1.

2.

Emily Cribas
Albert Einstein
July 7, 2015
College of Medicine,
NY
SSRP Journal Club
Harvard Medical

School
3. Ludwig Institute for
Cancer Research

Colon Cancer
Colon cancer
2nd leading cause
of cancer-related
death
Factors increase
risk
Diet

American Cancer Society

The colon
Absorbs vitamins
Stores and
eliminates waste
Breaks down
non-digestible
foods

Butyrate is Produced by Starch

Breakdown in the Colon

http://www.csiro.au/hungrymicrobiome/

Butyrate is Essential for Colon

Health
Main energy source of colon cells
Involved in cell differentiation and
proliferation
Useful in cancer differentiation
therapy
Done by histone deacetylase
inhibition

Differentiation Therapy Can Work as

a Cancer Treatment
Cancer cells stay in an immature state

http://www.csiro.au/hungrymicrobiome/

Butyrate Acts as a Histone

Deacetylase Inhibitor

ALPi is an Intestinal Differentiation

Marker
ALPi
Intestinal alkaline phosphatase
Has roles in:
Regulation of lipid absorption
Prevention of bacterial invasion
across the gut-mucus layer
Serves as a colon cell
differentiation marker
7

Butyrate Induces ALPi Gene

Expression
This relationship:
Occurs at the level of transcription
Requires new protein synthesis
Depends on a KLF/Sp regulatory
element in the ALPi promoter
Mechanism remains unclear

To further elucidate the mechanism by

which butyrate induces ALPi
expression in colon cancer cells

Q1: Which colon cells dont induce ALPi

expression by butyrate and which do?
Aim: figure out why or why not

10

Fig 1. Cell Lines Sensitive and

Resistant to Butyrate Induction of ALPi
were Identified

11

Q2: If sensitive cells have increased

ALPi activity with butyrate, is ALPi
protein amount increasing?

12

Fig 1C. ALPi Protein Amount

Confirms Sensitive and
Resistant Cell Identification

13

Q3: Does this ALPi protein increase

occur at the transcriptional (mRNA)
level?

14

Fig 1. Differential Induction of ALPi

Activity Correlates with ALPi mRNA
Expression

15

Q4: Does this increase in ALPi activity

in sensitive cells continue over time? Is
this a trend?

16

Fig 2. Induction of ALPi mRNA in a Sensitive

Cell Line Increased Over Time

17

Q5: Is gene induction by butyrate

specific to just ALPi or other HDACi
target genes as well?

18

Fig 2. Differential Induction of a

Gene by Butyrate is Specific to ALPi

19

Q6: Butyrate acts as an HDACi, so

another HDACi should exhibit the same
trend on ALPi induction

20

Fig 3. Results for Butyrate were

Similar to a Structurally Different
HDACi

21

Q7: HDACis should target other HDACi

target genes along with ALPi, unlike
butyrate

22

Fig 3. Results for Butyrate were

Similar to a Structurally Different
HDACi

23

Q8: Past literature says KLF/Sp

regulatory site is important for ALPi
promoter activity

24

Fig 4. Confirmed ALPi Promoter

Activity is Dependent on a
KLF/Sp Regulatory Element

25

Fig 4. Confirmed ALPi Promoter

Activity is Dependent on a KLF/Sp
Regulatory Element

26

Q9: Are the two types of cell lines

basally different in ALPi promoter
methylation?

27

Fig 5. KLF/Sp Element is Selectively

Methylated in Resistant Colon
Cancer Cell Lines

28

Fig 5. KLF/Sp Element is

Selectively Methylated in
Resistant Colon Cancer Cell Lines

29

Q7: Is methylation the sole cause of

differences in ALPi promoter activity?

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Fig 6. ALPi Gene Induction by Butyrate

is Dependent on Transcription
Factors

31

Q8: Which transcription factors affect

ALPi gene induction?

32

Fig 7. KLF5 is Differentially

Expressed Between Both Cell
Lines in Response to Butyrate

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Fig 7. KLF5 is Differentially

Expressed Between Both Cell Lines
in Response to Butyrate Over Time

34

Fig 7. KLF5 is Differentially Expressed

Between Both Cell Lines in Response
to Butyrate

35

Fig 7. KLF5 is Basally Differentially

Expressed Between Both Cell Lines
Overall

36

Q9: How important is this factor (KLF5)

in ALPi gene induction?

37

Fig 8. KLF5 Overexpression Induces

ALPi Promoter Activity

38

Fig 9. KLF5 Knockdown Attenuates

Basal and Butyrate Induction of ALPi
Expression

39

Conclusions
KLF5 is a key regulator of butyrate
induction of ALPi in colon cancer cells

40

Critique
Redundant
Methods were a little unclear
Couldve used newer methods

Some figures require more

explanation
Only tested one differentiation
marker

41

Future Work
Explore other transcriptional factors
that might play a role in ALPi
expression
Test relationship between butyrate
and differentiation phenotype
Look genome-wide

42

Fig 8. KLF5 Binds to The KLF/Sp

Site and Increases
Transcription of ALP

43