1477

Introduction
The traditional way to diagnose tumors is by histopathology
stains and analysis. The diagnosis of cancer relies primarily
on invasive tissue biopsy [1]. The conventional
histopathology based on light microscopy, however, has
recently been complemented with ultrastructure, immunohistochemistry
and molecular diagnostics [2].
Cancer of the uterine cervix is the most common form of
cancer in women developing countries as leading cause of
cancer-related deaths in women in the world as a whole
[3]. Cervical cancer is stay as the first for cancer caused
death in Indonesia and still be a problem of health [4].
There are four human viruses that cause cancer in human.
There are papilloma viruses (PV), epstein barr viruses
(EBV), hepatitis B virus (HBV), and kaposi sarcoma herpes
virus (KSHV) [3].
The E7 protein binds to the underphosphorelated form of
the tumor suppressor protein pRb and displaces the E2F
transcription factor that is normally bound by pRb
[3,5,6].
The latent membrane protein-1 (LMP-1) of EBV prevents
apoptosis of B cells by up regulating the expression of bcl2, and it activates growth promoting pathway that are normally
triggered by T cell-derivated signal [3,5,6].

etm
Introduction

Cervical cancer is the third most commonly diagnosed cancer and the fourth leading cause of
female mortality worldwide, with >85% of the associated deaths occurring in developing
countries (1). The American Cancer Society estimates ~12,360 new cases of invasive cervical
cancer and ~4,020 mortalities in the United States for 2014 (2). The one-year survival rate for
females with cervical cancer is 87% and the five-year survival rate is 68%. When detected
early, the five-year survival rate for patients with invasive cervical cancer is 91% (3).
Cervical cancer develops slowly, taking 10-15 years to develop into cancer from a
pre-cancerous condition called dysplasia. Although fully treatable in the early stages, once the
cancer has metastasized, patient outcome is poor.
Critical to tumor cell invasion are the processes of cell attachment, proteolytic degradation of
the extracellular matrix (ECM) and migration through the disrupted matrix (4). Rath and
Pauling (5) proposed that the use of nutrients, such as lysine and ascorbic acid, to target
plasmin-mediated connective tissue degradation should be considered as a universal approach
to control tumor growth and expansion. Binding to plasminogen active sites, lysine blocks
plasminogen activation into plasmin by tissue plasminogen activator; thus, it modulates the
plasmin-induced matrix metalloproteinase (MMP) activation cascade (6). We have previously
developed strategies to inhibit cancer growth and its spread using complex micronutrient
supplementation with select natural compounds, such as lysine, proline, ascorbic acid and
green tea extract (7). This nutrient mixture (NM) demonstrated pleiotropic synergistic
anticancer activity in vivo and in vitro in several cancer cell lines through the inhibition of
cancer cell growth, MMP secretion, invasion, metastasis and angiogenesis (7).
In previous studies we found that NM significantly inhibited the proliferation of cervical
cancer HeLa cells in vitro, the secretion of MMP-2 and -9, urokinase plasminogen activator
activity and Matrigel invasion, and enhanced tissue inhibitor of matrix metalloproteinases
2 activity (8,9). In the present study the in vivo effects of NM supplementation ontumor
growth and cancer markers in cervical cancer HeLa cell tumor xenografts in female nude
mice were investigated. The cancer cell markers studied by tumor immunohistochemistry
(IHC) were as follows: Ki67 (proliferation marker); MMP-2 and -9 (metastasis markers);
vascular endothelial growth factor (VEGF) (angiogenesis marker); terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) and B-cell lymphoma 2 (Bcl-2) (apoptosis
markers); cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) (inflammatory markers) and glutathione S-transferase (GST) (specific cancer marker).

ONCOTAGEN
Human papillomavirus (HPV) infection can lead to a variety of human cancers [1, 2]. It
has been shown that most cervical cancers are caused by HPV infection [3, 4]. Two
oncogenes encoded in the HPV genome, E6 and E7 play critical roles in cervical cancer
development [5]. Expression of HPV E6 leads to degradation of p53, which is a critical tumor
suppressor regulating cell growth and apoptosis. Furthermore, HPV E7 binds and deactivates
another important tumor suppressor, retinoblastoma protein (Rb). In most cases, expression
of both E6 and E7 are required for oncogenic transformation.
In addition to deactivating tumor suppressors p53 and Rb, HPV infection leads to many
cellular changes during cervical cancer development [6]. However, the impact of HPV
infection on host microRNA expression has not been well characterized. MicroRNAs
(miRNAs) are small non-coding RNA molecules (~23 nucleotides) that downregulate the
expression of their gene targets [7]. Both computational and experimental studies indicate
that thousands of human protein-coding genes are directly regulated by miRNAs. Thus,
miRNAs play important regulatory roles in many physiological processes as well as a variety
of disease states such as cancer [8]. Relevant to this study, altered miRNA expression profiles
have been reported in cervical carcinomas as compared with normal cervix [9-15]. In
addition, miRNA expression changes have been used as biomarkers for cervical cancer
prognosis [16]. However, it is not clear whether these cervical cancer-related miRNA changes
were directly caused by HPV infection, or simply indirect effects from cervical cancer
progression in general. To specifically characterize miRNA expression changes that are
related to HPV activity, we determined the expression status of HPV E6 and E7 transcripts in
101 cervical carcinomas. Transcriptional activity of HPV was then correlated to miRNA
expression profiles to identify HPV-associated miRNA changes. In this way, we have
identified miR- 9 as the most activated miRNA by HPV E6 in cervical cancer, and showed
that both the transcriptional activity of HPV E6/E7 and miR-9 were prognostic markers for
cervical cancer. Further target validation and functional cell biology analyses showed that
HPV-induced miR-9 activation led to significantly increased cell motility by downregulating
multiple gene targets that are involved in cell migration, which may contribute to the
progression of cervical cancer.

PONE 25
Cervical cancer ranks the third among the most common
cancers in women worldwide and is the most common type of
cancer in Eastern Africa, South-Central Asia and Melanesia [1].
The two major types are squamous cell carcinoma (SCC),
accounting for around 80% of the cases, and adenocarcinoma
(ADCA), for which the incidence has been increasing [24].
Persistent infection with high risk (HR) human papillomavirus
(HPV) is a major risk factor for the development of cervical cancer
and the two predominant types are HPV18 and HPV16, followed
by HPV45, 31, 33, 58, 52, 35, 59, 56, 6, 51, 68, 39, 82, 73, 66 and
70 in order of prevalence [5].
HR-HPV encodes two early proteins, E6 and E7, that target
two major cellular tumor suppressor pathways. E6 targets the p53
tumor suppressor for degradation, resulting in loss of p53dependent apoptosis and/or senescence [6,7]. E7 binds to the
pRb tumor suppressor, thereby disrupting G1/S transition control
[8]. Consequently, HR-HPV infection may lead to malignant
transformation and tumor development.
Genomic alterations such as gene amplifications are important
features of cervical carcinogenesis [9,10]. Copy number gain in the
long arm of chromosome 3 has been shown to be a biomarker of
progression from carcinoma in situ to invasive cancer [11]. The
amplified chromosome 3q region contains several genes with
relevance to cancer, such as the phosphoinositide-3-kinase
catalytic alpha polypeptide gene (PIK3CA) at 3q26.32 [12], the
telomerase RNA component gene (TERC) at 3q26.2 [13,14], the
retinol-binding protein 1 and 2 genes (RBP1-RBP2) at 3q23, the
TP63 gene at 3q28 [15], and the sex-determining region Y-box 2
gene (SOX2) at 3q26.33 [1618].
Human WIG-1 (wild type p53-induced gene 1; also named
ZMAT3) maps to 3q26.32 [19], which is in the critical 3q region.

WIG-1 is a bona fide p53 target gene [20]. It encodes a 288 aminoacid nuclear zinc finger
protein that binds to double-stranded
RNA with high affinity [19,21]. WIG-1 is highly conserved from
amoeba to human [22,23] and the Wig-1 protein has been shown
to bind to a U-rich element in the 39-UTR of TP53 mRNA which
is then stabilized. Thus, Wig-1 forms a positive feedback loop with
p53 that enhances p53 expression [24,25]. Wig-1 can also bind to
and stabilize the MYCN oncogene mRNA [26], and targets a
number of other mRNAs [27]. Moreover, Wig-1 has been shown
to destabilize CDKN1A mRNA (encoding p21) through recruitment
of the RISC complex [28]. WIG-1 is amplified and/or overexpressed
in human papillary thyroid carcinoma (www.ebi.ac.uk/
gxa/), lung squamous cell carcinoma [29], cervical SCC and other
human tumors (www.cbioportal.org), suggesting an oncogenic
function.
In this study we have examined the WIG-1 gene in cervical
carcinoma cell lines and Wig-1 expression in both cervical
carcinoma cell lines and tumor samples. We investigated the
expression of Wig-1 in relation to different clinical parameters
such as: histological tumor type, grade and stage, HPV status, age
at diagnosis, and survival. Our results indicate that high nuclear
Wig-1 expression is a marker for poor prognosis in cervical
carcinoma.

PONE 417
Cervical cancer is worldwide the second most common cancer in women with the majority of
squamous cell carcinoma (SCC) [1] resulting in 454,000 cases and 200,000 deaths per year in
2010. Frequent metastatic sites are the pelvic lymph nodes, para-aortic lymph nodes, lung,
extra-pelvic nodes, liver, and bones [2]. Approximately 11,000 new cases and 3,870
deathsoccur for cervical carcinoma in the U.S. [3]. Stage and nodal metastasis are related to
overall

survival [4]. Chemotherapy drugs used for cervical cancer include: paclitaxel, carboplatin,
cisplatinum,
bleomycin, mitomycin-C, vincristine and irinotecan [5]. Retinoids and interferon, in
combination with cytotoxic chemotherapy, have been shown to be effective [6]. However,
there is no standard treatment for metastatic cervical cancer. Therefore, a patient-like mouse
model of cervical cancer could be very useful.
Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse
model with the technique of surgical orthotopic implantation (SOI) [721]. Unlike
subcutaneoustransplant patient-derived xenograft (PDX) models, PDOX models metastasize. Most
importantly,
the metastasis pattern correlates to the patient.
Histologically intact human colon-cancer specimens derived surgically from patients were
implanted by SOI to the colon or cecum of nude mice. Extensive growth on the colon in 13 of
20 cases of implanted patient colon tumors was observed with subsequent regional,
lymphnode,
and liver metastasis, as well as general abdominal carcinomatosis [7].
SOI of histologically intact pancreatic-cancer specimens to the nude-mouse pancreas,
resulted
in a metastatic pattern that resembles the clinical pattern including local tumor growth,
extending to the stomach and duodenum, metastases to the liver and regional lymph nodes,
and distant metastases to the adrenal gland, diaphragm, and mediastinal lymph nodes. A
100%
take rate was demonstrated for 5 cases, of a total 17 mice transplanted, 15 supported tumor
growth. Immunohistochemical analysis of the transplanted human pancreatic tumors showed
a similar pattern of expression tumor-associated glycoprotein 72 and carcinoembryonic
antigen
in the transplanted tumors and the original surgical biopsy [8].
Histologically-intact patient specimens of ovarian cancer were developed by SOI under the
capsule of the nude mouse ovary. The tumors grew locally with a subsequent patient-like
metastatic
pattern, including the parietal peritoneum, colon, omentum, and ascites [10].
Histologically-intact patient breast tumor tissue was transplanted to the mammary fat
pad of nude mice by SOI where the tumor tissue grew extensively and metastasized to the

lung [11].
A patient-like metastatic model of human lung cancer constructed was developed with SOI
via thoracotomy in immunodeficient mice [9]. Tumors were transplanted into the left lung in
all these experimental animals. The left lung was used for tumor implantation for 2 reasons:
(1) the loss of lung function is smaller in the left lung than in right-lung during surgery. The
left-lung-operated animals survive the procedure better. (2) The left lung in mice has one
lobe, enabling tumors to readily develop after implantation [9].When a poorly-differentiated
large-cell squamous-cell patient tumor 2268 was implanted to the left lung by SOI directly
from surgery, 5 out of 5 mice produced locally-grown tumors, in an average time of 61 days.
Opposite-lung metastases occurred, as well as lymph-node metastases. The primary tumors
and metastases in the mice maintained their large-cell-squamous-cell morphology.When
subcutaneously implanted tumors grew only locally in 2 of 4 animals and no metastases were
observed [9].
In a clinical correlative study of 20 cases of stomach cancer that grew in nude mice, 5 had
clinical liver metastases and all 5 cases resulted in liver metastases in the nude mice. Of the
20
cases, 6 had clinical peritoneal involvement of their tumor, and of these, 5 resulted in
peritoneal
metastasis in the nude mice. There were statistically significant correlations for both liver
metastases
and peritoneal involvement between patients and mice [12].
In the present report, we describe the development of a PDOX model of HER-2-positive
cervical cancer with a metastatic pattern similar to the patient donor.

Kanker serviks adalah kanker yang paling banyak diderita oleh wanita diseluruh
negara dan merupakan salah satu penyebab utama kematian akibat kanker pada wanita di
dunia secara keseluruhan. Kanker serviks juga merupakan kanker yang menyebabkan
kematian utama pada wanita di Indonesia dan masih menjadi masalah kesehatan. (1477)
Tingkat kelangsungan hidup satu tahun kedepan untuk wanita dengan kanker serviks adalah
87% dan tingkat kelangsungan hidup lima tahun kedepan adalah 68%. Bila terdeteksi dini,
tingkat kelangsungan hidup lima tahun kedepan untuk pasien dengan kanker serviks invasif
adalah 91% . Kanker serviks berkembang lambat, sekitar 10-15 tahun untuk berkembang
menjadi kanker dari kondisi pra kanker (ETM)
Kanker serviks disebabkan oleh infeksi HPV. Dua onkogen dikodekan dalam genom
HPV, E6 dan E7 memainkan peran penting dalam perkembangan kanker serviks. Ekspresi
HPV E6 menyebabkan degradasi p53, yang merupakan penekan tumor yang mengatur
pertumbuhan sel dan apoptosis. Selain itu, HPV E7 mengikat dan menonaktifkan penekan
tumor lain yang penting seperti protein retinoblastoma (Rb). Dalam kebanyakan kasus,
ekspresi kedua E6 dan E7 yang diperlukan untuk transformasi onkogenik. Selain
menonaktifkan penekan tumor p53 dan Rb, infeksi HPV menyebabkan banyak perubahan sel
selama perkembangan kanker serviks (ONCOTAGEN).
Dua jenis utama kanker serviks adalah karsinoma sel skuamosa (SCC) sekitar 80% dari
kasus, dan adenokarsinoma (ADCA), yang angka kejadiannya telah meningkat. Infeksi
persisten dengan resiko tinggi human papillomavirus (HPV) merupakan faktor risiko utama
untuk pengembangan kanker serviks (Pone 25).

Kanker serviks di seluruh dunia mayoritas mengidap karsinoma sel skuamosa (SCC) sekitar
454.000 kasus dan 200.000 kematian per tahun di 2010. Metastasis tersering yaitu kelenjar
getah bening panggul, kelenjar getah bening para-aorta, paru-paru, node ekstra-panggul, hati,
dan tulang. Obat kemoterapi yang digunakan untuk kanker serviks meliputi paclitaxel,
carboplatin, Cisplatinum, bleomycin, mitomycin-C, vincristine dan irinotecan. Retinoid dan
interferon, di kombinasi dengan kemoterapi sitotoksik, telah terbukti efektif. Akan Tetapi,
tidak ada pengobatan standar untuk kanker serviks metastatik (Pone 417)