TMPRSS2 ERG or ETV1 Fusions in Prostate Cancer

1
TMPRSS2- ERG or ETV1 Fusions in Prostate Cancer

• By
• Brian A. Van Tine, MD.,PhD.
• 11/19/2007

2
Prostate Cancer

• Most commonly diagnosed cancer in American males
• 218,890 men in the United States will be
  diagnosed with prostate cancer this year and that
  27,050 deaths will occur
• Approximately 1 in 10 men will develop prostate
  cancer in their lifetime
• Prostate cancer is more frequent in patients with
  a strong family history
• Is the second leading cause of death from cancer
  in the United States for men
• Prostate cancer is more common in African
  American males
• The incidence of prostate cancer increases with
  age
• Deaths from prostate cancer continue to increase
  in other parts of the world, such as Australia,
  Europe, Japan, and Russia

www.uptodate.com
3
Androgen in Cancer

• In 1966 Charles B. Huggins (Surgeon) was awarded
  the Nobel Prize for Endocrine-Induced Regression
  of Cancers
• In the 1930s, Huggins discovered that castration
  of dogs prevented prostate cancer
• In the early 1940s he found he could retard the
  growth of prostate cancer by blocking the action
  of the patient's male.
• From his Nobel Lecture The prostatic cell does
  not die in the absence of testosterone, it merely
  shrivels. But the hormone-dependent cancer cell
  is entirely different. It grows in the presence
  of supporting hormones but it dies in their
  absence and for this reason it cannot participate
  in growth cycles.

"Huggins, Charles B.." Encyclopædia Britannica.
2007. Encyclopædia Britannica Online. 14  Nov. 
2007 http//nobelprize.org/nobel_prizes/medicine/l
aureates/1966/huggins-lecture.pdf
4
Androgen Receptor Signaling

• Prostatic cell survival and development depends
  on the androgen receptor
• Heat-shock proteins are bound to the receptor in
  the cytoplasm of cells
• The testical converts testosterone to its active
  form dihydrotestosterone
• This activation causes a dissociation from
  heat-shock proteins and translocation into the
  nucleus.
• The androgen receptor dimerizes in the nucleus
  and binds to androgen-response elements in DNA
  activating transcription of genes for growth and
  survival.

Debes JD and Tindall DJ NEJM (2004) 351 1488-1490
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First Line Therapy

• Standard first line therapy for metastatic
  disease includes androgen ablation with 80 of
  patients demonstrating a response
• Androgen deprivation causes come cells to undergo
  apoptosis while other cells arrest in G1
• Eventually further cellular changes allow the
  cancer to become unresponsive to hormonal
  manipulation and the cancer progresses.

www.uptodate.com
6
Types of Translocations Tumors

• Most translocations have been studied in
  Leukemias, Lymphomas and Sarcomas (Ewings). In
  general, there are two main types
• 1. A promoter and/ or enhancer elements of one
  gene are aberrantly juxtaposed to a
  proto-oncogene, thus causing altered expression
  of an oncogenic protein.
• - exemplified by the apposition of
  immunoglobulin (IG) and T cell receptor (TCR)
  genes to MYC, leading to activation of this
  oncogene in B and T cell malignancies
• 2. A rearrangement allowing for the fusion of
  two genes, resulting in a fusion protein that may
  have a new or altered activity.
• - example of this translocation is the BCR-ABL
  gene fusion in chronic myelogenous leukemia (CML)

J. D. Rowley, Nat. Rev. Cancer 1, 245 (2001).
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Solid Tumor Translocations

• Epithelial tumors (carcinomas) are harder to
  study genetically directly
• They display many nonspecific but few recurrent
  chromosomal rearrangements
• This karyotypic complexity is thought to reflect
  secondary genomic alterations acquired during
  tumor progression

HeLa Cell Spectral Karyotype
Tomlins, S.A. et al Science (2005) 310
664-648 Macville et al. 1999. Cancer Research 59
141-150.
8
Translocations in Cervical Cancer

• Chromosomal Gains and losses are found with
  progression to cancer
• Oncogenic transcriptional selection is also found
  with progression
• There is selection for a specific type of
  translocation for oncogenic transcription
• Viral promoter and viral E6/E7 oncogenes are
  fused to a host 3 poly A sequence allowing for
  greater transcript stability while inactivating
  certain viral genes.
• Regardless of the number of amplified DNA
  elements, selection for boarder transcription
  from a single DNA elements occurs

Van Tine, B.A., et al. Clonal Selection for J.
Virol. 2004 78(20)11172-86.
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The TMPRSS2- ETS Protein Fusion

• TMPRSS2 (transmembrane-serine protease)
• ETS Family of Transcription Factors
• ERG (ETS related gene)
• ETV1 (ETS variant gene 1)
• ETV4 (ETS variant gene 4)
• FLI1 (Friend leukemia virus integration 1)

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TMMPSS2

• Transmembrane protease, serine 2
• Located on chromosome 21q22.3
• It is androgen-regulated transmembrane-serine
  protease gene
• Also known as PRSS10
• The protein contains
• a type II transmembrane domain
• a receptor class A domain
• scavenger receptor cysteine-rich domain
• protease domain
• The protease domain is cleaved and secreted into
  cell media after autocleavage
• Function of this gene is unknown

http//www.ncbi.nlm.nih.gov/sites/entrez?DbgeneC
mdShowDetailViewTermToSearch7113ordinalpos1i
toolEntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.
Gene_RVDocSum
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ETS Family Members

• Originally identified in 1983 from v-ets oncogene
  of the avian leukemia virus protein E26
• Now are 25 human and 26 murine ETS family members
• ETS proteins are transcription factors that share
  a conserved winged helix-turn-helix DNA binding
  domain
• Recognize unique DNA sequences containing
  GGA(A/T)
• ETS factors act as positive or negative
  regulators of the expression many genes and that
  are implicated in
• cellular proliferation - differentiation
• hematopoiesis - apoptosis
• tissue remodeling - angiogenesis
• transformation

Hsu et al., JBC (2004) 91 896-903
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The Discovery of the Fusion

• Recurrent Fusion of TMPRSS2 and
• ETS Transcription Factor Genes in
• Prostate Cancer
• Scott A. Tomlins,1 Daniel R. Rhodes,1,2 Sven
  Perner,7,9
• Saravana M. Dhanasekaran,1 Rohit Mehra,1 Xiao-Wei
  Sun,7
• Sooryanarayana Varambally,1,6 Xuhong Cao,1 Joelle
  Tchinda,7
• Rainer Kuefer,10 Charles Lee,7 James E.
  Montie,3,5,6
• Rajal B. Shah,1,3,5,6 Kenneth J. Pienta,3,4,5,6
  Mark A. Rubin,7,8
• Arul M. Chinnaiyan1,2,3,5,6
• 28 OCTOBER 2005 VOL 310 SCIENCE

Tomlins, S.A. et al Science (2005) 310 664-648
13
Identification of a Target

• They hypothesized that rearrangements and
  high-level copy number changes that result in
  marked overexpression of an oncogene should be
  evident in DNA microarray data but not
  necessarily by traditional analytical
  approaches.
• To do this, they developed a method called cancer
  outlier profile analysis or COPA.
• It seeks to accentuate and identify outlier
  profiles by applying a simple numerical
  transformation based on the median and median
  absolute deviation of a gene expression profile.

Tomlins, S.A. et al Science (2005) 310 664-648
14
Identification of a Target

• They analyzed 132 gene expression data sets
  representing 10,486 microarray experiments
• Rank Score Gene Cancer
• 1 95 20.056 RUNX1T1 Leukemia
• 95 15.4462 PRO1073 Renal
• 1 90 12.9581 PBX1 Leukemia
• 1 95 10.03795 ETV1 Prostate
• 1 90 7.4557 WHSC1 Myeloma
• 1 75 5.4071 ERG Prostate
• 1 75 4.3628 ERG Prostate
• 1 75 4.3425 CCND1 Myeloma
• 1 75 3.4414 ERG Prostate
• 1 75 3.3875 ERG Prostate
• 3 95 13.3478 FGFR3 Myeloma
• 4 75 2.5728 ERBB2 Breast
• 6 90 6.6079 ERBB2 Breast
• 9 95 17.1698 ETV1 Prostate
• 9 90 6.60865 SSX1 Sarcoma
• 9 75 2.2218 ERG Prostate

Tomlins, S.A. et al Science (2005) 310 664-648
15
ETS

• COPA identified strong outlier profiles in six
  independent prostate cancer studies for ERG
  (21q22.3) and ETV1 (7p21.2)
• ETS are involved in oncogenic translocations in
  Ewings sarcoma and myeloid leukemias.
• (EWS-FLI1, EWS-ERG, and EWS-ETV1 )

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ETS

• ETS family members are functionally redundant in
  oncogenic transformation
• Only one translocation is typically observed in
  each case of Ewings sarcoma
• They hypothesized that if ERG and ETV1 were
  involved in the development of prostate cancer,
  each tumor should overexpress only one of the two
  genes.

Tomlins, S.A. et al Science (2005) 310 664-648
17
mRNA Profiling

• mRNA quantitative PCR was done to measure the
  overexpression of either ETV1 or ERG Transcripts
• CPP pooled benign prostate tissue
• VCaP and DuCaP are ERG overexpressing cell lines
• LNCaP is an ETV1 overexpressing cell line
• MET30/28-LN
• MET26-RP hormone refractory primary carcinoma
• MET26-LN a lymph node met from the same patient

Tomlins, S.A. et al Science (2005) 310 664-648
18
Missing Exons

• They did not find consistent DNA amplification in
  there samples that correlated with ERG and ETV1
  overexpression
• They performed QPCR on mRNA by Exon Walking on
  ETV1
• CPP pooled benign prostate tissue
• LNCaP is an ETV1 overexpressing cell line
• MET30/28-LN
• MET26-RP hormone refractory primary carcinoma
• MET26-LN a lymph node met from the same patient
• They found gt90 reduction in expression of exons
  2 and 3 compared to exons 4 and 7 in the MET
  samples

Tomlins, S.A. et al Science (2005) 310 664-648
19
Race to the Beginning

• 5 Race combined with exon walking was done on
  samples and fusions were found with a protein
  called TMRPSS2
• Sequencing of the cloned products revealed
  fusions of the prostate-specific gene TMPRSS2
  with ETV1 in MET26-LN and with ERG in MET28-LN.

Tomlins, S.A. et al Science (2005) 310 664-648
20
Validation Assays

• 42 nonrandom cases of clinical localized prostate
  cancer and metastatic prostate cancer were
  analyzed by quantitative PCR for EGR/ETV1
  Expression and TMPRSS2-ERG/TV1 expression.
• TMPRSS2ERG/ETV1 fusions were only found in cases
  that overexpressed ERG/ETV1

Tomlins, S.A. et al Science (2005) 310 664-648
21
Interphase FISH in Tissue Sections

• Rearrangements at the chromosomal level on
  formalin-fixed paraffin-embedded (FFPE) specimens
  and normal peripheral lymphocytes (NPLs) were
  next performed.
• TMPRSS2ETV1fused signals are yellow because they
  are on different chromosomes. (T721)
• Probes spanning the 5 and 3 regions of the ERG
  locus were used to assay for gene rearrangements
  for ERG because only 3 megabases separate TMPRSS2
  to ERG on chromosome 21.
• FISH analyses was done on serial samples from
  tissue microarrays sample cores from 13 cases of
  localized prostate cancer and 16 cases of
  metastatic prostate cancer.
• Of 29 cases, 23 (79.3) showed evidence of
  TMPRSS2ETV1 fusion (7 cases) or ERG
  rearrangement (16 cases).

ETV1 (red) Chr 7 TMPRSS2 (Green) Chr 21
ERG 5 (Red) ERG 3 (Green)
ETV1 (red) Chr 7 TMPRSS2 (Green) Chr 21
ERG 5 (Red) ERG 3 (Green)
Tomlins, S.A. et al Science (2005) 310 664-648
22
Androgen regulation of ERG is present only after
translocation

• Casodex is and Flutamide are androgen receptor
  antagonists
• LNCaP (ETV1) is do not express the ERG fusion
  transcript
• VCaP does express the fusion transcript

• TMPRSS2 is expressed in normal and neoplastic
  prostate tissue and is strongly induced by
  androgen in androgen-sensitive prostate cell
  lines.
• These results suggest that the fusion with
  TMPRSS2 may explain the aberrant expression of
  ERG or ETV1 in specific subsets of prostate
  cancer.

Tomlins, S.A. et al Science (2005) 310 664-648
23
ETV4 another ETS

• Continued analyzation demonstrated ETV4
  overexpression in 2 of 98 cases.
• Using quantitative PCR and fluorescence in situ
  hybridization, they demonstrated a TMPRSS2
  (21q22) ETV4 (17q21) translocation in a single
  case.
• They hypothesis that fusions of ETS family
  members with TMRPSS2 may be an initiating event
  in prostate cancer development.

• Tomlins et al. Cancer Res (2006)66 3396-400.

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Frequency of Translocations done by other groups

• Soller et al using RT-PCR for TMPRSS2ERG
  amplified cDNA fragments in 14 (78) of 18
  prostate adenocarcinomas whereas no PCR products
  were obtained when using TMPRSS2 forward and ETV1
  reverse primers
• Yoshimoto et al. analyzed of 15 cases by RT-PCR
  and FISH and detected six TMPRSS2ERG-related
  gene fusions.
• Perner et al analyzed Samples from
  hospital-based cohorts of 237 clinically
  localized PCA (TMPRSS2ERG 48.5), 34 hormone
  naive metastases (TMPRSS2ERG 30), 9 hormone
  refractory metastases (TMPRSS2ERG 33).
• Merhra et al analyzed 96 American men surgically
  treated for clinically localized prostate cancer
  and identified rearrangements in TMPRSS2, ERG,
  ETV1, and ETV4 in 65, 55, 2, and 2 of cases,
  respectively. Overall, 54 were TMPRSS2ERG and
  2 were TMPRSS2ETV1 fusions.

Soller et al. GENES, CHROMOSOMES CANCER
45717719 (2006) Yoshimoto et al. Neoplasia.
2006 Jun8(6)465-9. Perner et al Am J Surg
Pathol. 2007 Jun31(6)882-8. Mehra et al. Mod
Pathol. 2007 May20(5)538-44
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TMPRSS2 ERG is increased in poorly
differentiated Tumors

• A total of 106 cases were analyzed by FISH.
• The TMPRSS2ERG fusion was found more frequently
  in moderate to poorly differentiated tumors
  (35/86, 40.7) than in well differentiated tumors
  (1/15, 6.7, p 0.017).
• TMPRSS2ETV1 gene fusions were not detected in
  any of the cases tested.

Rajput et al. J Clin Pathol 20076012381243.
26
There are morphological features associated with
TMPRSS2ERG fusion

• Mosquera et al. analyzed 253 prostate cancer
  samples.
• detected significant associations with common
  morphological features
• Five morphological features were found to be
  associated with TMPRSS2ERG fusion prostate
  cancer
• blue-tinged mucin
• cribriform growth pattern
• Macronucleoli
• intraductal tumor spread
• signet-ring cell features
• all with p-values lt0.05
• This may have pathologists identify TMPRSS2-ERG
  fusion samples and help to identify higher risk
  prostate cancers

Mosquera et al. J Pathol 2007 212 91101
27
Watchful Waiting Might be Bad if you have this
Translocation

• Demichelis et al. examined the outcomes of
  patients with ETS fusion proteins in a watchful
  waiting cohort of 111 men.
• 17/111 has TMPRSS2ERG Translocations and
  overexpression of ERG
• TMPRSS2-ERG positive tumors had an almost 3 times
  higher risk of dying of prostate (cumulative
  incidence ratio2.7, Plt0.01, 95 confidence
  interval1.3-5.8).

Demichelis et al. Oncogene 20072645969
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Model

• Fusion of untranslated sequences of TMPRSS2ETS.
• Other molecular changes include loss of PTEN, a
  tumor suppressor.
• Increased expression of an ETS transcription
  factor in response to activated androgen receptor
  then occurs.
• The ETS transcription factor would then induce
  transcription of genes that block checkpoints
  triggered indirectly by inactivation of PTEN.
• This allows for down regulation of receptor
  tyrosine kinases (RTKs)allowing for unchecked
  activity of Akt, which promotes cell
  proliferation and survival.

Shaffer Pandolfi, NATURE MEDICINE (2006)
114-15.
29
Future Directions

• The use of the translocation in stratification
  for treatment decisions
• Randomized prospective studies of the incidence
  of this translocation and its importance in
  progression and survival
• TMPRSS2ETS is now a target for small molecules
  to inhibit ERG and ETV1 over-expression.
• Determination of further aspects of the biology
  of the fusion protein itself may yield even more
  drug targets