it is about the transcription factor family KLF4 and their expression in the colon cancer at different stage.which is help full in diagnosis of such type of cancer

1. Altered expression of
the KLF4 in colorectal
cancers
Byung Joon Choi et. al.,2006
By
Avinash tiwari
M. Tech 1st year
201710902010002

2. Introduction:
• The mammalian gastrointestinal epithelium is a dynamic system in
which cell proliferation is coupled to differentiation.
• Aberrations in this process frequently lead to colorectal cancer.
• colorectal cancer is one of the leading primary cancers

3. cause
Minor reason
(inherited predisposition)
Hereditary non-polyposis
c.c.
familial adenomatous
polyposis
Major reason
Somatic mutation
or
Altered expression of
tumor- associated protein

4. KLF-4 in colorectal cancer:
• Kru¨ppel-like factor 4 (KLF-4) is family of zinc finger containing
transcription factor
• Highly expressed in epithelial of gastrointestinal tract, skin and
vascular endothelial cells.
• Play a important roll during the differentiation of gut epithelial cell.
• KLF4 activates the promoter of the negative cell-cycle-regulatory
cyclin-dependent kinase inhibitor p21WAF/Cip1 gene in a p53-
dependent manner

5. • Induces cell cycle arrest at the transition from G1 to S phase
• Altered expression of KLF4 may be involved in the tumorigenesis of
several kinds of human cancers, including colon, breast, gastric,
esophageal, and bladder cancers.
• the level of KLF4 and the KLF4 mRNA expression is significantly
decreased in colonic adenomas & in sporadic colonic adenomas and
carcinoma patients with familial adenomatous polyposis when
compared with normal colonic tissues

6. Fig.- different function of KLF4
(form research gate)

7. Material and method:
Method & material
Tissue samples
Immunohistochemistry
for the KLF4:

8. A. Tissue samples
1.
• Formalin-fixed and paraffin-embedded samples were
obtained from surgical resections of 123 colorectal cancers
2.
• Classification of tumor according Duke’s criteria.
3.
• There were 12, 47, 56, and 8 cases with stage A, B, C, and
D, respectively.

9. Tissue samples cont.
4.
• Screened the histological sections and selected areas of the
representative tumor cells.
5.
• Tissue cores were taken from each tumor sample and placed in a
paraffin block using a commercially available microarray instrument
6.
• One cylinder of normal colonic mucosa adjacent to each tumor was
also transferred to the recipient block.

10. B. Immunohistochemistry for the KLF4:
1.
• 2 mm sections were cut the day before use and stained
according to standard protocols.
2.
• To maximize the signal on immunohistochemistry, two
strategies were used
=
• antigen retrieval in citrate buffer
• Signal amplification with biotinylated tyramide

11. 3.
• heat-induced epitope retrieval was conducted in citrate buffer (pH
6.0) and boiling the buffer for 30 min and cool
4.
• rinsing with PBS, the slides were treated with 1% H2O2 in PBS for 15
min at room temperature to abolish endogenous peroxidase activity.
5.
• washing with TNT buffer (0.1 mol/L Tris-HCl, pH for 20 min the slides
were treated with TNB buffer.
6.
• incubated overnight at 4 degree C with the antibody (1/100 dilution)
for KLF4 protein

12. 7.
• detection was carried out using biotinylated goat anti-rabbit antibody,
followed by incubation with peroxidase-linked avidin–biotin complex.
8.
• Diaminobenzidine was used as chromogen, and the slide was
counterstained with Mayer’s hematoxylin.
9.
• As negative controls, the slide was treated by replacement of primary
antibody with non-immune serum.

13. Results:
Immunohistochemistry for the KLF4:
• In immunohistochemistry, normal colonic mucosa showed moderate
to strong expression of KLF4 protein mainly in the nucleus of the
glandular epithelial cells.
• Inflammatory cells, including lymphocytes
in lamina propria of colonic mucosa, also
Demonstrated focal, weakly positive staining
Colorectal normal cells

14. • Expression of KLF4 in tumor cells shows Moderate to strong
immunopositivity for KLF4 protein was clearly marked on the nucleus
of colorectal tumor cells compared with normal colonic mucosa.
• Colorectal cancer, well differentiated
adenocarcinoma, displayed moderate
to strong immunostaining in nucleus
of cancer cells.
Colorectal cancer stage B

15. • Another cancer showed immunonegativity for the KLF4 protein in the
nucleus of the cancer cells
• KLF4 protein was detected in 30 (24.4%)
of 123 colorectal cancers
Colorectal cancer stage C

16. • KLF4 protein was detected in 30 (24.4%) of 123 colorectal cancers
• KLF4 expression was detected in 100% (6/6),73.2%
(82/112), and 100% (5/5) of well, moderately,and poorly
differentiated colorectal cancers
• In this classification, the expression of the KLF4 protein
was not associated with the differentiation of tumor
cells (w2 test, P40.05).
• Loss of KLF4 expression was seen in 4 (33.3%) of 12
cases corresponding to stage A, 9 (19.1%) of 47 to
stage B, 16 (28.6%) of 56 to stage C, and 1 (12.5%) of 8
to stage D.
• In addition, KLF4 expression was lost in 16 (27.1%) of
59 cases with lymph node metastasis.
• There was a tendency that loss of KLF4 expression
was more common in left-sided colon cancer (27.5%)
than in right-sided colon cancer (12%).