 0.0008 T2 151 15.8 93 61.6 58 38.4 T3 681 71.2 477 70.0 204 30.0 T4 92 9.6 54 58.7 38 41.3

pN N0 484 50.5 297 61.4 187 38.6 0.0006 N1 303 31.7 225 74.3 78 25.7 N2 170 17.8 118 69.4 52 30.6

pM M0 849 87.1 558 65.7 291 34.3 0.1437 M1 126 12.9 91 72.2 35 27.8

TNM Stage I 127 13.1 68 53.5 59 46.5 0.0012 II 328 33.8 210 64.0 118 36.0 III 389 40.1 277 71.2 112 28.8 IV 126 13.0 91 72.2 35 27.8

Dukes’ Stage A 128 13.3 69 53.9 59 46.1 0.0006 B 325 33.7 210 64.6 115 35.4 C 386 40.0 280 72.5 106 27.5 D 126 13.0 91 72.2 35 27.8

Differentiation Well differentiated 102 10.3 61 59.8 41 40.2 0.0083 Moderate differentiated 786 79.1 545 69.3 241 30.7 Poor differentiated 105 10.6 59 56.2 46 43.8

MSI-IHC MSI-H 96 9.7 38 39.6 58 60.4 <0.0001 MSS 896 90.3 629 70.2 267 29.8

Smad4 Mutation Yes 52 12.6 43 82.7 9 17.3 0.0032 No 361 87.4 227 62.9 134 37.1

Smad4 FISH Monosomy/Deletion 577 60.8 427 74.0 150 26.0 <0.0001 Normal 372 39.2 215 57.8 157 42.2

Survival OS 5 Years 71.6 78.9 0.0068




24

Figure Legends

Figure 1. TGFβ induces Smad4 dependent apoptosis in CRC cells. (A) Smad4

expression in a panel of CRC cell lines. Proteins were isolated from nine CRC cell lines

and immunoblotted with antibodies against Smad4 and GAPDH. (B) Representative

images of fluorescence immunostaining for Smad4 in four-selected CRC cell lines and

confirm results from western blot analysis. (C) CRC cells were treated with indicated

doses of TGFβ for 48 hours and cell viability was measured by MTT assay (D-E) CRC

cells were treated with indicated doses of TGFβ for 48 hours and clonogenic assay

were performed. Cells (6x102) after post treatment were seeded into each of three

dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal

violet (E). Colony numbers in the entire dish were counted. (F) CRC cells were treated

with indicated doses of TGFβ for 48 hours and cells were analyzed for apoptosis by flow

cytometry. Data presented in the bar graphs are the mean ± SD of three independent

experiments. *Indicates a statistically significant difference compared to control with p <

0.05.

Figure 2. TGFβ induces EMT followed by apoptosis. (A) Cell morphology under

microscope after treatment with TGFβ at different time-periods. (B)

Immunofluorescence analysis of EMT marker E-cadherin after treatment with TGFβ at

different time-periods. (C) CRC cells were treated with TGFβ at the indicated times and

cells were stained with flourescein-conjugated annexin-V and propidium iodide (PI) and

analyzed by flow cytometry. (D) CRC cells were treated with TGFβ at the indicated




25

times and proteins were isolated and immunoblotted with antibodies against E-cadherin,

caspase-3, Smad4 and GAPDH.

Figure 3. Snail and Zeb1 promote EMT and apoptosis. (A-B) TGFβ induces

upregulation of Snail1 and Zeb1 expression in smad4 proficient cells but not in smad4

deficient CRC cells. CRC cells were treated with TGFβ for 48 hours and proteins were

isolated and immunoblotted with antibodies against Snail1, Zeb1 and GAPDH. (C)

Silencing of Snail1 and Zeb1 inhibits the TGFβ induced down-regulation of E-cadherin

in smad4 proficient cells. Smad4 proficient cells were transfected with Snail1 and Zeb1

shRNA’s and were treated with TGFβ for 48 hours. Proteins were isolated and

immunoblotted with antibodies against E-cadherin and GAPDH. (D) Cell morphology

under microscope after 48 hours of TGFβ treatment in smad4 proficient cells expressing

control shRNA’s, or shRNA’s targeting Snail1 and Zeb1. (E-F) Silencing of Snail1 and

Zeb1 inhibits the TGFβ induced apoptosis in smad4 proficient cell. Smad4 proficient

cells after transfection with Snail1 and Zeb1 shRNA’s were treated with TGFβ for 48

hours. Cells were stained with flourescein-conjugated annexin-V and propidium iodide

(PI) and analyzed for apoptosis by flow cytometry. Data presented in the bar graphs are

the mean ± SD of three independent experiments. *Indicates a statistically significant

difference compared to control with p < 0.05.

Figure 4. Sox4 is essential for TGFβ induced apoptosis. (A) Silencing of Sox4

inhibits the TGFβ induced apoptosis in smad4 proficient cells. Smad4 proficient cells

were transfected with Sox4 shRNA’s and were treated with TGFβ or TGFβ and 5-FU




26

combination for 48 hours and cells were analyzed for apoptosis. (B-C) Silencing of Sox4

has no effect on TGFβ induced EMT in smad4 proficient cells. Smad4 proficient cells

were transfected with Sox4 shRNA’s and were treated with TGFβ for 48 hours. (B) Cell

morphology was photographed under microscope. (C) Proteins were isolated and

immunoblotted with antibodies against E-cadherin, Sox4 and GAPDH. (D) Silencing of

Snail1 and Zeb1 has no effect on Sox4. Smad4 proficient cells were transfected with

Snail1 and Zeb1 shRNA’s and were treated with TGFβ for 48 hours. Proteins were

isolated and immunoblotted with antibodies against Sox4 and GAPDH. (E) Isolation of

spheroid-forming cells from Smad4 proficient cells. Sphere forming assay was

performed by culturing cells (5 x 102 cells/well) in sphere medium for 14 days in 24-well

ultra-low attachment plates. Stemness property of the isolated spheroids was verified

using cancer stem cell markers by immunoblotting. (F) Sox4 expression in spheroids

and adherent cells after TGFβ for 48 hours treatment (G-H) Silencing of Sox4 inhibited

self-renewal ability of spheroids. Smad4 proficient cells were transfected with Sox4

shRNA. After transfection, cells were subjected to sphere forming assay. Spheroids in

the entire well were counted. (I) Silencing of Sox4 inhibited stemness of spheroids as

confirmed by immunoblotting using stem cell markers. Data presented in the bar graphs

are the mean ± SD of three independent experiments. *Indicates a statistically

significant difference compared to control with p < 0.05.

Figure 5. Silencing of KLF5 increases apoptosis in Smad4 deficient CRC cells. (A)

Downregulation of KLF5 expression after TGFβ treatment in smad4 proficient cells but

not in smad4 deficient cells. Smad4 proficient and smad4 deficient cells were treated




27

with TGFβ for 48 hours. Proteins were isolated and immunoblotted with antibodies

against KLF5 and GAPDH. (B) Silencing of KLF5 sensitize smad4 deficient cells to 5-

FU with and without TGFβ treatment. Smad4 deficient cells were transfected with KLF5

shRNA’s. After transfection, cells were treated with 5-FU and TGFβ either alone or in

combination for 48 hours and analyzed for apoptosis. (C) Spheroids were generated

from Smad4 deficient cells and stemness of the isolated spheroids was verified using

cancer stem cell markers by immunoblotting. (D-E) Silencing of KLF5 decreases self-

renewal ability of spheroids. Smad4 deficient cells were transfected with KLF5 shRNA.

After transfection, cells were subjected to sphere forming assay. Spheroids in the entire

well were counted. (F) Silencing of KLF5 suppressed stemness of spheroids as

confirmed by immunoblotting using stem cell markers. (G) Overexpression of KLF5

inhibits TGFβ induced downregulation E-cadherin in smad4 proficient cells. Smad4

proficient cells were transfected with KLF5 cDNA and treated with TGFβ for 48 hours.

Proteins were isolated and immunoblotted with antibodies against E-cadherin, KLF5

and GAPDH. (H) Overexpression of KLF5 inhibits TGFβ-induced apoptosis in smad4

proficient cells. Cells after KLF5 overexpression were treated with TGFβ for 48 hours

and analyzed for apoptosis. (I) Depletion of KLF5 induces apoptosis whereas depletion

of Sox4 inhibits apoptosis after TGFβ treatment in smad4 deficient cells. Smad4

deficient cells were transfected with KLF5 and Sox4 shRNA’s. After transfection, cells

were treated with 5-FU and TGFβ for 48 hours and analyzed for apoptosis. Data

presented in the bar graphs are the mean ± SD of three independent experiments.

*Indicates a statistically significant difference compared to control with p < 0.05.




Published OnlineFirst May 3, 2019.Mol Cancer Ther Abdul K Siraj, Poyil Pratheeshkumar, Sasidharan Padmaja Divya, et al. in CRC

Induced SMAD4 Dependent Apoptosis Proceeded by EMTβTGF

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