The relationship between Calprotectin and Gastric Cancer

1. NEW INSIGHT INTO GASTRIC
CANCER
By: Samieh Asadian
Ph.D Candidate of Molecular Medicine
School of Medicine, QUMS
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2. CALPROTECTIN AND INFLAMMATION
 Transition metals are necessary for all forms of life including microorganisms, evidenced
by the fact that 30% of all proteins are predicted to interact with a metal cofactor.
 Through a process termed nutritional immunity, the host actively sequesters essential
nutrient metals away from invading pathogenic bacteria.
 Neutrophils participate in this process by producing several metal chelating proteins,
including lactoferrin and calprotectin (CP).
 As neutrophils are an important component of the inflammatory response directed
against the bacterium Helicobacter pylori, a major risk factor for gastric cancer, it was
hypothesized that CP plays a role in the host response to H. pylori.
 Utilizing a murine model of H. pylori infection and gastric epithelial cell co-cultures, the
role CP plays in modifying H. pylori -host interactions and the function of the cag Type IV
Secretion System (cag T4SS) was investigated.
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3.  Studies indicates elevated gastric levels of CP are
associated with the infiltration of neutrophils to the H.
pylori-infected tissue.
 In vitro data demonstrate that culturing H. pylori with
sub-inhibitory doses of CP reduces the activity of the
cag T4SS and the biogenesis of cag T4SS-associated
pili in a zinc-dependent fashion.
 Taken together, these data indicate that zinc
homeostasis plays a role in regulating the
proinflammatory activity of the cag T4SS.
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4.  The cag T4SS is a macromolecular assembly that is responsible for translocating the
oncogenic effector molecule, CagA and peptidoglycan, into host cells.
 These translocated effectors elicit a variety of host cell responses, including activation
of nuclear factor kB (NFkB) and secretion of the proinflammatory cytokines, IL-1b and
IL-8.
 gastric CP levels are elevated in H. pylori-infected humans and rodents, and that most
of the CP localizes to neutrophils in H. pylori-infected tissues.
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5.  CP affects growth and viability of H. pylori
 CP has been demonstrated to inhibit bacterial growth via sequestration of nutrient
manganese and zinc.
 CP affects H. pylori colonization and H. pylori-induced
inflammation in a mouse model.
 Because CP can inhibit the growth of H. pylori in vitro, there is hypothesized that this host
protein would also contribute to control of the bacterial burden in vivo.
 CP inhibits activity of the H. pylori cag T4SS.
 A functional cag T4SS translocates the effector molecule, CagA, into host cells, where it
is then phosphorylated.
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6. NFKB ACTIVATION AND IL-8 SECRETION IN RESPONSE TO CO-CULTURE WITH H. PYLORI IS DEPENDENT ON ZINC AVAILABILITY. BACTERIA WERE GROWN IN MEDIUM ALONE OR
MEDIUM SUPPLEMENTED WITH THE SYNTHETIC ZINC CHELATOR TPEN (TPEN), WILD-TYPE CP (CP) AT 200 MG/ML, OR MUTANT FORMS OF CP [DS1 OR DS2 AT 200
MG/ML OR 600 MG/ML, OR A DOUBLE-SITE MUTANT (DS) AT 1200 MG/ML] ALONE OR IN THE PRESENCE OF 100 MM ZINC CHLORIDE (+ZINC) PRIOR TO CO-CULTURE WITH
AGS CELLS. A) NFKB ACTIVATION IN HUMAN GASTRIC EPITHELIAL CELLS CO-CULTURED WITH H. PYLORI FOR 4 HOURS WAS QUANTIFIED USING A LUCIFERASE REPORTER
ASSAY. B) IL-8 SECRETION BY HUMAN GASTRIC EPITHELIAL CELLS WAS QUANTIFIED USING AN IL-8 ELISA ASSAY. BARS REPRESENT THE MEAN +/2 SEM PER EACH GROUP
(N = 3–5 BIOLOGICAL REPLICATES). ASTERISKS INDICATE *P,0.05, ** P,0.01, *** P,0.001, **** P,0.0001, COMPARED TO MEDIUM ALONE (STUDENT’S T TEST).
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7.  Metal sequestration by CP inhibits cag T4SS pilus biogenesis.
 co-culture experiments demonstrated that metal sequestration by CP leads to abrogated
phosphorylation of CagA and inhibition of downstream cellular activation in gastric
epithelial cells.
 Results from these studies indicate that zinc homeostasis
plays an important role in regulating the cag T4SS in H. pylori.
 Previous reports have shown that when H. pylori interacts with gastric epithelial cells,
the cag T4SS translocates CagA into host cells leading to activation of c-Src and
changes in the cytoskeleton.
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8.  A functional cag T4SS also increases the activation of NFkB , which ultimately results
in the production of IL-8 and the recruitment of neutrophils.
 In response to H. pylori infection, neutrophils are recruited to the site of infection. CP is
deposited, and nutrient manganese and zinc are sequestered.
 Sequestration of zinc by CP represses cag T4SS pilus formation and CagA
translocation, and results in diminished NFkB activation and IL-8 secretion.
 With reduced IL-8, less inflammation develops and the end result of CP-dependent zinc
sequestration is increased bacterial persistence.
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9.  These data suggest that whether CP is present or absent, there is cross
regulation between the bacteria and the host immune response, leading to a
level of inflammation which controls bacterial burden but does not necessarily
induce enough inflammation to completely clear the infection, and therefore,
the bacteria persist.
 In addition to indicating that CP restricts the growth of H. pylori, our work
reveals that CP represses the activity of the cag T4SS, an important virulence
factor that has been associated with carcinogenesis.
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10. H. PYLORI CAG T4SS PILUS PRODUCTION IS MODULATED BY THE ZINC
SEQUESTRATION ACTIVITY OF CALPROTECTIN.
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11. THE NUCLEAR FACTOR ΚB PATHWAY: A LINK TO THE
IMMUNE SYSTEM IN THE
RADIATION RESPONSE
 Exposure to ionizing radiation modulates immune responses in a
complex dose-dependent pattern, with possible anti-inflammatory
effects in the low dose range, expression of pro-inflammatory cytokines
at moderate doses and immunosuppression after exposure to higher
doses due to precursor cell death together with concomitant
exacerbated innate immune responses.
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12.  A central regulator in the immune system is the transcription factor Nuclear Factor κB
(NF-κB).
 NF-κB is involved in the regulation of cellular survival,immune responses and
inflammation, resulting in eminent importance in cancerogenesis.
 At high radiation doses, products released fromdamaged or dying cells represent
damage-associated molecular patterns (DAMPs) and bind to pattern recognition
receptors (PRR) such as TLR , resulting in NF-κB activation and production of
proinflammatory cytokines and type I interferon (IFN).
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13. THE GENOTOXIC STRESS INDUCED NF-ΚB SUB-PATHWAY. THIS PATHWAY STARTS IN THE CELL NUCLEUS WITH ACTIVATION OF ATM AND
PARP-1 BY IONIZING RADIATION INDUCED DNA DSB. A NUCLEOPLASMIC SIGNALOSOME FORMS, WHICH CONTAINS ATM AND NEMO;
FACULTATIVE BINDING PARTNERS ARE PIDD AND RIP1. PIASY SUMOYLATES NEMO IN THE COMPLEX. SUMOYLATED NEMO AND
ATM SHUTTLE TO THE CYTOPLASM, WHERE THEY ASSEMBLE THE IKK COMPLEX INCLUDING ELKS. PHOSPHORYLATION OF IΚB BY IKK
RESULTS IN IΚB UBIQUITINATION AND SUBSEQUENT PROTEASOMAL DEGRADATION. THE LIBERATED NF-ΚB, IN CASE OF IONIZING
RADIATION MOSTLY A P50:P65 HETERODIMER, TRANSLOCATES TO THE NUCLEUS AND ACTIVATES TARGET GENE EXPRESSION.
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14. NFKB AND EMT
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15. HELICOBACTER PYLORI-INDUCED CELL DEATH IS
COUNTERACTED BY NF-ΚB-MEDIATED TRANSCRIPTION
OF DARPP-32
 DARPP-32 is a frequently amplified and overexpressed gene that promotes several
oncogenic functions in gastric cancer.
 Researcher investigated the relationship between Helicobacter pylori infection,
proinflammatory NF-κB activation and regulation of DARPP-32.
 H. pylori infection increased the DARPP-32 mRNA and protein levels in gastric cancer
cell lines and gastric mucosa of mice.
 H. pylori infection increased the activity of NF-κB reporter and p-NF-κB (S536) protein
level in vitro and in vivo.
 Immunohistochemistry analysis demonstrated a significant positive correlation between
NF-κB and DARPP-32 expression levels in gastric cancer tissues.
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16.  Given the high frequency of DARPP-32 overexpression and its
prosurvival oncogenic functions, the induction of DARPP-32
expression following H. pylori infection and activation of NF-κB
provides a link between infection, inflammation and gastric
tumourigenesis.
 H. pylori induces DARPP-32 expression in gastric epithelial cells in vitro
and in vivo.
 NF-κB transcriptionally upregulates DARPP-32 expression.
 H. pylori regulates DARPP-32 promoter activity and mRNA
expression through NF-κB-P65 binding.
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17. MICRORNAS IN TUMORIGENESIS, METASTASIS, DIAGNOSIS AND
PROGNOSIS OF GASTRIC CANCER
 MicroRNAs (miRNAs), which are small single-stranded RNA molecules
composed of 18–20 nucleotides, act as oncogenes or tumor suppressor
genes playing important roles in tumor formation, Infiltration and metastasis.
 In gastric cancer, many studies have detected abnormal expression forms of
miRNAs and confirmed their involvement in its tumorigenesis, progression,
invasion and metastasis.
 They may become valuable diagnostic markers and therapeutic targets for
gastric cancer.
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18. MIRNAS AND TUMORIGENESIS OF
GASTRIC CANCER
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19. MIRNAS AND THE INVASION AND METASTASIS OF
GASTRIC
CANCER
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21. FUTURE DIRECTIONS
o In the past decade, important advances regarding the expression, structure and
signalling of S100 proteins have improved our understanding of normal cell physiology as
well as the pathophysiology of cancer.
o The development of molecular probes — such as antibodies and small-molecule
inhibitors — will be instrumental for deciphering the in vivo functions of specific S100
proteins, as well as distinguishing the contribution of intracellular and extracellular S100
proteins.
o Moreover, these probes may also have therapeutic potential for cancer or other diseases.
o Despite considerable progress in S100 protein biology, we currently have little
information on how post-translational modifications or heterodimer formation affect S100
signalling.
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22. Finally …
 A mechanistic examination of both S100 protein
biology and biochemistry will be required to define
how each family member contributes to the
proliferation, metastasis, angiogenesis and immune
evasion of cancers and other diseases.
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