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Mitotic Stress in Cancer: Tipping the Fine Balance
SUSANTA ROYCHOUDHURY
Saroj Gupta Cancer Center and Research Institute
Former, CSIR-Indian Institute of Chemical Biology
Kolkata
81st Annual Meeting,
Indian Academy of Sciences
6-8 November 2015
IISER, Pune
THEODOR BOVERI (1862-1915)
Born
12 October 1862
Died
15 October 1915
Nationality German
Fields
Genetics, Cell biology
Embryonic development
Known for Boveri-Sutton chromosome theory
Centrosome
He reasoned in 1902 that a cancerous tumor
begins with a single cell in which the makeup of
its chromosomes becomes scrambled, causing
the cells to divide uncontrollably. He proposed
carcinogenesis was the result of aberrant mitoses
and uncontrolled growth caused by radiation,
physical or chemical insults or by microscopic
Pathogens.
Wikipedia
Chromosomal Abnormalities in Human Cancer
Science, 19 August 2011
Cell Cycle and Mitosis
The Spindle Assembly Checkpoint (SAC)
Metaphase
Anaphase
Bub3
BubR1
C-M2
APC/C
Cdc20
Securin
Separase
C-M2: Closed Mad2
CyclinB
CDK1
Adapted from Silva et al., Cell Proliferation, 2011
Fine Balance is the Key
“…..few cancer-associated mutations in these or other
mitotic regulators have been described thus far and many
of these molecules do not fit into the classical definition
of oncogenes or tumor suppressor genes.
In
some
expression
cases,
of
both
these
over-expression
genes
result
in
and
decreased
mitotic
arrest.
Moreover, some mitotic regulators such as MAD2 are
either up- or down-regulated depending on the tumor
types and, in both cases, these alterations result in
chromosomal imbalances and tumor development.”
Perez de Castro et al., Carcinogenesis, 2007
Agenda
 Specific molecular defects in SAC leads to aneuploidy in cancer
 Chromosome abnormality in cancer cells can be exploited
for its own destruction.
Overexpression of Cdc20 Causes Aneuploidy
Cdc20
low
Cdc20
high
Cdc20
low
Cdc20
high
Comparison between -Noc and +Noc: p= 0.2671 for Cdc20 low; p= 0.0063 for Cdc20 high
Mandal et al, Carcinogenesis, 2006
How does the SAC Switch Function?
IN PROMETAPHASE
PLK1
Ub
Ub
Bub3
P
Mad2
BubR1
Cdc20
APC/C
UbcH10
P
CDK1
Co-regulated overexpression of Cdc20 and UbcH10 causes
chromosome non-disjunction
Cdc20
Cdc20
Excess
Cdc20
Chromosome
Non-disjunction
Spindle assembly
checkpoint active
APC/C
Active APC/C
UbcH10
UbcH10
A novel transcription regulatory function of APC/CCdc20 complex
APC/C
Cdc20
E2F1
CBP/p300
DP1
+1
Ac
Ac
UBCH10
Response element
UBCH10 promoter
Nath et al, J. Biol. Chem, 2011
Nath and Chowdhury et al, Mol Cell Biol (2015)
Forcing premature cell division
Control
Control
+Noc
Cdc20
+Noc
UbcH10
+Noc
E2F1
+Noc
pCDNA5-CDC20
transfected
pCDNA3E2F1
transfected
pCS2-UBCH10
transfected
Mock
transfected
Deregulation
of Cdc20-E2F1
mediated
UBCH10 expression
leads
to ANEUPLOIDY
Premature
chromosome
segregation
causes
aneuploidy
Proposed mechanism for aneuploidy
APC/C
CBP/p300
E2F1
DP1
E2F1
DP1
Cdc20
Cdc20
+1
Rb
Response element
UbcH10
UbcH10
UBCH10 promoter
UbcH10
p53
UBCH10
UbcH10
UbcH10
Premature
anaphase entry
Spindle assembly checkpoint active
Banerjee et al, Nucleic Acid Res (2009) Nath et al, J. Biol. Chem (2011)
Aneuploidy
Nath and Chowdhury et al, Mol Cell Biol (2015)
SAC as a system
??
Transcriptional
regulation
Regulation of
SAC proteins
Epigenetic
regulation
Micro RNA Biogenesis and Function
miR-125b induces chromosomal instability
in a Mad1-specific manner
UPCI:SCC084
Excess miR-125b decreases cell viability
120
800
100
600
Percent viability
Number of colonies
700
500
400
300
200
Empty vector
60
miR-125b
40
miR125b+Mad1
20
100
0
pRNAU6.1miR-125b
(mg)
80
0
0.25
0.5
1
2
0
Hours
0
after
transfection
UPCI:SCC084
24
48
72
90
Proposed model illustrating miR-125b mediated
regulation of SAC and cell fate
miR-125b
miR-125b
MAD1
MAD1
Mad1
Mad1
TEMPORARY SAC ON
SAC OFF
MITOTIC DELAY AND CIN
CELL DEATH
PREMATURE MITOTIC EXIT AND CIN
CELL PROLIFERATION
Bhattacharjya et al., Cell Death Differ., 2012
Witheferin A kills cancer cells by degrading Spindle Assembly Checkpoint complex
Das et al., Biochem Pharmacol., 2014
SAC inactive
Mitotic
delay
Premature
anaphase entry
Level of aneuploidy
SAC hyperactive
Death
Aneuploidy
Survival
Cell death
Cell death
Cell
proliferation
Cell
proliferation
Summary

Mechanistic insight into the cause of chromosomal instability in cancer
through SAC deregulation has been generated.

A novel transcriptional role of APC/CCdc20 Complex has been established.

Two tumor suppressor genes RB and p53 have been implicated in
SAC regulation.

miRNA mediated SAC regulation has been documented.

SAC proteins as potential target for the development of anti-cancer
biologics and naturally occurring small molecules has been demonstrated.
PAST PhD STUDENTS & RESEARCH ASSOCIATES
Sumana
Tania
Sanjib
Damayanti
PRESENT PhD STUDENTS AND RESEARCH ASSOCIATES
Arindam
kayum
Debrup
Abhishek
Kumar
Dishari
Chetan
Pijush
Sangeeta
Ruma
Acknowledgement
The Basic Scientist Collaborators:
The Clinical Collaborators:
Dr. Chinmay K. Panda, CNCI
Prof. Nitai P. Bhattacharyya, SINP
Prof Partha P. Majumder, NIBMG
Prof. Bidyut Roy, ISI
Dr. Shantanu Chowdhury, IGIB
Dr. Dibyendu Bhattacharyay, ACTREC
Dr. Saurabh Ghosh, ISI
Prof. Siddhartha Roy, IICB
Dr. Santu Bandyopadhyay, IICB
Dr. Hemanta K. Majumder, IICB
Prof. Dev Mukhopadhyay, Mayo Clinic
Dr. Maitrayee Dasgupta, CU
Dr. Sanghamitra Sengupta, CU
Dr. Arunabha Sengupta
Dr. Arun Roy
Dr. Jayanta Chakrabarty, CNCI
Prof. Abhijit Chowdhury, IPGMER
Dr. Mrinalini Moghe, DMH
Dr. Sultan Pradhan, PAKH
Financial support:
CSIR
DBT
DST
ICMR
WBDST
 At the end of 1951 some 40,000 men and women on
the British Medical Register replied to a simple questioner
relating to their smoking habits.
Sir Richard Doll
(1912-2005)
 On that basis they were divided into non-smokers and
three groups of smokers (including ex-smokers) according
to the amount they smoked at that time
(or when they gave up).
 This preliminary report is confined to the deaths among
the 24,389 men over the age of 35.
A. Bradford Hill
(1897-1991)
 Though the numbers of deaths at present available
are small the resulting rates reveal a significant and
steadily rising mortality from deaths due to cancer of
the lung as the amount of tobacco smoked increases.
Aneuploidy Caused CIN
or
CIN Caused Aneuploidy ?
Experiments:
+ chr. 3
1. MIN+
Stable (2n+1) cells
2. MIN+ x MIN+
Fused stable cell (4n)
3. CIN+ x CIN+
Fused cell, unstable
4. MIN+ x CIN+
Fused cell: MIN- : CIN+
•Abnormal no. of chrs. in a cell by itself could not account for high level of
CIN found in the aneuploid cells
•CIN phenotype may be acting dominantly at the cellular level.
Expression analysis of ‘mitotic hits’
Mutation analysis of ‘mitotic hits’
2-7% mutation
5%
Underexpressed
1%
Overexpressed
15%
1-2%
mutation
11%
Data not found
1%
< 1% mutation
84%
Unchanged
83%
Total mitotic genes analyzed
in 8 HNSCC datasets
557
Total mitotic genes
analyzed
525
Genes over expressed
82
Genes with <1% mutation
440
Genes under expressed
8
Genes with 1-2% mutation
60
Genes with 2-7% mutation
25
Genes that remain unchanged
461
No data obtained
6
Cancer Gene Census
Catalogue of Somatic mutations in cancer (COSMIC) v74 Release
Slide updated 14 October 2015
Other Statistics
Total Number
Somatic Mutation
534
Germline Mutation
88
Total Genes
573
Source: http://www.sanger.ac.uk/genetics/CGP/Census/
PARADOX




Spontaneous mutation frequency for human is 1.4x10-10
nucleotides/cell/division or 2.0x10-7 mutations/gene/cell division.
Clonal theory suggests that cancer arise in one or, few cells.
Thus, only two or three mutations per tumor are accountable from
background mutation frequency calculation.
In contrast, four to eight mutations in growth regulatory genes were
observed in most tumors.
Cancer cells show increased genomic instability.
Loeb LA, Cancer Res. 34: 2311 (1974)
Arial view of carcinogenesis
Vogelstein et al, Science, 339, 1546 (2013)
DNA repair Defects and Human Cancer
CBP/p300
E2F1
E2F1
APC/C
Cdc20
Cdc20
+1
UBCH10
Rb
p53
UbcH10
Premature
anaphase entry
Spindle assembly
checkpoint active
Nath and Chowdhury et al, Mol Cell Biol (2015)
Aneuploidy
Banerjee et al, Nucleic Acid Res (2009)