Introduction and bronchus, and colon and rectum with prostate

Introduction

The
American Cancer society looks at the number of cases of Cancer in North America
every year and compiles the data to provide statistics for the coming year. At
the beginning of 2017, the three types of cancer expected to be most commonly
diagnosed in women was breast, lung and bronchus, and colorectal. Breast cancer
was expected to account for 30% of all new cancer diagnoses in women. In men
the cancers to be most commonly diagnosed were prostate, lung and bronchus, and
colon and rectum with prostate expected to account for 19% of new cases. (Siegel
et al., 2017). In Ireland, Cancer is the second biggest killer with
over 9,000 deaths a year. (Irish Cancer Society, 2017). Focusing on Breast,
Colon and Prostate Cancer as they are the leading killers and most studied, we
see that an increased amount of knowledge could lead to better therapies. Studies
have shown PDZ_LIM proteins are highly associated with regulating certain
cancers. PDLIM2 is a protein coding gene also known as Mystique which is
located on the 8p21.2 chromosome which encodes the PDZ_LIM protein. Only one
protein isoform, Mystique 2 was found in cell lines. This protein has
numerous  amounts of cellular functions
including cell migration and adhesion, epithelial mesenchymal transition (EMT)
and cell polarization. (Loughran
et al., 2005)

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Main Body

PDLIM2
and Breast Cancer

It
has been shown that NF-?B (nuclear factor kappa-light-chain-enhancer of
activated B cells) plays a major role in Breast Cancer and that PDLIM2 is
majorly repressed in breast cancer cells. It was therefore hypothesised and
proven that PDLIM2 may be involved in breast cancer pathogenesis. NF- ?B is a proinflammatory
signalling pathway which is due to the nuclear factors high expression in
cytokines, adhesion molecules and chemokines which are all proinflammatory
genes (Lawrence,
2009). NF-?B has other functions also including cell
proliferation and expressing I?B?, the activated I?B? then enters the nucleus
and transports the active NF-?B back to the cytoplasm. P65 is a member of NF-?B
 and was shown to play a role in breast
cancer development and progression allowing it to resist therapeutic treatments
of cancer. PDLIM2 was found to target P65 specifically for polyubiquitination-mediated
proteasomal degradation which in turn terminates the activation of NF-?B. It
has been shown that the C-terminal Lim domain is responsible for promoting the
ubiquitination of P65 and that the N-terminal PDZ domain is responsible for
transporting the P65 into intranuclear compartments for the proteasomal
degradation. The activation of NF-?B isn’t well
understood but it’s thought that PDLIM2 plays a role. It was found that when
PDLIM2 was expressed, NF-?B activation was inhibited as well as malignant tumor
cells tumor formation in vivo and anchorage-independent growth in vitro. PDLIM2
mutants which lost the NF-?B inhibiting ability also lost the ability to
suppress breast cancer. The protein level of PDLIM2 was tested in many breast
cancer cell lines including the two most used model human breast cancer cell
lines, MCF-7 and MDA-MB-231. It was proven that the breast cancer cell lines
express much less PDLIM2 irrespective of ER expression in comparison with the
nontumorigenic breast cells, HMT3522 and MCF10A, (Fig 1A) (Qu
et al., 2010a). A study also showed that the PDLIM2 repression was
actually at the mRNA level in colon cancer (Yan
et al., 2009). Qu et al. also discovered a PDLIM2 decrease at the
mRNA level in breast cancer cell lines compared to normal cells, (Fig 1B).

As
mentioned above, PDLIM2 expression inhibited NF-?B activation therefore the
data suggested that the repression of PDLIM2 may then take part in the breast
cancer cell NF-?B activation. A luciferase gene reporter assay was carried out and
showed that PDLIM2 which when transiently expressed provided a dose dependant
suppression of activated NF-?B. A permanent expression of PDLIM2 resulted in
inhibition of NF-?B activation and inhibited Bcl-2 and cyclin D1 expression,
these are NF-?B targeted genes in which are associated with tumors and found in
the cell lines used to carry out the assay. As PDLIM2 inhibits NF-?B activation
by transporting p65 for proteasomal degradation, the impact that PDLIM2 has on
the expression of p65 was examined. MDA-MB-231 cells were used to carry this examination
out. PDLIM2 overexpression caused the p65 expression to decrease in the soluble
nuclear area but caused an increase of p65 in the insoluble nuclear area.
MG132,  a cell permeable proteasome inhibitor
was used to treat the cells resulting in a build up of p65 at the insoluble
fraction and a slight p65 recovery in the soluble fraction. The MG132 caused a
decrease in PDLIM2 in the nuclear soluble fraction but and increase in the
insoluble. This showed that PDLIM2 cannot be released until the proteasomal degradation
of p65 is carried out. These results further the suggestion of the repression of
PDLIM2 being part of the activating mechanism of NF-?B in human breast cancer
cell lines.

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PDLIM1
in Breast Cancer

 

PDLIM2
in Colon Cancer

The
risk of colon cancer is increased with chronic inflammatory disorders like inflammatory
bowel diseases including ulcerative colitis and Crohn’s Disease. These result
in constant recurring inflammation elevating the risk of colorectal cancer
causing it to be the second most common source of cancer related deaths (Burstein
and Fearon, 2008). The activation of the NF-?b transcription factor
also plays a major role in colon tumorigenesis and colon inflammation. PDLIM2 a
terminator of the activation of NF-?b and was proven to be repressed in human
colorectal cancer cell lines in comparison to a non-cancer cell line (Figure
1A), methylation is involved in the repression of the PDLIM2 protein. The
activity of NF-?b is closely regulated under physiological conditions (Xiao,
2007). PDLIM2 inhibits NF-?b activation as mentioned above
which leads to tumour supressing activity, demonstrated through the decreased
expression of PDLIM2 in colon cancer cells. How PDLIM2 is repressed was
suggested by linking DNA methylation regulation and PDLIM2 expression. A DNA
methyltransferase inhibitor (5-aza-dC) replenished the PDLIM2 expression in
colon cancer cell lines suggesting that DNA methylation is responsible for
PDLIM2 repression (Fig. 1B). More data from a bisulfite genomic DNA sequencing furthered
the belief that methylation is directly responsible for the expression of
PDLIM2 (Qu
et al., 2010b). 5-aza-dC is toxic to cancer cells and is non-toxic
to normal cells which makes it a hopeful therapy for colon cancer (Garcia-Manero,
2008).

 Qu et al. then looked at PDLIM2 re-expression
and its relationship with NF-?b activation in colon cancer cells. Luciferase gene
reporter assays were carried out to research how the restoration of PDLIM2
effects NF-?b activation in these colon cancer cells. The re-expression of
PDLIM2 caused a dose-dependent suppression of the NF-?b activation in a number
of colon cancer cell lines, this was linked with a decreased expression of p65.
Further studies showed that similarly to the p65 in breast cancer cell lines, PDLIM2
is responsible for the shuttling of p65 for proteasomal degradation in the
nuclear matrix. The data again implies that the repression of PDLIM2 is a
contributing mechanistic factor of NF-?b activation but this time, also in colon
cancer cells. To look further at PDLIM2 re-expression, stable expression of
PDLIM2 was done in HCT116 and DLD1whcih are colorectal cancer cell lines. The
soft agar colony formation activity was measured compares to a control. The
cell lines which expressed PDLIM2 formed smaller and less colonies in the soft
agar. This showed that in colon cancer cells, tumorigenicity is supressed by
PDLIM2 in vitro. To establish that the same would happen in vivo, a control and
colon cancer cells expressing PDLIM2 were injected subcutaneously into differing
flanks of  SCID mice. As you can see in Figure
3 below, the control and PDLIM2 both developed tumors at the site of the
injection but the tumors at the site including PDLIM2 were noticeably smaller
which indicates that PDLIM2 does suppress colon cancer tumorigenicity in vivo.

 

PDLIM2
in Human Castration-Resistant Prostate Cancer

Prostate
cancer is the most common occurring cancer in males, it accounts for nearly 1
in 5 new diagnoses in males. The rate of cases is dropping due to an increase
in prostate-specific antigen (PSA) testing (Siegel
et al., 2017). Contrary to above where the ability of PDLIM2 to
suppress tumors was mentioned, in this case, a prostate cancer cell line
(DU145) showed a high level of expression of PDLIM2. It’s clear that PDLIM2 has
a multifunctional role amongst various cancers.

 

Discussion