Frequent overexpression of cyclin D1 in sporadic pancreatic
`endocrine tumours
`
`S S Guo, X Wu, A T Shimoide, J Wong, F Moatamed1 and
`M P Sawicki
`Department of Surgery, West Los Angeles VA Medical Center and the UCLA School of Medicine, Los Angeles, California 90095, USA
`1Department of Pathology, West Los Angeles VA Medical Center and the UCLA School of Medicine, Los Angeles, California 90095, USA
`(Requests for offprints should be addressed to M P Sawicki, Division of General Surgery, UCLA School of Medicine, Room 72-215,
`CHS10833 Le Conte Avenue, Los Angeles, CA 90095-6904, USA; Email: msawicki@ucla.edu)
`
`73
`
`Abstract
`
`Pancreatic endocrine tumours (PETs) occur sporadically or
`are inherited as part of the multiple endocrine neoplasia
`type-1 syndrome. Little is known about the molecular
`events
`leading to these tumours. Cyclin D1, a key
`regulator of
`the G1/S transition of
`the cell cycle,
`is
`overexpressed in a variety of human cancers as well as
`certain endocrine tumours. We hypothesized that similar
`to other endocrine tumours, cyclin D1 is overexpressed in
`human sporadic PETs. Cyclin D1 protein overexpression
`was found in 20 of 31 PETs (65%) when compared with
`normal pancreatic tissue. Furthermore, Northern blot
`analysis suggests that cyclin D1 up-regulation occurs at the
`post-transcriptional level in some PETs. Because the key
`cell growth signalling pathways p42/p44/ERK (extra-
`cellular signal-regulated kinase), p38/MAPK (mitogen-
`activated protein kinase), and Akt/PKB (protein kinase B)
`
`can regulate cyclin D1 protein expression in other cell
`types, pancreatic endocrine tumours were analysed with
`phospho-specific antibodies against the active forms of
`these proteins to elucidate a tissue-specific regulatory
`mechanism of cyclin D1 in PETs. We found frequent
`activation of
`the p38/MAPK and Akt pathways, but
`down-regulation of
`the ERK pathway,
`in cyclin D1
`overexpressing PETs. This study demonstrates that cyclin
`D1 overexpression is associated with human sporadic PET
`tumorigenesis, and suggests that this up-regulation may
`occur at the post-transcriptional level. These findings will
`direct future studies of PETs towards cell cycle dysregu-
`lation and the identification of key growth factor pathways
`involved in the formation of these tumours.
`Journal of Endocrinology (2003) 179, 73–79
`
`Introduction
`
`(PETs) develop from
`Pancreatic endocrine tumours
`neuroendocrine cells residing in and around the pancreas.
`These rare tumours occur either
`sporadically or are
`inherited as part of
`the multiple endocrine neoplasia
`type-1 syndrome (MEN-1). While PETs are well known
`for characteristic hormonal production related syndromes,
`such as Zollinger Ellison syndrome, they may also cause
`symptoms from local invasion as well as metastatic spread.
`There are few effective therapies for patients with PETs
`besides
`surgical management,
`and prognosis varies
`between excellent for those without metastasis to poor for
`those with metastasis. Knowledge of the molecular mech-
`anisms leading to PET tumorigenesis would greatly facili-
`tate the development of rational anti-tumour therapy, as
`well as provide diagnostic and prognostic markers.
`Little is known regarding the molecular pathogenesis of
`PETs. The gene responsible for the MEN-1 syndrome,
`MEN1, was cloned in 1997 (Chandrasekharappa et al.
`
`1997), and is mutated in 23% of PETs (Guo & Sawicki
`2001). The tumour suppressor Smad4/DPC4 (Bartsch
`et al. 1999) is mutated in 50% of nonfunctional tumours.
`Other tumour suppressors and oncogenes implicated in
`PET tumourigenesis include HER-2/neu (Evers et al.
`1994, Terris et al. 1998), p16INK4 (Muscarella et al. 1998),
`p27 (Guo et al. 2001), and p53 (Evers et al. 1994, Lin
`et al. 1997). Loss-of-heterozygosity studies have also
`suggested the involvement of tumour suppressor genes
`from chromosomes 1 (Ebrahimi et al. 1999, Guo et al.
`2002b), 3p (Chung et al. 1997), 3q (Guo et al. 2002a), 11p,
`16p (Chung et al. 1998), 17p (Beghelli et al. 1998) and 22q
`(Chung et al. 1998).
`Cyclins are the regulatory subunits of the cyclin/Cdk
`(cyclin-dependent kinase) complexes. Cyclin D1 binds
`to Cdk4/6, and the resultant complex phosphorylates
`the retinoblastoma susceptibility gene product, pRb.
`Sequential phosphorylation of pRb by cyclin D/Cdk4/6
`and cyclin E/Cdk2 inactivates pRb and allows cell-cycle
`progression through G1 (Fagan et al. 1994). Once this
`
`Journal of Endocrinology (2003) 179, 73–79
`0022–0795/03/0179–073  2003 Society for Endocrinology Printed in Great Britain
`
`Online version via http://www.endocrinology.org
`
`Roxane Labs., Inc.
`Exhibit 1011
`Page 001
`
`

`
`74
`
`S S GUO and others
`
`· Overexpression of cyclin D1 in PETs
`
`restriction-point is traversed, cells lose their mitogen-
`dependence and progress through the cell cycle until the
`restriction point of the next cell cycle is reached. Although
`there is variability amongst different cell systems, the
`synthesis and accumulation of the regulatory cyclins, in
`particular cyclin D1,
`is an important control of pRb
`phosphorylation (Grana & Reddy 1995) (reviewed in
`Hatakeyama & Weinberg 1995). Not surprisingly, many
`human cancers (e.g. breast, parathyroid, head and neck,
`gastric, oesophageal) have increased cyclin D1 expression
`(reviewed in Hunter & Pines 1994), and cyclin D1 is a
`prognostic marker for many cancers (Bova et al. 1999,
`Keum et al. 1999, Samejima et al. 1999, Cuny et al.
`2000, Itoi et al. 2000). Moreover, forced overexpression
`of cyclin D1 results in a shortened G1 phase, reduced
`cellular dependence on exogenous mitogens, and cell
`transformation (Bodrug et al. 1994, Wang et al. 1994).
`Finally, activation of Cdk4 causes pancreatic ♢-islet pro-
`liferation while inactivation of Cdk4 results in ♢-islet
`hypoplasia and insulin-dependent diabetes (Rane et al.
`1999).
`Cyclin D1 expression is regulated by transcriptional and
`post-transcriptional mechanisms. At
`the transcriptional
`level, kinase pathways such as p38/MAPK (mitogen-
`activated protein kinase), c-Jun N-terminal kinase/SAPK
`(stress-activated protein kinase),
`and p42/p44/ERK
`(extracellular signal-regulated kinase) transmit exogenous
`growth signals to up-regulate cyclin D1 (Lavoie et al.
`1996, Lee et al. 1999). Similarly, activation of the Akt/
`PKB (protein kinase B) pathway can increase cyclin
`D1 expression through either transcriptional (Gille &
`Downward 1999) or post-transcriptional mechanisms
`(Diehl et al. 1998, Muise-Helmericks et al. 1998). There
`is, however, tremendous tissue specificity in cyclin D1
`regulation and conflicting data regarding the effects of
`these pathways on cyclin D1 expression between cell
`types. Such studies have not been performed on human
`pancreatic endocrine tissues.
`We hypothesized that sporadic PETs have dysregulation
`of cyclin D1 similar to parathyroid adenomas and other
`endocrine tumours. Western blot analysis demonstrated
`frequent overexpression of cyclin D1 in sporadic PETs
`compared with normal pancreas tissues. Cyclin D1 over-
`expression was confirmed by immunohistochemistry
`(IHC) analysis on a subset of 5 sporadic PETs. Northern
`analysis suggests that cyclin D1 up-regulation in these
`tumours occurs at the post-transcriptional
`level. Using
`phospho-specific antibodies, we also show frequent
`activation of
`the p38/MAPK and Akt pathways, but
`down-regulation of
`the ERK pathway,
`in cyclin D1
`overexpressing sporadic PETs. Our findings are strength-
`ened by a recent report of
`frequent cyclin D1 over-
`expression in human sporadic PETs (Chung et al. 2000).
`Together, these two studies strongly suggest the import-
`ance of cyclin D1 up-regulation in the tumourigenesis of
`sporadic PETs.
`
`Materials and Methods
`
`Tissue isolation
`Tumour samples were obtained from patients who have
`received extensive pre-operative evaluations at exploratory
`laparotomy. Tissues were snap-frozen in liquid nitrogen
`and stored at 80 C.
`
`Institutional review and informed consent
`This study was performed under the auspices of the West
`LA, VA, Institutional review board, with appropriate
`institutional oversight. Informed consent was obtained
`from all patients from whom tissue samples were harvested.
`
`Protein isolation
`Tissues were homogenized using a Kontes pellet pestle
`(Fisher Scientific, Pittsburg, PA, USA) in CHAPS lysis
`buffer (10 mM Tris–HCl, pH 7·5, 1 mM MgCl2, 1 mM
`EGTA, 0·1 mM benzamidine, 5 mM ♢-mercaptoethanol,
`0·5% CHAPS, 10% glycerol). After 30 min incubation,
`centrifugation was performed and cellular debris discarded.
`The supernatant was stored at 80 C until use. Protein
`concentration was determined using the Bichinchonic
`method (Pierce Biotechnology, Rockford, IL, USA).
`
`Western blot analysis
`Protein samples were denatured in loading dye (62·5 mM
`Tris–HCl, 2% SDS, 10% glycerol, 0·1% Bromophenol
`Blue, 50 mM dithiothreitol) and boiled at 100 C. Equal
`amounts (25 µg) of protein were loaded into each well and
`resolved in 10% SDS-PAGE gel. The protein samples
`were transferred to Hybond-C Extra protein membrane
`(Amersham, Piscataway, NJ, USA), agitated in blocking
`solution (20 mM Tris, 140 mM NaCl, 5% dry milk,
`0·1% Tween 20), and probed with primary antibodies in
`primary antibody dilution buffer (20 mM Tris, 140 mM
`NaCl, 5% bovine serum albumin, 0·1% Tween 20). After
`hybridization with the appropriate secondary antibody
`(1:2000 dilution), blots were developed with ECL reagents
`according to the manufacturer’s instructions (Amersham).
`Equal protein loading and transfer were reconfirmed with
`Ponseau S staining (Sigma, St Louis, MO, USA) of the
`blots. The expression level of cyclin D1 was normalized to
`that of ♢-tubulin, and the expression levels of phospho-
`Akt, phospho-p38, and phospho-ERK were normal-
`ized to those of
`total-Akt,
`total-p38, and total-ERK
`respectively.
`
`Antibodies
`(Santa Cruz
`anti-cyclin D1
`Monoclonal mouse
`Biotechnology, Santa Cruz, CA, USA) was used at 1:500
`anti-♢-tubulin (Sigma
`dilutions. Monoclonal mouse
`Scientific) was used at 1:2000 dilutions. Rabbit polyclonal
`anti-total p38, anti-phospho-specific p38, anti-total Akt,
`
`Journal of Endocrinology (2003) 179, 73–79
`
`www.endocrinology.org
`
`Roxane Labs., Inc.
`Exhibit 1011
`Page 002
`
`

`
`anti-phospho-specific Akt, and anti-total ERK (New
`England Biolabs, Beverly, MA, USA) were used at 1:1000
`dilutions. Mouse monoclonal anti-phospho-specific ERK
`(New England Biolabs) was used at 1:1000 dilutions. For
`IHC, monoclonal cyclin D1 (NeoMarkers, Fremont, CA,
`USA) was used at 1:50 dilutions.
`
`Immunohistochemistry
`Tissues (3 PETs with cyclin D1 overexpression – 14T,
`78T, 111T; 2 PETs without cyclin D1 overexpression –
`226T, 250T) were taken from the frozen tissue bank, and
`fixed in formalin, ethanol, and xylene in an automated
`tissue processor (LX300 Tissue Processor, Fisher Scientific,
`Tustin, CA, USA). After tissues were embedded in
`paraffin,
`they were cut
`into 4 µm thick slices and
`mounted onto slides. Target retrieval was performed with
`DAKO Target Retrieval Solution (DAKO Corporation,
`Carpenteria, CA, USA) at 95 C for 20 min in a steamer,
`then cooled to room temperature. Slides were then placed
`into the DAKO Autostainer and stained using the
`DAKO LSAB2 System, Peroxidase (DAKO Corporation).
`Primary antibody was diluted in DAKO Antibody
`Diluent, and blocking was performed in DAKO Protein
`Block Serum-Free (DAKO Corporation). Signals were
`obtained using DAKO Liquid DAB Large Volume
`Substrate-Chromogen System (DAKO Corporation).
`Specificity of the antibody was confirmed by peptide
`blocking using the cyclin D1 peptide (NeoMarkers).
`Appropriate positive and negative controls were used.
`
`Northern blot analysis
`Tissues (2 PETs with cyclin D1 overexpression – 67T,
`113T; 2 PETs without cyclin D1 overexpression – 7T,
`43T) were homogenized (see above) and RNA was
`isolated according to the RNeasy Mini protocol (Qiagen,
`Santa Clarita, CA, USA). RNA was resolved in 1·2% FA
`Gel (1·2% agarose, 20 mM MOPS, 5 mM sodium acetate,
`1 mM EDTA, adjusted to pH 7·0), and transferred to a
`nitrocellulose membrane (Schleicher & Schuell, Keene,
`NH, USA) by capillary transfer (Sambrook et al. 1989).
`Cyclin D1 cDNA was labelled by a random primed
`DNA labelling kit (Boehringer Mannheim, Mannheim,
`Germany) using [♡-32P]dCTP (ICN Biomedical, Costa
`Mesa, CA, USA). Hybridization was performed with
`ExpressHyb Solution (Clontech, Palo Alto, CA, USA)
`according to the manufacturer’s protocol.
`
`Results
`
`Overexpression of cyclin D1 in sporadic PETs
`Cyclin D1 is a key regulatory protein for the G1-S
`transition of the cell cycle, and was first shown to be
`up-regulated in human endocrine tumours (Motokura
`et al. 1991). We hypothesized that
`similar to other
`
`Overexpression of cyclin D1 in PETs
`
`·
`
`S S GUO and others
`
`75
`
`Table 1 Activation of growth signalling pathways in sporadic PETs
`with increased cyclin D1 expression. Western blots with tumour
`protein were probed with antibodies to cyclin D1 and
`phospho-specific as well as total forms of Akt/PKB, p38/MAPK,
`and p42/p44/ERK. The Western data were normalized to the
`pancreas control band to eliminate the effect of different exposure
`times between the different Western blots, and quantified to a
`scale between 3 to 3+ by two independent reviewers.
`3+ represents the strongest signal while 0 denotes a signal
`comparable to that of the control. Clinical phenotype and the
`presence of hepatic involvement in each tumour are also shown.
`There is up-regulation of the Akt and p38 pathways, but
`down-regulation of the ERK pathway in sporadic PETs. There is no
`particular correlation between growth pathway activation and
`clinical behaviour or tumour type
`
`Phenotype
`
`Hepatic
`involvement
`
`Akt
`
`p38
`
`ERK
`
`Case
`1T
`4T
`8T
`14T
`39T
`67T
`78T
`83T
`111T
`113T
`119T2
`124T
`200TL3
`201T2C
`202T
`206T1
`208T
`213T
`214T2
`225T
`
`Gastrinoma
`Gastrinoma
`Gastrinoma
`Gastrinoma
`VIPoma
`Gastrinoma
`Gastrinoma
`Non-functional
`Non-functional
`Gastrinoma
`Gastrinoma
`Insulinoma
`Gastrinoma
`Gastrinoma
`Gastrinoma
`Gastrinoma
`Gastrinoma
`Gastrinoma
`Gastrinoma
`Gastrinoma
`
`No
`Yes
`No
`Yes
`No
`No
`No
`Yes
`No
`No
`No
`No
`Yes
`No
`No
`No
`No
`No
`No
`No
`
`1+
`0
`1+
`1+
`0
`3+
`1+
`0
`0
`0
`2+
`1+
`0
`2+
`1+
`2+
`1+
`1+
`2+
`1+
`
`3+
`0
`0
`2+
`0
`0
`0
`2+
`0
`0
`1+
`0
`1+
`1+
`2+
`2+
`1+
`0
`1+
`0
`
`2
`3
`3
`0
`3
`3
`2
`3
`3
`3
`1+
`0
`3
`1
`2
`1
`3
`0
`0
`2
`
`VIPoma, vasoactive intestinal peptide tumour.
`
`endocrine tumours there is dysregulation of cyclin D1 in
`sporadic PETs. To assess the expression of cyclin D1 at the
`protein level, Western blot analysis was performed on 31
`sporadic PETs. As shown in Table 1, 20 of 31 sporadic
`PETs (65%) demonstrated overexpression of cyclin D1 by
`Western blot analysis compared with two normal pancreas
`controls. A representative Western blot is shown in Fig. 1.
`Protein concentration of tumour samples was performed
`by the Bichinchonic assay, and equal loading was demon-
`strated with Ponseau S staining (data not shown) as well as
`by stripping the blots and re-probing with ♢-tubulin
`antibody (Fig. 1).
`To validate the Western blot data, IHC was performed
`in 5 sporadic PETs for which we had sufficient tumour
`material
`for analysis (Fig. 2 and data not shown). Of
`these 5 PETs analyzed by IHC, all three PETs found to
`have cyclin D1 overexpression by Western blot analysis
`(tumours 14T, 78T, and 111T) also demonstrated both
`nuclear and cytoplasmic staining of cyclin D1 by IHC.
`
`www.endocrinology.org
`
`Journal of Endocrinology (2003) 179, 73–79
`
`Roxane Labs., Inc.
`Exhibit 1011
`Page 003
`
`

`
`76
`
`S S GUO and others
`
`· Overexpression of cyclin D1 in PETs
`
`(Diehl et al. 1998, Muise-Helmericks et al. 1998, Lee et al.
`1999). The active regulatory pathway is cell-type specific
`and context sensitive. No studies have been performed on
`human pancreatic endocrine tumour tissues.
`We therefore investigated which Ras effector pathways
`might play an important role in cyclin D1 overexpression
`in sporadic PETs. Using phospho-specific antibodies and
`total-form antibodies against activated- and total-Akt,
`-p38/MAPK, and -p42/p44/ERK respectively, the acti-
`vation status of these growth pathways in sporadic PETs
`was examined. Protein gel loading was equalized accord-
`ing to the total forms of these antibodies, and Western
`bands were quantified on a scale between 3 and 3+ by
`two independent reviewers. As shown in Table 1, within
`the group of sporadic PETs that show cyclin D1 over-
`expression,
`there is activation of
`the Akt and/or the
`p38/MAPK growth pathways in 80% (16 of 20). In
`contrast,
`there is down-regulation of
`the ERK path-
`way in 75% (15 of 20) of the same group of samples. A
`representative Western blot is shown in Fig. 4.
`
`Cyclin D1 expression does not correlate with tumour
`aggressiveness or differentiation in sporadic PETs
`Because other studies have shown cyclin D1 to be a
`prognostic factor in human cancers (Bova et al. 1999,
`Keum et al. 1999, Samejima et al. 1999, Cuny et al. 2000,
`Itoi et al. 2000), cyclin D1 expression levels were analyzed
`for correlation with clinical tumour behaviour of these
`sporadic PETs. Tumours were categorized as benign or
`malignant based on the presence or absence of hepatic
`metastases. Of the six sporadic PETs that metastasized to
`the liver, four expressed increased levels of cyclin D1
`(67%) versus two that did not (Table 1 and data not
`shown). Conversely, of the 25 sporadic PETs without
`hepatic involvement, 16 expressed increased levels of
`cyclin D1 (64%). Analysis according to tumour hormonal
`status (gastrinoma vs insulinoma vs nonfunctional tumours,
`etc.) also did not reveal any association with cyclin D1
`overexpression. Thus there is no correlation between
`cyclin D1 overexpression and sporadic PET clinical
`aggressiveness or differentiation.
`
`Discussion
`
`In this study, 31 sporadic pancreatic endocrine tumours
`were analysed for the expression of cyclin D1 and 20 PETs
`(65%) showed overexpression by Western blot analysis.
`These findings were validated by IHC in a subset of
`tumours. Although our sample size is limited, this is one of
`only two such studies in these rare and generally small
`tumours.
`Previous studies showed no gross chromosomal rear-
`rangement or DNA amplification in the region of cyclin
`D1 in sporadic PETs. This was confirmed by our
`
`Figure 1 Cyclin D1 expression by Western blot in sporadic PETs.
`Tumours were snap-frozen at exploratory laparotomy, and stored
`at 80 C. After protein isolation (see Materials and Methods),
`PETs were probed with cyclin D1 and representative Western
`blots are shown. Protein from two normal pancreases was used
`for control. Results from both pancreases were similar, and data
`from one is shown. The same blot was stripped and re-probed
`with ♢-tubulin to demonstrate similar protein loading among
`samples.
`
`In contrast, two PETs without cyclin D1 overexpression by
`Western blot analysis (226T and 250T) showed undetect-
`able cyclin D1 staining by IHC. Peptide blocking con-
`firmed the specificity of our cyclin D1 antibody (Fig. 2a
`and b). IHC demonstrated minimal contamination in the
`surrounding normal tissue in our samples.
`
`Cyclin D1 overexpression occurs at the post-transcriptional
`level in sporadic PETs
`One mechanism for cyclin D1 overexpression in many
`human tumours is through DNA amplification or rear-
`rangement (e.g. breast cancer and parathyroid tumours),
`resulting in transcriptional up-regulation. However, pre-
`vious studies did not reveal gross cytogenetic rearrange-
`ment or DNA amplification in the region of cyclin D1 in
`sporadic PETs (Sawicki et al. 1992, Terris et al. 1998,
`Speel et al. 1999). Cyclin D1 expression in sporadic PETs
`is therefore likely to occur through a transcriptional or a
`post-transcriptional mechanism. We investigated these
`possibilities by Northern blot analysis of 4 sporadic PETs
`(2 PETs with cyclin D1 overexpression - 67T, 214T2;
`2 PETs without cyclin D1 overexpression - 7T, 43T).
`Figure 3 shows that although the level of cyclin D1 is
`dramatically different between these 4 sporadic PETs,
`their cyclin D1 mRNA expression is not significantly
`altered. This suggests that cyclin D1 overexpression may
`occur at the post-transcriptional level in sporadic PETs.
`
`Activation of MAP kinase pathways in pancreatic endocrine
`tumours
`Ras and its multiple effector pathways
`such as Akt,
`p38/MAPK, and p42/p44/ERK are known to have
`regulatory roles, through either transcriptional or post-
`transcriptional mechanisms, in the expression of cyclin D1
`
`Journal of Endocrinology (2003) 179, 73–79
`
`www.endocrinology.org
`
`Roxane Labs., Inc.
`Exhibit 1011
`Page 004
`
`

`
`Overexpression of cyclin D1 in PETs
`
`·
`
`S S GUO and others
`
`77
`
`Figure 2 Cyclin D1 expression by IHC in sporadic PETs. Cyclin D1 overexpression was confirmed by IHC in 5 tumours (3 tumours with
`cyclin D1 overexpression – 14T, 78T, 111T; 2 PETs without cyclin D1 overexpression – 226T, 250T). Representative photomicrographs are
`shown. (a) Tumour (14T) found to have cyclin D1 overexpression by Western analysis has nuclear as well as cytoplasmic staining of cyclin
`D1 by IHC. (b) Specificity of the antibody used in immunohistochemistry is demonstrated with peptide blocking. Cyclin D1 staining in the
`same tumour is completely abolished with peptide blocking. (c) Large dysmorphic tumour cells can also be seen in another tumour with
`cyclin D1 overexpression. (d) A PET found to have no cyclin D1 overexpression (226T) by Western analysis also has no significant staining
`of cyclin D1.
`
`Northern blot analysis that suggests in some sporadic
`PETs, post-transcriptional mechanisms are responsible
`for the up-regulation of cyclin D1 expression. Current
`studies
`in other cellular contexts demonstrated that
`the control of cyclin D1 expression by key growth
`regulatory pathways may be cell-type specific. We found
`activation of either the p38/MAPK or the Akt pathway
`in 80% (16 of 20) of sporadic PETs with cyclin D1
`
`overexpression. In contrast, the ERK pathway is down-
`regulated in 75% of these tumours (15 of 20). These
`observational studies on these rare human tumours may
`provide important clues
`for growth dysregulation in
`sporadic PETs.
`In addition, no correlation between cyclin D1 over-
`expression and clinical behaviour or tumour phenotype
`was found. Studies of various human cancers suggest that
`
`www.endocrinology.org
`
`Journal of Endocrinology (2003) 179, 73–79
`
`Roxane Labs., Inc.
`Exhibit 1011
`Page 005
`
`

`
`78
`
`S S GUO and others
`
`· Overexpression of cyclin D1 in PETs
`
`Figure 3 Cyclin D1 mRNA expression in sporadic PETs. Two
`cyclin D1 overexpressing tumours (67T, 214T2) and 2 tumours
`with normal cyclin D1 expression (7T, 43T) were analysed by
`Northern blot analysis. RNA was isolated from snap-frozen PETs
`and probed with radioisotope-labelled cyclin D1 cDNA. Northern
`blot bands and cyclin D1 Western blot bands of the same
`tumours are shown. The lack of increased signal on Northern blot
`analysis suggests that the increased cyclin D1 level occurred at the
`post-transcriptional level.
`
`Figure 4 Activation of growth signalling pathways in sporadic
`PETs. Western blots were probed with phospho-specific and
`total forms of antibodies against Akt/PKB, p38/MAPK, and
`p42/p44/ERK. Protein loading was equalized according to the
`signal from the total forms of these antibodies. Representative
`blots are shown.
`
`cyclin D1 dysregulation may occur before the tumour
`diverges into the different functional lineages or develops
`invasive behaviour. It may also be possible that cyclin D1
`expression is a key indicator of generalized islet growth
`rather than tumour-specific changes. This is suggested by
`the findings of Rane et al. (1999) where activation of Cdk4
`resulted in islet hyperplasia, but loss of Cdk4 led to
`insulin-deficient diabetes. Resolution of this issue will
`require functional studies in a suitable islet-specific model.
`Recently, Chung et al. (2000) also found frequent
`overexpression of cyclin D1 in human PETs. While the
`frequency of overexpression was lower in their series (43%
`vs 65% in this study), they also found that cyclin D1
`overexpression did not correlate with the hormonal sub-
`types of
`the PETs. Although a trend was
`suggested
`between cyclin D1 overexpression and tumour aggressive-
`ness, this was not statistically significant. Similarly, we
`found no correlation between cyclin D1 overexpression
`and hepatic metastasis. Finally, while their study suggested
`that cyclin D1 up-regulation may be at the transcriptional
`level, we failed to show differences in cyclin D1 mRNA
`levels between cyclin D1 overexpressing and non-
`overexpressing tumours. This incongruity may be attri-
`buted to the small sample sizes in both of our studies (nine
`vs four tumours), and warrants further study.
`In summary, we have shown that cyclin D1 is over-
`expressed in 65% of sporadic PETs. We have also shown
`that this cyclin D1 overexpression may be at the post-
`transcriptional
`level, and that the Akt, p38, and ERK
`pathways may play important roles in sporadic PETs.
`Further characterization of the G1/S transition dysregu-
`lation, as well as validation of the roles of these pathways by
`functional studies in suitable models, will yield a more
`complete understanding of
`the molecular mechanisms
`involved in the tumorigenesis of these rare tumours.
`
`Acknowledgements
`
`We express sincere gratitude to Drs Edward Passaro Jr
`and Gregory Brent for their intellectual discussion, support
`and guidance.
`
`Funding
`
`This research was funded by a VA MERIT award (to
`M P S) and a VA REAP grant (to S S G). There is no
`conflict of interest involved in this research.
`
`cyclin D1 overexpression may act either as a marker of
`tumour growth and aggressiveness, or as a marker of
`tumour differentiation.
`In sporadic PETs, however,
`neither aggressiveness (malignant vs benign behaviour)
`nor tumour differentiation (functional vs non-functional)
`correlate with cyclin D1 expression. It is possible that
`
`References
`
`Bartsch D, Hahn SA, Danichevski KD, Ramaswamy A, Bastian D,
`Galehdari H, Barth P, Schmiegel W, Simon B & Rothmund M
`1999 Mutations of the DPC4/Smad4 gene in neuroendocrine
`pancreatic tumors. Oncogene 18 2367–2371.
`
`Journal of Endocrinology (2003) 179, 73–79
`
`www.endocrinology.org
`
`Roxane Labs., Inc.
`Exhibit 1011
`Page 006
`
`

`
`Overexpression of cyclin D1 in PETs
`
`·
`
`S S GUO and others
`
`79
`
`Beghelli S, Pelosi G, Zamboni G, Falconi M, Iacono C, Bordi C &
`Scarpa A 1998 Pancreatic endocrine tumours: evidence for a
`tumour suppressor pathogenesis and for a tumour suppressor gene
`on chromosome 17p. Journal of Pathology 186 41–50.
`Bodrug SE, Warner BJ, Bath ML, Lindeman GJ, Harris AW & Adams
`JM 1994 Cyclin D1 transgene impedes lymphocyte maturation and
`collaborates in lymphomagenesis with the myc gene. EMBO Journal
`13 2124–2130.
`Bova RJ, Quinn DI, Nankervis JS, Cole IE, Sheridan BF, Jensen MJ,
`Morgan GJ, Hughes CJ & Sutherland RL 1999 Cyclin D1 and p16
`INK4A expression predict reduced survival in carcinoma of the
`anterior tongue. Clinical Cancer Research 5 2810–2819.
`Chandrasekharappa SC, Guru SC, Manickam P, Olufemi SE, Collins
`FS, Emmert-Buck MR, Debelenko LV, Zhuang Z, Lubensky IA,
`Liotta L et al. 1997 Positional cloning of the gene for multiple
`endocrine neoplasia-type 1. Science 276 404–407.
`Chung D, Smith A, Louis D, Graeme-Cook F, Warshaw A & Arnold
`A 1997 A novel pancreatic endocrine tumor suppressor gene locus
`on chromosome 3p with clinical prognostic implications. Journal of
`Clinical Investigation 100 404–410.
`Chung D, Brown S, Graeme-Cook F, Tillotson L, Warshaw A, Jensen
`R & Arnold A 1998 Localization of putative tumor suppressor loci
`by genome-wide allelotyping in human pancreatic endocrine
`tumors. Cancer Research 58 3706–3711.
`Chung DC, Brown SB, Graeme-Cook F, Seto M, Warshaw AL,
`Jensen RT & Arnold A 2000 Overexpression of cyclin D1 occurs
`frequently in human pancreatic endocrine tumors. Journal of Clinical
`Endocrinology and Metabolism 85 4373–4378.
`Cuny M, Kramar A, Courjal F, Johannsdottir V, Iacopetta B, Fontaine
`H, Grenier J, Culine S & Theillet C 2000 Relating genotype and
`phenotype in breast cancer: an analysis of the prognostic
`significance of amplification at eight different genes or loci and of
`p53 mutations. Cancer Research 60 1077–1083.
`Diehl JA, Cheng M, Roussel MF & Sherr CJ 1998 Glycogen synthase
`kinase-3♢ regulates cyclin D1 proteolysis and subcellular
`localization. Genes and Development 12 3499–3511.
`Ebrahimi S, Wang EH, Wu A, Schreck RR, Passaro E Jr & Sawicki
`MP 1999 Deletion of chromosome 1 predicts prognosis in
`pancreatic endocrine tumors. Cancer Research 59 311–315.
`Evers BM, Rady PL, Sandoval K, Arany I, Tyring SK, Sanchez RL,
`Nealon WH, Townsend CM Jr & Thompson JC 1994 Gastrinomas
`demonstrate amplification of the HER-2/neu proto-oncogene.
`Annals of Surgery 219 596–601.
`Fagan R, Flint KJ & Jones N 1994 Phosphorylation of E2F-1
`modulates its interaction with the retinoblastoma gene product and
`the adenoviral E4 19 kDa protein. Cell 78 799–811.
`Gille H & Downward J 1999 Multiple ras effector pathways
`contribute to G(1) cell cycle progression. Journal of Biological
`Chemistry 274 22033–22040.
`Grana X & Reddy EP 1995 Cellcycle control in mammalian cells:
`role of cyclins, cyclin dependent kinases (CDKs), growth suppressor
`genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 11
`211–219.
`Guo SS & Sawicki MP 2001 Molecular and genetic mechanisms of
`tumorigenesis in multiple endocrine neoplasia type-1. Molecular
`Endocrinology 15 1653–1664.
`Guo SS, Wu X, Shimoide AT, Wong J & Sawicki MP 2001
`Anomalous overexpression of p27(Kip1) in sporadic pancreatic
`endocrine tumors. Journal of Surgical Research 96 284–288.
`Guo SS, Arora C, Shimoide AT & Sawicki MP 2002a Frequent
`deletion of chromosome 3 in malignant sporadic pancreatic
`endocrine tumors. Molecular and Cellular Endocrinology 190 109–114.
`Guo SS, Wu AY & Sawicki MP 2002b Deletion of chromosome 1,
`but not mutation of MEN-1, predicts prognosis in sporadic
`pancreatic endocrine tumors. World Journal of Surgery 26 843–847.
`Hatakeyama M & Weinberg RA 1995 The role of RB in cell cycle
`control. Progress in Cell Cycle Research 1 9–19.
`
`Hunter T & Pines J 1994 Cyclins and cancer. II: Cyclin D and CDK
`inhibitors come of age. Cell 79 573–582.
`Itoi T, Shinohara Y, Takeda K, Nakamura K, Takei K, Sanada J,
`Horibe T, Saito T, Kasuya K & Ebihara Y2000 Nuclear cyclin D1
`overexpression is a critical event associated with cell proliferation
`and invasive growth in gall bladder carcinogenesis. Journal of
`Gastroenterology 35 142–149.
`Keum JS, Kong G, Yang SC, Shin DH, Park SS, Lee JH & Lee JD
`1999 Cyclin D1 overexpression is an indicator of poor prognosis in
`resectable non-small cell lung cancer. British Journal of Cancer 81
`127–132.
`Lavoie JN, L’Allemain G, Brunet A, Muller R & Pouysségur J 1996
`Cyclin D1 expression is regulated positively by the p42/p44MAPK
`and negatively by the p38/HOGMAPK pathway. Journal of Biological
`Chemistry 271 20608–20616.
`Lee RJ, Alanese C, Stenger R, Watanabe G, Inghirami G, Haines III
`GK, Webster M, Muller WJ, Brugge JS, Davis RJ & Pestell RG
`1999 pp60v-src induction of cyclin D1 requires collaborative
`interactions between the extracellular signal-regulated kinase, p38,
`and Jun kinase pathways. Journal of Biological Chemistry 274
`7341–7350.
`Lin HJ, French SW, Reichenbach D, Wan YJ, Passaro E Jr & Sawicki
`MP 1997 Novel p53 mutation in a malignant tumor secreting
`vasoactive intestinal peptide. Archives of Pathology and Laboratory
`Medicine 121 125–128.
`Motokura T, Bloom T, Kim HG, Juppner H, Ruderman JV,
`Kronenberg HM & Arnold A 1991 A novel cyclin encoded by a
`bcl1-linked candidate oncogene. Nature 350 512–515.
`Muise-Helmericks RC, Grimes HL, Bellacosa A, Malstrom SE,
`Tsichlis PN & Rosen N 1998 Cyclin D expression is controlled
`post-transcriptionally via a phosphatidylinositol 3-kinase/
`Akt-dependent pathway. Journal of Biological Chemistry 273
`29864–29872.
`Muscarella P, Melvin WS, Fisher WE, Foor J, Ellison EC, Herman
`JG, Schirmer WJ, Hitchcock CL, DeYoung BR & Weghorst CM
`1998 Genetic alterations in gastrinomas and nonfunctioning
`pancreatic neuroendocrine tumors: an analysis of p16/MTS1 tumor
`suppressor gene inactivation. Cancer Research 58 237–240.
`Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, Reddy EP
`& Barbacid M 1999 Loss of Cdk4 expression causes insulin-deficient
`diabetes and Cdk4 activation results in beta-islet cell hyperplasia.
`Nature Genetics 22 44–52.
`Sambrook J, Fritsch EF & Manistis T 1989 Molecular Cloning, A
`Laboratory Manual, edn 2. New York: Cold Spring Harbor
`Laboratory Press.
`Samejima R, Kitajima Y, Yunotani S & Miyazaki K 1999 Cyclin D1
`is a possible predictor of sensitivity to chemoradiotherapy for
`esophageal squamous cell carcinoma. Anticancer Research 19
`5515–5521.
`Sawicki MP, Wan YJ, Johnson CL, Berenson J, Gatti R & Passaro E
`Jr 1992 Loss of heterozygosity on chromosome 11 in sporadic
`gastrinomas. Human Genetics 89 445–449.
`Speel EJ, Richter J, Moch H, Egenter C, Saremaslani P, Rutimann K,
`Zhao J, Barghorn A, Roth J, Heitz PU & Komminoth P 1999
`Genetic differences in endocrine pancreatic tumor subtypes detected
`by comparative g