The Role of Insulin and IGF2 Signalling on Metabolic Pathways in Prostate Cancer Progression Amy Anne Lubik Bachelors of Molecular Biology and Biochemistry (Hon), Simon Fraser University, British Columbia, Canada Institute of Health and Biomedical Innovation Faculty of Science and Technology Australian Prostate Cancer Research Centre – Queensland (APCRC-Q) Queensland University of Technology, Brisbane, Australia Submitted for the fulfilment of the Requirements for the Degree of Doctor of Philosophy ~2011~
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The Role of Insulin and IGF2 Signalling on Metabolic Pathways in Prostate
Cancer Progression
Amy Anne Lubik
Bachelors of Molecular Biology and Biochemistry (Hon), Simon Fraser University,
British Columbia, Canada
Institute of Health and Biomedical Innovation
Faculty of Science and Technology
Australian Prostate Cancer Research Centre – Queensland (APCRC-Q)
Queensland University of Technology, Brisbane, Australia
Submitted for the fulfilment of the Requirements for the Degree of
1.2 Prostate cancer ................................................................................................ 25
1.3 Androgen receptor signalling in prostate cancer ............................................ 27
1.4 Steroidogenesis and prostate cancer ............................................................... 29
1.4.1 Steroidogenesis enzyme expression in prostate cancer ........................... 29
1.4.2 Intraprostatic androgens/ de novo steroidogenesis .................................. 32
1.4.3 AR and steroidogenesis inhibitors in prostate cancer treatment .............. 35
1.5 Lipid/ fatty acid contribution to prostate cancer and sterol response element binding protein...................................................................................................... 39
1.5.1 Lipogenesis and prostate cancer .............................................................. 39
1.5.2 Sterol regulatory element binding protein and prostate cancer ............... 40
1.5.3 Fatty acids and steroidogenesis ............................................................... 43
1.6 Metabolic syndrome and prostate cancer ....................................................... 44
1.6.1 Metabolic syndrome correlates with prostate cancer progression in epidemiological studies .................................................................................... 44
Chapter 3: Insulin Directly Increases de novo Steroidogenesis in Prostate Cancer Cells ......................................................................................................................... 83
3.3.1 Insulin upregulates expression of enzymes necessary for steroidogenesis at the mRNA and protein levels ........................................................................ 89
3.3.2 Insulin increases intracellular steroids in prostate cancer cells ............... 96
3.3.3 Insulin increases secretion of steroids from prostate cancer cells ........... 96
3.3.4 PSA expression and secretion are increased by insulin ......................... 103
3.3.5 In LNCaP xenografts mice which showed an increase in both PSA and RDH5 expression at 28 days post castration also displayed an increase in INSR-A and IRS2 mRNA .............................................................................. 107
Chapter 4: The Effect of Insulin Analogs on Steroidogenesis and Insulin Effect on Breast Cancer Steroidogenesis ............................................................................... 115
4.3.1 Insulin and analogs upregulate enzymes necessary for steroidogenesis at the mRNA level .............................................................................................. 122
4.3.1 Insulin analog effect on de novo steroidogenesis in LNCaP and VCaP medium ........................................................................................................... 124
4.3.3 Insulin effects on steroidogenesis in breast cancer ................................ 126
5.3.3 Analysis of de novo fatty acid synthesis and lipid profile after insulin treatment ......................................................................................................... 145
5.3.4 Insulin effects on fatty acid metabolism, as demonstrated by microarray analysis ........................................................................................................... 148
Chapter 6: Drugs Used in Metabolic Syndrome, Metformin and Simvastatin, Inhibit Fatty Acid Synthesis and Steroidogenesis in Prostate Cancer ............................... 159
Chapter 7: Insulin-like Growth Factor 2 Increases de novo Steroidogenesis in Prostate Cancer Cells ............................................................................................. 193
7.2.5 Western blotting ..................................................................................... 200
7.2.6 Steroid analysis in LNCaP cells ............................................................ 201
7.2.7 Radio-labelled acetate analysis of de novo steroidogenesis in LNCaP and VCaP cells ...................................................................................................... 201
8.2 Prostate cancer and metabolic syndrome: reducing risk by nutritional and pharmacological means ...................................................................................... 224
8.3 The effect of insulin on breast cancer: background and prospects for study/ intervention ......................................................................................................... 228
8.3.1 The similarities between breast and prostate cancer .............................. 228
8.3.2 Breast cancer and hyperinsulinemia ...................................................... 230
8.3.3 Insulin effects on steroidogenesis in breast cancer ................................ 231
8.4 IGF2, steroidogenesis, and bone metastases ................................................ 231
8.4.1 Androgens and bone metastases ............................................................ 231
8.4.2 IGF2 may promote steroidogenesis contributing to growth of bone metastases ....................................................................................................... 232
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8.4.3 Insulin and IGF2 may promote prostate cancer progression ................. 234
8.5 Final Conclusion and Summary ................................................................... 235
increased levels of the first steroid converted from cholesterol in the pathway,
pregnenolone, 2.5 fold (figure 3.3A; p<0.05), and we observed a 15-fold increase in
total intracellular 17-OH-progesterone levels (figure 3.3A; p<0.05), which is
converted from progesterone by CYP17A1, an enzyme shown in figure 3.2 to be
significantly increased with insulin. CYP17A1 also catalyses the final reaction in
the synthesis of DHEA, which was substantially increased, 18-fold, by insulin
(figure 3.3A; p<0.05). Notably, testosterone levels were also increased
approximately 60-fold in LNCaP cells following treatment with insulin (p<0.05).
Intracellular levels of testosterone were calculated to increase from approximately
0.011 to 0.65ng/g cells when treated with 10nM insulin for 16hrs (calculations and
spectra are shown in figure 3.4A,C). These levels are consistent with the
testosterone levels of our previous findings (Locke et al. 2008). Previously,
Gregory et al. have demonstrated DHT concentrations as low as 1x10−14 mol/L
(2.92 × 10−6 ng/g) to transactivate AR in prostate cancer cell lines (Gregory et al.
2001a). Titus et al. (2005b) report 1.25 pmol/g tissue (0.498ng/g) of DHT in
recurrent prostate cancer tissue specimens, and 0.4ng/g tissue was found in clinical
samples of prostate epithelium by Liu et al. (1985). Mosteghel found that DHT
levels in castrate patients are 0.2 to 1.78 ng/g (Drejer 1992); therefore, the
androgen concentrations detected in our study and increased by insulin treatment in
LNCaP cells were consistent with levels needed for activation of the AR.
3.3.3 Insulin increases secretion of steroids from prostate cancer cells
As steroidogenic cells differentially secrete specific steroids, we measured steroid
levels in the medium of prostate cancer cells cultured in serum-free medium versus
medium supplemented with 10nM insulin for 16 hours. HPLC-MS analysis of
steroids in LNCaP medium (figure 3.3B) was consistent with our intracellular
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steroid data. As expected, it was the steroids in the latter part of the steroidogenic
pathway that appeared in the medium. Of greatest significance 17-OH progesterone
was 2-fold increased; p<0.05, testosterone 1.3-fold and DHT, 1.5-fold increased;
p<0.05, and androstenedione was increased 3-fold; p<0.05.
Importantly, these data indicate that insulin-stimulated intracellular steroidogenesis
by prostate cancer cells could provide steroids, including androgens, to the tumour
microenvironment. The concentrations of DHT and testosterone secreted into the
medium by LNCaP cells after 16 hours of insulin treatment in our studies were
calculated to be approximately 0.0249 and 0.037nM (calculations and spectra are
shown in figure 3.4B,C), with the baseline levels consistent with our previous
studies (Locke et al. 2008) and within the range necessary to activate AR (Gregory
et al. 2001a; Titus et al. 2005b).
To investigate de novo steroidogenesis, alongside steady state levels, LNCaP and
VCaP cells were treated with 14C labelled acetate in the presence and absence of
insulin for 72hrs before radiometric analysis of cell culture medium. Increased
steroids were persisting in samples treated with insulin compared to vehicle control,
consistent with steady-state data from LNCaP cells. In LNCaPs (figure 3.3C), we
demonstrated significant increases in testosterone (2-fold, p<0.05), androstenedione
(1.5-fold, p<0.05), and androsterone (2-fold), p<0.05), and pregnan-3,20-dione
(1.7-fold, p<0.05), as well as several other peaks within the steroid region which
did not correspond to Mix 10 standards (Spectrum shown in figure 3.5). Included in
these peaks was a peak with a retention time between that of progesterone and
pregnan-3,20-dione, 34 min, which was significantly increased (2.4-fold, p<0.05).
Steroid peaks were also significantly increased in VCaP extracts following insulin
treatment as well as a 4-fold increase in cholesterol synthesis (figure 3.3D).
Furthermore, steroids beyond androstenedione and androsterone in the pathways
were not detected in VCaP cells (spectra shown in figure 3.5) suggesting rates of
steroid synthesis differ between the cell lines. In 22RV1 cells, DHT secretion into
the medium was significantly increased from 0.23nM to 0.36nM (figure 3.3E),
following 48-hour treatment with 10nM insulin. This is comparable to DHT
secreted by LNCaP cells (calculations in figure 3.4) and sufficient to activate the
AR (Gregory et al. 2001a; Titus et al. 2005b).
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(Figure 3.3, page over)
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Figure 3.3: Insulin treatment increases steroid production in prostate cancer cells.
LNCaP cells were treated with 10nM insulin for 16 hours and steroids extracted.
LC-MS-MS was used to identify and quantitate (A) intracellular steroids showing a
significant increase in pregnanolone, 17-OH progesterone (17-OH P), DHEA and
testosterone (T) (*p<0.05). (B) Medium was collected and also analyzed to identify
and quantitate extracellular steroids. Statistically significantly increased steroid
levels were also seen in the medium for androstendione (Andr), 17-OH
progesterone (17-OH P), DHT and testosterone (T). Pregnanolone (Preg) was
increased, but did not reach significance. Steroid levels were adjusted to cell pellet
weight and recovery of deuterated testosterone used to calculate extraction
efficiency. Results were compared to the vehicle control. Error bars represent SE
(*p<0.05). De novo steroid synthesis was measured by incubating cells with
6µCi/ml radiolabelled acetate. 14C-labelled steroids were extracted from cell culture
medium after 72hr incubation with insulin (to allow accumulation to quantitative
levels) and HPLC and radiometric detection used to identify and quantitate
extracellular steroids from (C) LNCaP cells. Increased magnitude of persisting
steroid peaks was measured in insulin treated versus control samples with
significant increases in testosterone, androstenedione, androsterone and pregnan-
3,20-dione as well as a peak at 34 minutes which falls within the steroid range but
with no corresponding retention time amongst the standards. (D) In VCaPs,
androstendione, the step before testosterone in the classical steroid pathway,
increased approximately 3-fold (p<0.05); whereas, androsterone, a steroid of the
backdoor pathway, increased approximately 2-fold (p<0.05). The 34min steroid
peak which elutes at a time between progesterone and pregnan-3,20-dione, and
pregan-3,30-dione steroid increased 1.75 and 1.5-fold, respectively (p<0.05).
Cholesterol was detected in VCaP cells and furthermore, was increased
approximately 4-fold by insulin treatment. Intriguingly, steroids beyond
androstenedione and androsterone in the pathways were below the limit of
detection in VCaP cells at 72hrs. (E) DHT was measured by ELISA in medium
collected from 22RV1 cells after 48hr incubation with insulin and compared to
vehicle control. Statistically significantly increased DHT levels were demonstrated.
Error bars represent SE (*p<0.05) n≥3.
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(Figure 3.4, page over)
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Figure 3.4: Calculations and steroid spectra for insulin induction of
steroidogenesis. (A) Table of values from a representative LC/MS/MS spectrum
shown in ng/ml isolated from approximately 0.060g of LNCaP cells. Cellular
steroids were extracted by MTBE extraction into 95µl 0.2M hydroxylamine HCl,
and concentrations of that volume were given by the LC/MS/MS. The amount for
the whole sample (ng) was calculated, and then divided by approximately 0.06g to
give ng/g cell pellet. (B) For medium concentrations, 3mL of medium were
extracted into 95µl with MTBE. The total grams of steroid was calculated then
converted to moles and divided by the original volume to get the original
concentration of steroids in the medium. The concentrations of T (cells), and T and
DHT (medium) have been included in the paper. (C) This method measures
unlabelled steady-state steroid concentrations (in contrast to de novo synthesised
steroids measured in figure 3.3D and 3.3E), therefore a spectrum of peaks has been
provided. Spectrum from vehicle control appear in the top panel and insulin treated
in the bottom panel.
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Figure 3.5: Representative spectra of steroids isolated from 2mL of medium
following treatment with 14C acetate for 72hours in the +10nM insulin of VCaP
(A,B) or LNCaP cells (C, D). For VCaP cells, the control (A) was compared to
10nM insulin treated (B) to calculate fold change. The same was done for LNCaPs,
vehicle-treated control cells (C) being used to normalise insulin induced peaks (D).
Steroid retention times were comparable in both LNCaP and VCaP cells and are
identified from 1 to 7, with testosterone the first to elute from the column and
cholesterol the last. De novo synthesised testosterone was detected in LNCaPs. The
total amount of radiolabelled steroids after 72 hours insulin treatment in the
presence of 14C acetate was substantially lower in VCaPs. Peaks were also present
in the steroid region which could not be definitively identified (Peak 5; 34 mins) as
there was no corresponding peak in the Mix 10 standards.
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3.3.4 PSA expression and secretion are increased by insulin
Serum PSA is the biomarker for CaP recurrence and for progression to CRPC
following ADT. We and others have shown that a key mechanism underlying
CRPC progression is the activation of androgen driven pathways through the
androgen receptor (AR) (Huang et al. 1999; Mostaghel et al. 2007); therefore, we
have used PSA as a functional surrogate of AR reactivation via increased androgen
production and measured the effect of insulin on PSA production from LNCaP
cells. To directly compare the level of insulin stimulation of PSA with DHT,
LNCaP cells were exposed to 10nM insulin or 10nM DHT for 16, 24, or 48 hours.
As shown in figure 3.6A, 24hr insulin treatment induced a 10-fold increase in PSA
mRNA, compared to a 20-fold increase by DHT, whereas the mRNA levels
decreased by 48hrs, likely due to the metabolism of these hormones. In contrast,
the non-metabolizable and more potent androgen R1881 continued to increase PSA
mRNA levels over this time course (figure 3.6B). To equate levels of response
between 10nM insulin and DHT, we performed a DHT titration; at 24 hours, as
shown in figure 3.4C, PSA induction by 10nM insulin was equivalent to the level
of induction seen with treatment of approximately 0.16(+/-0.29)nM DHT, as
calculated by linear regression. Mean concentration of PSA with vehicle did not
change over time (data not shown). PSA secreted into the medium was increased
due to insulin treatment after 16hrs (figure 3.6C), which suggests a lag in PSA
response which is both consistent with requisite steroid production and the lag in
PSA response profile seen directly with androgen treatment in figure 3.6A. These
data clearly show that PSA was increased at 16 hours and significantly accumulated
in the medium by 48hrs to 1.8 fold of baseline following insulin exposure (p<0.05).
This is supported by further data showing intracellular androgen levels induced by
insulin are sustained to 48hrs (figure 3.7). Induction of PSA expression following
24-hour insulin treatment also occurred in VCaP cells (figure 3.6E), with an
increase of approximately 40% from baseline (p<0.05). Furthermore, insulin
induced a 2-fold increase in PSA mRNA in 22RV1 cells at 48hr (figure 3.6F).
Treatment with the AR antagonist, bicalutamide attenuated the insulin induced
increase in PSA expression in LNCaP and 22RV1 cells (figure 3.6G-H) directly
implicating insulin activation is mediated by the AR.
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(Figure 3.6, page over)
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Figure 3.6: Insulin treatment increases expression of PSA. (A) Insulin-induced
changes in PSA mRNA expression from LNCaP cells were compared to 10nM
DHT at 16, 24 and 48hrs by QRT-PCR, ΔΔCt method as described, normalized to
rpl32 control gene. Changes in PSA expression by insulin are not detected until the
24 hour time point. The effects of DHT are reduced after 48 hours of culture. In
contrast (B), increased expression of PSA at 2 and 24 hours in LNCaP cells is
maintained at 48 hours by non-metabolizable AR agonist R1881. (C) To compare
the PSA response of insulin and DHT, LNCaP cells were treated with DHT
concentrations from 10nM to 0.1pM for 24hrs and the values compared to 10nM
insulin (normalized to rpl32 control gene). (D) Medium was collected from LNCaP
cells treated with insulin (10nM) for 5, 16 and 48 hours and 48hr control and PSA
analyzed by ELISA. Control is shown at 48hrs. Increased expression of PSA
mRNA was measured following insulin (10nM) compared to vehicle following (E)
24hr treatment of VCaP cells and (F) 48hrs treatment of 22RV1 cells. Treatment
with the AR-inhibitor bicalutamide (25µM) attenuated the insulin induced increase
in PSA expression in (G) LNCaP cells and (h) 22RV1 cells by ~50% (†p<0.05). All
results are shown +SE (*p<0.05) n≥3.
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Figure 3.7: LNCaP cells were treated with 10nM insulin for 48hr and steroids were
extracted and prepared for quantitation by LC/MS/MS. Graphs depict the difference
in intracellular steroids, shown in ng/ml (controlled to weight of cell pellet), + SE,
n≥3, and show accumulation of androgens over time.
107
3.3.5 In LNCaP xenografts mice which showed an increase in both PSA and
RDH5 expression at 28 days post castration also displayed an increase in INSR-A
and IRS2 mRNA
CRPC progression can be modelled in vivo using LNCaP tumours injected
subcutaneously into immunocompromised male mice; tumour growth is followed
by monitoring tumour-derived PSA levels in the serum. Typically, after a 6-week
period of growth the mice are castrated, PSA levels fall to a Nadir (N) within 7
days. In most mice PSA levels will begin to increase again by day 28 post-
castration and this is referred to as castrate resistance (CR) in this model. However,
in some mice there is a greater lag of PSA production not arising until after 35
days. In a blinded study for PSA level following castration, tumours were grown
for 28 days post castration, and then analysed for the expression of markers relevant
to steroidogenesis. From the isolated LNCaP tumours, QRT-PCR was performed
on RNA for PSA, insulin receptor isoform A (INSR-A), IRS2, and RDH5. As
mentioned above, RDH5 is a key enzyme of the backdoor pathway to DHT
synthesis, and tumours expressing this enzyme may be more steroidogenic (Auchus
2004; Locke et al. 2008). Our previous studies have shown that the mRNA of most
steroidogenesis enzymes, of which RDH5 is an example, increase during
progression (Locke et al. 2008). In mice that exhibited a CRPC increase in PSA
levels 28 days following castration, PSA, RDH5 and IRS2 showed significantly
higher gene expression levels (figure 3.8A; p<0.05) and INSR-A isoform showed a
trend towards increased expression (figure 3.8A). This was in contrast to mice
bearing LNCaP tumours that did not show a serum PSA increase by 28 days (non-
progressed), where the genes were unchanged (figure 3.8B). Therefore, increased
steroidogenesis correlates to increased androgen activation (PSA production) in
vivo. Furthermore, the changes in key insulin signalling molecules suggest that
insulin may act via INSR-A and IRS2 in this model.
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Figure 3.8: In vivo tumour xenograft model. In LNCaP tumour xenografts were
collected from athymic nude mice at castration (pre-Cx), at the PSA nadir (8 days
post castration – N) and at castrate-resistant stage (28 days post castration – CR).
QRT-PCR analyses of PSA, IRS2, INSR-A, and RDH5 mRNA in tumours show
statistically significant increase in expression in mice that experienced PSA
recurrence (A). In contrast, the expression of these genes was not changed in mice
that did not progress to castrate resistance (B). Error bars represent SE (*p<0.05),
n=8.
INSR‐A
INSR‐A
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3.4 Discussion
Reactivation of the androgen receptor following ADT, defined by rising serum PSA
is a hallmark of castrate resistant prostate cancer progression. Several mechanisms
including hypersensitivity of the receptor to low steroid concentrations and ligand
promiscuity arising from mutations in the receptor ligand binding domain can play
a role AR reactivation (Gregory et al. 2001a; Mostaghel et al. 2007). We and
others have previously identified that intratumoural androgen production is also
associated with activation of AR (Heinlein et al. 2004; Titus et al. 2005b). We have
demonstrated that the LNCaP prostate cancer cell line expresses all of the enzymes
required for de novo androgen synthesis (Locke et al. 2008), and these observations
have been extended into VCaP and 22RV1 prostate cancer cells. Following ADT,
androgen levels continue to be substantial in prostate tissue, compared to the
dramatic and continued decrease of androgens in sera. These low levels are
sufficient to activate the AR (Stanbrough et al. 2006; Locke et al. 2008). These
studies indicate that the synthesis of androgens plays an important role in CRPC
progression; however, the biological factors inducing and regulating
steroidogenesis during prostate cancer progression have not been largely explored.
Androgen deprivation therapy is associated with a pattern of metabolic alterations
consistent with insulin resistance and the metabolic syndrome including an increase
in fat mass and fasting plasma insulin (hyperinsulinemia) (Basaria et al. 2006;
Smith et al. 2006). Emerging evidence suggests that both body mass index and high
serum insulin levels are independently predictive of poorer patient outcomes
including increased disease aggression and increased cancer mortality (Ma et al.
2008); obese men are more likely to have higher grade cancers, high recurrence
rates, and high prostate cancer-specific mortality (Buschemeyer et al. 2007).
However, recent studies have also identified a correlation between serum C-peptide
levels and prostate cancer mortality (Smith et al. 2006; Fowke et al. 2008; Isbarn et
al. 2008; Ma et al. 2008; Cox et al. 2009), suggesting a role for insulin in disease
progression. Moreover, mouse studies have shown that diet-induced
hyperinsulinemia leads to more aggressive tumour growth (Venkateswaran et al.
2007) and insulin has long been known to stimulate proliferation in breast and
prostate cancer cells (Lann et al. 2008; Pollak 2008b). In contrast, men with low
insulin levels due to diabetes appear to have a decreased risk of CaP development
110
(Zangeneh et al. 2006; Hsing et al. 2007; Kasper et al. 2008). Furthermore, recent
studies have shown increased insulin receptor (INSR) expression in neoplastic
prostate specimens as opposed to non-neoplastic prostate tissue (Cox et al. 2009)
suggesting increased insulin signalling in these cells.
Although there is mounting epidemiological evidence linking hyperinsulinemia and
CRPC, the direct action of insulin on prostate cancer cells has not been
investigated. Insulin is able to promote steroidogenesis through upregulation of
steroidogenic enzymes (Ogishima et al. 1989; Stattin et al. 2000; Tsilchorozidou et
al. 2003; Munir et al. 2004; Seto-Young et al. 2007; Diamanti-Kandarakis et al.
2008) in conditions such as polycystic ovarian syndrome (PCOS), and insulin
receptors have been reported on prostate cancer cell lines and prostate tumour tissue
(Cox et al. 2009). The ability for prostate cell lines to produce steroids has been
demonstrated (Nestler 1997; Soronen et al. 2004; Dillard et al. 2008; Locke et al.
2008; Leon et al. 2010). Therefore, we investigated whether insulin plays a role in
prostate cancer progression through the promotion of de novo steroidogenesis. We
show for the first time that insulin upregulates steroidogenesis in AR-responsive
prostate cancer cell lines, LNCaP, VCaP and 22RV1 cells, leading to increased cell
survival and likely exacerbation of CRPC progression.
We demonstrated that many enzymes required for steroidogenesis, via both the
classical and backdoor pathways, are upregulated following insulin treatment at
both the RNA and protein levels. Expression of the insulin signalling molecule,
IRS-2 is significantly increased at the RNA level in LNCaPs; increased expression
of IRS-2 has been associated with increased steroidogenesis in both ovarian thecal
and breast cancer cells (Wu et al. 2000; Cui et al. 2003). Importantly, we showed
an increase in expression of mRNA and protein for SREBP, the transcription factor
which is responsible for coordinating the initiation of cholesterol synthesis in
LNCaP cells, following 10 hours insulin treatment and 48hr treatment of 22RV1
cells. There was an increase in the level of CYP11A1 in all cell types and of StAR
in LNCaPs and 22RV1 cell lines; these enzymes are responsible for the importation
of cholesterol into the mitochondria for steroidogenesis and pregnenolone
synthesis. The enzymes which catalyse more than one step in the steroidogenesis
pathway including CYP17A1, HSD3B2, HSD17B3 and SRD5A1 (figure 3.1) were
all significantly upregulated by insulin treatment. All three prostate cancer cell lines
111
respond to insulin with upregulation of CYP17A1, HSD3B2 and HSD17B3.
Significantly increased expression of SRD5A1 was seen in LNCaP cells only but
RDH5 expression was increased in all cell lines; these enzymes convert
testosterone and androstenediol into DHT, respectively. Taken together our data
suggests both pathways of de novo androgen synthesis are upregulated in CaP cells
following insulin treatment, allowing for versatile means of synthesis of potent
androgens as seen in our previous studies (Kohn et al. 2007).
Insulin consistently stimulated an increase in intracellular steroids and steroids
released into the medium including androgens in all cell lines indicating the
enzymes are functionally active. The release of steroids by prostate cancer cells
may provide paracrine activity of the steroids within the microenvironment. Rising
PSA following ADT is considered the sentinel for CRPC progression most likely
driven by AR reactivation. We observed increased mRNA expression at 24hrs in
LNCaP and VCaP cells and 48hrs in 22RV1 cells, as well as increased PSA
secretion following 48hrs insulin treatment in LNCaPs which demonstrates there is
adequate AR activation in all 3 cell lines to stimulate PSA expression (Mostaghel et
al. 2007; Labrie et al. 2008; Locke et al. 2008) and this can be inhibited by
bicalutamide treatment. An increase in structurally related steroids may still be
relevant in cancer progression, in the cases where the AR has acquired mutations
leading to promiscuous activation by steroids and compounds other than
testosterone and DHT (Monge et al. 2006). The mutation of the LNCaP AR ligand
binding domain (T877A) also makes the AR susceptible to activation by non-
androgenic steroids, which may also contribute to activation by substrates and by
products of de novo synthesis (Hagedorn et al. 1936). Recent studies have
specifically identified a correlation between elevated insulin/ C-peptide levels, a
surrogate measure of insulin levels, with high grade CaP and worse patient
prognosis (Smith et al. 2006; Fowke et al. 2008; Isbarn et al. 2008; Ma et al. 2008;
Nandeesha et al. 2008; Cox et al. 2009). Major findings from recent studies of men
receiving ADT demonstrated a relationship between elevated C-peptide levels and
more rapid progression to castrate resistance (Huggins 1942; Neuhouser et al.
2010).
In summary, our research has shown that insulin increases steroidogenesis in AR
positive prostate cancer cell lines by increasing the mRNA and protein levels of
112
steroidogenic enzymes, leading to an increase in steroid production, including
androgens. Subsequent increases in PSA secretion suggest insulin can affect
prostate cancer cell survival and CRPC progression. Increased expression of the
insulin receptor in the LNCaP xenograft model during progression of cancer to
castrate resistance provides further evidence that insulin may be acting directly on
prostate cancer cells through the INSR. There are multiple studies correlating high
insulin levels and CaP progression (Hsing et al. 2001; Basaria et al. 2006; Hsing et
al. 2007; Venkateswaran et al. 2007; Nandeesha et al. 2008; Vieira et al. 2008;
Albanes et al. 2009). The significance of cholesterol synthesis (steroid precursor)
and steroidogenesis in CaP progression suggests treatments which target these
pathways are pertinent for the treatment of patients with CRPC, particularly in the
context of hyperinsulinaemia and the metabolic syndrome (Soronen et al. 2004;
Stanbrough et al. 2006; Mostaghel et al. 2007; Dillard et al. 2008; Labrie et al.
2008; Locke et al. 2008; Montgomery et al. 2008; Mostaghel et al. 2008; Leon et
al. 2010). Of note, Abiraterone, an inhibitor of CYP17A1, has shown promising
results in clinical trials with men who are no longer responsive to androgen ablation
(Goodwin et al. 2002; Allen et al. 2005). This is one pathway by which insulin may
contribute to cancer progression; however, in addition to upregulating
steroidogenesis, insulin is expected to activate multiple pathways in cancer cells
(Pollak 2008c). Further understanding of the direct action of insulin on prostate
cancer cells may provide important insight into new therapeutic strategies to
prevent progression of castrate resistant prostate cancer.
The results of our study suggest that the metabolic dysfunction of prostate cancer
patients should also be addressed. There are a number of pharmacological agents
currently available for the treatment of insulin resistance which can improve
(reduce) circulating insulin levels including metformin and recent studies suggest
targeting insulin resistance can have positive effects on cancer patient outcomes
including prostate cancer (Sahra et al. 2008; Algire et al.). Upstream inhibitors of
cholesterol synthesis such as the thiazolidinediones (TZDs) have been shown to be
effective insulin sensitizers in patients with metabolic syndrome. In a cancer
context, TZDs have been shown to decrease androgen production in H295 cells by
down-regulation of CYP17A1 and HSD3B2 (Kempná et al. 2007) and to reduce
proliferation of cancer cells (Krishnan et al. 2007; Luconi et al. 2010).
113
Furthermore, CaP patients who are undergoing cholesterol lowering treatment with
the class of HMG CoA inhibitors, statins, show markedly lower PSA and tumour
volumes than non-users (Loeb et al. 2009). Currently, ADT-induced
hyperinsulinaemia is not addressed in prostate cancer patients, despite an
significantly increased risk of cardio-vascular mortality in these patients (Redig et
al. 2010a); however, we provide further evidence that management of the metabolic
consequences of ADT may be as important as treatment of the cancer itself.
.
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Chapter 4: The Effect of Insulin Analogs on
Steroidogenesis and Insulin Effect on Breast Cancer
Steroidogenesis
116
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4.1 Introduction
Since 1922, insulin monotherapy has been used to treat type 1 diabetes (T1D) and
type 2 diabetes (T2D) (Carlbor 1938). Fredrich Banting and Charles Best first
isolated pancreatic insulin and administered it to diabetic dogs before successful
human treatment (Banting et al. 1922). Because regular insulin was so short lived,
interest soon followed to find a longer acting, more stable formulation. One of the
most crucial discoveries in this field happened at the University of Toronto, where
Scott and Fischer found that adding zinc acetate or sulfate and other metallic salts
to solution would change the chemical structure of insulin, decreasing the
solubility, slowing the absorption, and therefore prolonging the effect (Carlbor
1938; Scott et al. 1938). In 1936, Hagedorn et al. discovered that adding a basic
protein (protamine) to the formulation localized the insulin to the injection site,
which prolonged its action (Hagedorn et al. 1936); this was deemed Neutral
Protamine Hagedorn [insulin] (NPH). NPH begins action after an hour and a half
and takes 4-12hrs to reach peak serum concentrations.
Most insulin was obtained from porcine or bovine species until the 1980s, when
biosynthetic human insulins were produced by recombinant DNA technologies
(Gualandi-Signorini et al. 2001). This new era of insulin synthesis led to interest in
additional long or short acting insulin analogs, with the benefit of acting faster at
meal times in the case of T2D (short acting) or more constantly for T1D (long
acting) (Werner et al. 2011). Use of analogs in humans has only come about
relatively recently, the most utilized being Asp B28 (discovered 1990) and Lys Pro
(1992), which are short acting, and the long acting insulin glargine (2000), which
has residues of arginine inserted into the β-chain, and a glycine substituted for
histidine in the α-chain, and insulin determir (1997), which has a myristic acid
chain added to bind to albumin and slow absorption to areas beyond the injection
site (Werner et al. 2011).
Most of the modifications of insulin analogs occur around the β-chain, so as to not
alter insulin binding; however, it has recently become apparent that some insulin
analogs actually have a much higher affinity for the IGF1R than “normal” (wild-
type) insulin does (100 fold less than IGF1), which is of concern to some of the
medical community, as IGFs are known to increase risk of certain cancers, such as
breast and prostate (Kurtzhals et al. 2000; Pollak et al. 2010; Pollak 2010a). One of
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the first insulin analogs, Asp B10, which differed from regular insulin by increased
affinity for IGF1R, was shown to have carcinogenic properties in female rats
(Werner et al. 2011).
T2D patients have an increased risk of cancer mortality (Currie et al. 2009), and
mice on high carbohydrate diets (T2D) had greater tumour growth and increased
IGF1R and insulin receptor (INSR) signalling. It has also been shown that an
increase in C-peptide levels correlates with poor outcome in breast and prostate
cancer (Pollak et al. 2010). Studies on the effect of insulin analogs on cancer have
been varied, with many conflicting opinions. In a large scale population study,
Hemkens et al. showed that patients treated with glargine have higher incidence of
cancers (Hemkens et al. 2009). Jonasson et al. demonstrated an increased incidence
of breast cancer in glargine treated patients (Jonasson et al. 2009). In a study in
Scotland, glargine was not associated with site specific cancer, but patients on
glargine did appear to have higher risk overall; however, this study had a small
sample size (Colhoun 2009). Another study showed glargine was associated with
cancer more than other insulins (Mannucci et al. 2010). Conversely, Rosenstock et
al. compared glargine to NPH in T2D patients over 4 years and saw no differences
in cancer incidence (Rosenstock et al. 2009). Analysis of Sanofi-Aventis’ clinical
data showed no difference in cancer incidence between glargine and other insulins
(Werner 2011).
Weinstein et al. (2009) are highly cited for their study on the differences in effects
of multiple analogs on various cancer cells, though some of the data has been
interpreted as incomplete (as most experiments were only done on colorectal cancer
cells), or super-physiological concentrations (100nM) (Weinstein et al. 2009). They
treated colorectal cancer (HCT-116), prostate cancer (PC3), or breast cancer
(MCF7) cells with long or short acting insulins, for 96hrs. Cell mitogenicity of
HCT-116 was increased 26% with 100nM IGF1 compared with glargine, lispro, or
detemir, 22, 20, or 17%, respectively. In PC3 cells, IGF1 increased mitogenicity by
25%, with glargine and determir at 16 and 14%, In the MCF7 breast cancer cell line
IGF1 displayed 22% increase, with glargine and determir increasing by 14% and
6% respectively compared to control. Insulin itself only increased mitogenicity by
7%. Both IGF1 and glargine increased cell proliferation in HCT-116 cells in a dose
dependent manner, whereas insulin did not. The remaining experiments, in HCT-
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116 cells, showed a decrease in apoptosis with IGF1, glargine and determir, though
not with insulin. Signalling studies showed most analogs caused signalling with
greater similarity to IGF1R activation than insulin mediated INSR signalling. In
other studies of MCF7 cells, Kurtzhals et al. saw that glargine had 7.83 times
greater mitogenic potential than “normal” (wild-type) insulin, and both Kohn et al.,
and Mayer et al. demonstrated a 4-fold increase in proliferation of human
mammary epithelial cells with glargine compared to insulin (Kurtzhals et al. 2000;
Kohn et al. 2007; Mayer et al. 2008). Kurtzhals et al. and Shukla et al.
demonstrated increased mitogenic potential occurs primarily through aberrant
IGF1R signalling (Kurtzhals et al. 2000; Shukla et al. 2009). Sciacca et al.
demonstrated that signalling responses to short acting insulin analogs were more
similar to normal insulin than those of long acting analogs (Sciacca et al. 2010).
Because of the interest in insulin analogs and cancer, and the fact that we have
recently shown (chapter 3) that insulin upregulates both steroidogenesis enzymes
and steroid hormones themselves (Lubik et al. 2011), it was important to
investigate whether there are differences in the steroidogenic potential of long-
acting insulin analogs and regular insulin at equimolar ratios (10nM). Analogs used
were Glargine (trade name Lantus), and insulin X10, also known as B10Asp. A
single aspartate is substituted for histidine in the dimer forming region of the
insulin X10 peptide, to prevent aggregation of insulin and prolong its systemic
effects (Brange et al. 1988), and it has twice the receptor affinity for INSR
compared to insulin, and approximately 10 times the affinity for IGF1R. Glargine
has had two amino acid substitutions to shift the solubility in order to make small
precipitates in the blood at the injection site, which would slow absorbance for
potentiating insulin effects with one daily dose (Wang et al. 2003). Glargine has
less affinity for INSR (0.86 compared to insulin), but more than 6-times greater
affinity than insulin for IGF1R (Kurtzhals et al. 2000). In this study, it has been
demonstrated that these analogs are not more steroidogenic than insulin.
Furthermore, the effect of insulin on steroidogenesis in breast cancer cells was also
investigated, because much of the data on the effects of insulin and analogs on
cancer growth was attained from breast cancer studies (Jonasson et al. 2009;
Kurtzhals et al. 2000; Kohn et al. 2007; Mayer et al. 2008). Also, breast cancer and
120
CaP are similar in hormonal dependence and treatment (Risbridger et al. 2010). In
breast cancer patients, obesity and hyperinsulinemia are associated with recurrence
and fatality (Goodwin et al. 2002). Also, high serum insulin levels are associated
with risk of developing breast cancer. Insulin receptor A (INSR-A) is almost
ubiquitously expressed and overexpressed in breast cancer, and is not down
regulated by exposure to ligand, as it is in normal tissue (Crettaz et al. 1984;
Goodwin 2008). There is much interest in the inhibition of many enzymes in the
steroidogenesis pathway for breast cancer therapy, not only aromatase, but the
HSD17B enzymes (Miyoshi et al. 2001; Day et al. 2008; Day et al. 2009; Poirier
2010), AKR1C3 (Rizner et al. 2006), and sulfotransferase, which bypasses
aromatase to convert less potent steroids to estrogens (Woo et al. 2010). There is
also interest in the use of abiraterone, which inhibits CYP17A1 (Risbridger et al.
2010). As of yet, it has been assumed that breast cancer can only synthesize
estrogens from exogenous steroids and precursors, as it was for CaP before the
ability of tumours to perform de novo steroidogenesis was demonstrated by our
group (Locke et al. 2008). It may be that breast cancer cells are also able to produce
de novo estrogens, and if so, the triggers for this would be essential to understand.
For these reasons, the expression of steroidogenesis enzymes in MCF7 AR and ER
positive breast cancer cells, in the presence and absence of insulin, has been
examined. Findings in this chapter demonstrate that breast cancer cells may in fact
be capable of de novo estrogen synthesis, which may contribute to breast cancer
progression. Moreover, it has been shown herein that insulin increases mRNA
levels of steroidogenesis enzymes and secreted levels of estradiol in MCF-7 breast
cancer cells.
4.2 Materials and Methods
4.2.1 In vitro model: LNCaP cells were cultured in phenol red-free RPMI 1640
(Invitrogen) and 5% fetal calf serum (FBS; Hyclone). VCaP and MCF7 cells were
cultured in DMEM and 10% FBS. For modelling of androgen deprivation, all cell
lines were cultured in charcoal-stripped serum (CSS; Hyclone) as follows: cells
were plated in FBS and at 60% confluence were changed to 5% CSS medium for
24hrs, followed by 24hr starvation in serum-free medium, after which, LNCaP and
VCaP cells were treated with 10nM insulin (Novo Nordisk), glargine (Sanofi-
Aventis), obtained from Vancouver General Hospital Pharmacy by prescription, or
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X10 (Novo Nordisk, Denmark) for various times (48hrs for mRNA collection,
72hrs for steroid measurement) or with vehicle control. MCF7 cells were treated for
10 or 48hrs for mRNA collection and 48hr for estradiol extraction. Insulin was
refreshed every 24hrs.
4.2.2 QRT-PCR: QRT-PCR was carried out as follows: RNA was extracted from
prostate cancer cells using Trizol (Invitrogen, Burlington, Ontario, Canada), before
reverse transcription with Superscript III reverse transcriptase (Invitrogen) as
described in chapter 2.5-2.7. Subsequent QRT-PCR using Applied Biosystems
7900HT Fast Real Time PCR System used SYBR Green detection (Applied
Biosystems). Primers were designed by Primer 3 software from coding segments of
genes, obtained from the NCBI data bank and ordered from Integrated DNA
Technologies (San Diego, California, USA). Primers used for prostate cells were
SREBP, StAR, CYP11A1, CYP17A1, HSD3B2, AKR1C3, HSD17B3, and RDH5.
Additionally, HMGR, HMGS, and aromatase were used in MCF7 cells. For Primer
sequences, see Appendix A. Gene expression was normalized to the housekeeping
gene rpl32, then expressed relative to the vehicle control at the same time point.
Cycling conditions are described in chapter 2.7. Data was analyzed with SDS 2.3
software by means of the ∆∆Ct method (Livak et al. 2001). Experiments were
repeated a minimum of 5 times for CaP cells, 3 times for MCF7 cells.
4.2.3 De novo steroid analysis using radiometric detection: LNCaP cells were
grown in 6 well plates and treated as described in chapter 4.2.1. At the time of
insulin treatment, 6µCi/ml 14C-acetate (PerkinElmer, Woodbridge, Ontario) was
added to each plate for co-incubation. Steroids from 2ml of medium were extracted
with 75/25 hexane/ ethanoloacetate (water equilibrated), dried down and
resuspended into 75ul 50% methanol. Detailed method is described in chapter
2.12.These samples were analysed on the Waters Alliance 2695 HPLC System and
Packard Radiomatic Detector 150TR Flow Scintillation Analyzers. Peaks were
identified by comparison of retention times to Mix 10 steroid standard (Sigma).
4.2.4 Total levels of MCF7 estradiol as measured by LC/MS/MS:
Steroid analysis was performed as previously described, chapter 2.11. Briefly, cells
were grown in 15cm plates and treated with 10nM insulin for 10 and 48hr. Two
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plates of cells were washed with PBS and pooled to give one sample. Medium was
collected and likewise combined. Steroids were extracted from the cell pellet and
medium with a 70/30 hexane/ EtOAc solution. Once the steroids extracted from
cells and medium and dried down, steroids were derivatized with 2-fluoro-1-methyl
pyridinium qp-toluenesulfonate (FMP). All samples were run on the Waters
Acquity Liquid Chromatography system and the Waters Quattro Premier
LC/MS/MS, identified using standards of known retention times and analyzed
using Quanlynx Software (Waters Corp, USA). Readings were adjusted using cell
pellet weight and normalized to vehicle-treated samples.
4.2.5 Statistics: All statistical analysis was carried out using the two-tailed
student’s t-test assuming equal variance on Graphpad Prism 5 software. P-value of
0.05 or less was considered significant.
4. 3 Results
4.3.1 Insulin and analogs upregulate enzymes necessary for steroidogenesis at
the mRNA level
In chapter 3, it was shown that insulin upregulates the mRNA transcripts of
steroidogenesis enzyme in LNCaP and VCaP cells (Lubik et al. 2011); therefore,
LNCaP and VCaP cells were treated with insulin, glargine, or X10 (figure 4.1A). In
VCaP cells, insulin significantly upregulated mRNA for SREBP, CYP11A1,
CYP17A1, HSD3B2, HSD17B3 and RDH5 (p<0.05), whereas X10 upregulated
SREBP (p<0.1), HSD3B2, and HSD17B3 significantly (p<0.05). Glargine did not
seem to induce mRNA expression of most enzymes; however, it did show
significant increase in CYP17A1 and HSD17B3 mRNA (p<0.05).
In LNCaP cells (figure 4.1B), insulin upregulated SREBP, StAR, CYP11A1,
CYP17A1, HSD3B2, and HSD17B3 (p<0.05). Induction with X10 only
demonstrated significant induction with CYP17A1, and HSD3B2 (p<0.05), but
trended toward induction of StAR, CYP11A1, and RDH5. Finally, glargine
increased the mRNA levels of StAR, HSD3B2, and RDH5 (p<0.05). Neither analog
showed extensive similarity to the insulin induction pattern of steroidogenic
enzyme mRNA.
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Figure 4.1: Insulin and analogs regulate expression of steroidogenic enzymes at the
mRNA level. (A) Following 48 hour insulin (IN), X10, or glargine (Glar) treatment
(10nM), RNA was extracted from VCaP cells and used for QRT-PCR analysis of
steroidogenesis enzymes. Results are analyzed by ΔΔCt method and normalized to
RPL32 as a control gene, before normalization to vehicle-treated controls for the
equivalent time point.(B) Following 48 hour insulin, X10, or glargine (Glar)
treatment (10nM), RNA was extracted from LNCaP cells and used for QRT-PCR
analysis of steroidogenesis enzymes as per VCaP cells in (A). Error bars represent
SE, * = p<0.05 from control unless otherwise indicated in text n≥5. Brackets
represent statistical difference over all treatments. (B) LNCaP cells were analysed
as VCaPs.
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4.3.1 Insulin analog effect on de novo steroidogenesis in LNCaP and VCaP
medium
By treating LNCaP or VCaP cells for 72hrs with 14C-labeled acetate and subsequent
HPLC and radiometric detection, increases in de novo steroidogenesis with insulin
and analogs were demonstrated (figure 4.2A). In VCaPs, androstenedione was
increased approximately 2.5 to 3.5-fold from control with all analogs (p<0.05), and
androsterone increased approximately 2-fold with insulin and glargine, though not
significantly with X10. There was also a 1.75-2.5-fold increase in pregnan-3,17-
dio-20-one across the insulins (p<0.05). A 1.5 to 1.8-fold increase in a steroid peak
with retention time of 34 minutes, most closely resembling progesterone in
retention time, was seen with insulin and X10 (p<0.05), though not with glargine.
In VCaPs, an increase in de novo cholesterol, which is the building block of
steroids, was also seen (4-10 fold, p<0.1) with all compounds; however, because
the extraction method utilized was not specific for cholesterol, increases are more
qualitative than quantitative.
In LNCaP cells (figure 4.2B) androsterone appeared to increase 1.5-fold with
insulin, but not with either of the analogs, where decrease was seen. Pregnan-3,17-
dio-20-one was static with insulin but decreased with the analogs. The
progesterone-resembling peak at 34 minutes was upregulated 2.5-fold with insulin
(p<0.1) and glargine 2-fold (p<0.1). There was no change with X10. Insulin
showed a trend toward increase in testosterone (4.5 fold), and a 7-fold increase was
demonstrated with X10 (p<0.1), though no induction was demonstrated with
glargine. The differences between the effects on steroids and precursors in VCaP
and LNCaP cells could be a result of their different lineages. It may be that
LNCaPs, where accumulation is of those steroids further along in the pathway to
androgens, process acetate and cholesterol to steroids/ androgens faster than
VCaPs, where acetate appears to accumulate in cholesterol. We have previously
demonstrated that cancer cells adapt to their microenvironment for their
steroidogenic needs (Locke et al. 2009a), which could be the reason for differences
in expression of steroidogenic enzyme mRNA and steroid induction between the
LNCaP and VCaP cells, as the metastases they were derived from came from
different microenvironments.
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Figure 4.2: Insulin analogs increase de novo steroidogenesis in prostate cancer
cells. Medium was collected from VCaP and LNCaP cells after 72hr incubation
with 10nM insulin, X10, or glargine (Glar) and 6µCi/ml 14C-acetate before HPLC
and radiometric detection were used to identify and quantitate extracellular steroids.
(A) In VCaPs, all analogs demonstrated a similar steroid profile. In LNCaPs (B),
insulin and X10 showed similar inductions of testosterone, where insulin and
glargine had similar effects on a peak resembling progesterone. Error bars
represent SE (*p<0.1), n=3.
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4.3.3 Insulin effects on steroidogenesis in breast cancer
The expression of steroidogenesis enzymes in MCF7 cells, including HMGR and
HMGS cholesterol synthesis enzymes, was investigated after 10 and 48hrs insulin
treatments (10nM) (figure 4.3A). At 10hrs, an increase in the cholesterol synthesis
enzymes was apparent, with approximately a 1.5 and 2-fold upregulation for
HMGR and HMGS mRNA, respectively, with little change in other steroidogenic
enzymes (data not shown). At 48hr, the presence of mRNA transcripts for all
necessary enzymes for steroidogenesis was detected with appreciable Ct values
(between 19- 33 cycles). No change was demonstrated for SREBP or StAR at these
times. This does not rule out a role for these factors in breast cell steroidogenesis as
activation; protein level and activation state maybe more informative for these
enzymes, as was the case in VCaP cell steroidogenesis (chapter 3). Significant 25%
increases in mRNA were demonstrated for CYP17A1 and HSD3B2. This may be of
interest to the groups studying the effects of abiraterone on breast cancer. A 2-fold
increase in HSD17B3 mRNA was apparent (p<0.05). There was little change in
SRD5A1 or RDH5. A striking increase in AKR1C3 was demonstrated (p<0.05),
which may indicate that targeting this enzyme in hyperinsulinemic breast cancer
patients would be beneficial, as AKR1C3 converts androstenedione to testosterone,
which can then be converted into estrogens (Ishikawa et al. 2006). Interestingly,
there appears to be an unanticipated decrease in aromatase; however, there are 2
enzymes responsible for the formation of estradiol, the most potent estrogen, and
the other is sulfotransferase. At the time of these experiments, this enzyme was not
examined, but in hyperinsulinemic patients it has been demonstrated that aromatase
inhibitors are not as effective (Goodwin 2008); therefore, a shift towards active
sulfotransferase may occur and should be investigated, as this may actually be the
driving force for estradiol synthesis in some breast cancers (Santen et al. 1986;
Pasqualini et al. 1996; Woo et al. 2010).
Of most importance, in treating steroid and insulin starved MCF7 cells with insulin
for 48hr in the presence of non-labelled acetate, a 50% decrease in intracellular
estradiol occurs in the presence of insulin, with a corresponding 2.3-fold (p<0.05)
increase in estradiol concentration in the medium (figure 4.3B,C). These are
sufficient concentrations to activate ER, at approximately 2.5pM; as 0.26pM
estradiol is required for minimum ER activation (Mattick et al. 1997).
127
4.4 Discussion
Much recent interest has been shown in the effects of insulin and analogs on cancer
(Pollak et al. 2010). Conflicting studies report that analogs may have more
carcinogenic potential than normal insulin, and most evidence supports the theory
that this may be through dysregulated IGF1R or INSR signalling; in fact, most
evidence of increases in mitogenic potential is demonstrated to be through IGF1R
(Kurtzhals et al. 2000; Shukla et al. 2009; Weinstein et al. 2009).
Herein it has been revealed that induction of steroidogenesis enzyme mRNA and
steroid levels in VCaP cells appears to be dissimilar between insulin, X10 and
glargine (long acting insulin). Interestingly, glargine, for which most evidence of
increased cancer risk has been reported, seems to show less steroidogenic potential
than the others. In LNCaPs, neither analog appears to have equal activity to insulin
for induction of steroidogenesis enzyme mRNA; X10 trends toward more similarity
than glargine. Significantly, though insulin induced androsterone production,
neither analog did. Increases in a progesterone-like peak were seen with insulin and
glargine, but not X10; whereas, both insulin and X10 seemed to increase
testosterone levels, with Glargine having no effect.
These findings indicate that clinical use of insulin analogs may not have any more
consequences for patients with prostate cancer than normal insulin, at least in terms
of stimulating steroidogenesis. It is interesting to note that X10 and insulin had
more effects on the cells, where glargine seemed to be less potent. As glargine has
been shown in breast cancer to implement signalling similar to IGF1, it may be that
glargine is affecting other proliferative and/ or mitogenic pathways (Varewijck et
al. 2010), which are beyond the scope of this study. It has been suggested that
insulin and analogs do not cause cancer transformation, but increase the growth of
precancerous lesions (Miller 2007), which would suggest these compounds should
all be used cautiously in patients predisposed to cancer. The difference in effects of
the insulin analogs on VCaP and LNCaP cells suggests that the analogs may have
differential effects on various cancer stages. It is important to note that the benefits
of insulin use for T1D and T2D patients far outweigh the risks (Pollak et al. 2010).
More studies will have to be done to determine if there is more cancer risk with
analogs, especially glargine. However, at the present time, the connection is
unconvincing. Interestingly, any increased risk of cancer growth with glargine or
128
other insulins in colorectal or pancreatic cancer was suppressed with metformin use
(Currie et al. 2009), a promising new finding for metabolic syndrome sufferers,
which will be further discussed in chapter 6.
Furthermore, metabolic syndrome has been shown to exacerbate breast cancer
progression, as well as CaP (Redig et al.2010a; Goodwin 2010a). Data shown in
this chapter supports the hypotheses that (a) breast cancer cells may be capable of
de novo steroidogenesis (b) and insulin may increase de novo steroidogenesis in
breast cancer cells, and exacerbate the conversion of exogenous precursors to
estradiol. These pathways should be further explored, in the presence and absence
of metformin; the effect of metformin on steroidogenesis will be demonstrated in
chapter 6.
129
0
1
2
3
4
5
6
AKR1C3
0
0.5
1
1.5
2
2.5
MCF7 mRNA Fold Steroid Enzyme Chan
ge from
Control (48hr)
Control
Insulin
0
0.5
1
1.5
2
2.5
MCF7 mRNA Fold Steroid Enzyme Change
from Control (10hr)
*
*
* *
**
0
0.5
1
1.5
2
2.5
3
Control Insulin
Change
in Ediolin M
edia (pM) (48h
rs) Ediol
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
0.05
Control Insulin
Chan
ge in In
tracellu
lar Ed
iol(ng/g pellet) (48hr)
Ediol *
A
B C
(Figure 4.3, page over)
130
Figure 4.3: Insulin regulates expression of key steroidogenic enzymes at the
mRNA level and increases estradiol secretion. A) Following 10 and 48 hour insulin
treatment (10nM), RNA was extracted from MCF7 cells and used for QRT-PCR
analysis of steroidogenesis enzymes. Results are analyzed by ΔΔCt method and
normalized to RPL32 as a control gene, before normalization to vehicle-treated (no
insulin) for the equivalent time point. Error bars represent SE (*p<0.05). MCF7
cells were treated with 10nM insulin for 48 hours and estradiol (Ediol) was
extracted from B) cell pellet and C) media. LC-MS was used to quantitate
intracellular steroids showing a significant decrease in intracellular Ediol, and a
significant increase in secreted Ediol. Steroid levels were adjusted to cell pellet
weight and deuterated testosterone for extraction efficiency and compared to the
vehicle time point control. Error bars represent SE (*p<0.05), n=3.
131
Chapter 5: Insulin increases Fatty Acid Synthesis in
Prostate Cancer Cells.
132
133
5.1 Introduction
Humans can derive necessary fatty acids from either their diet or de novo synthesis.
In most diets, the exogenous fat intake is sufficient and fatty acid synthase (FASN),
which is responsible for converting malonyl-CoA and acetyl-CoA into long-
chained fatty acids, is expressed at low or undetectable levels in normal tissues
(Menendez et al. 2006). In normal cells, fatty acids are converted into triglycerides
for storage. In cancers, however, FASN over-expression has been noted for the
better part of a century (Menendez et al. 2006), and FASN over-expression has
been shown by immunohistochemistry in prostate epithelial tumours and prostate
intraepithelial neoplasia (PIN). Over-expression of FASN protein/ mRNA has also
been demonstrated in breast, colorectal, bladder, ovarian, oesophageal, stomach,
lung, thyroid, and endometrial cancers as well as oral squamous carcinoma, head
and neck carcinoma, mesothelioma, nephroblastoma, retinoblastoma, soft tissue
sarcomas and cutaneous melanocytic neoplasms (Menendez et al. 2006).
Furthermore, FASN over-expression is associated with more aggressive cancers
and bone metastasis, as well as poor patient outcomes in CaP (Swinnen et al. 2000;
Baron et al. 2004; Horiguchi et al. 2008). Finally, it has been suggested that it
may be a marker for poor differentiation in breast cancer (Alo et al. 1999).
In in vivo and in vitro cancer models, inhibition of FASN or enzymes associated
with lipogenesis, such as sterol regulatory element binding protein (SREBP) and
acetyl CoA carboxylase (ACC), has been shown to cause apoptosis or to halt cell
growth (Alo et al. 1999; Swinnen et al. 2006; Beckers et al. 2007; Ho et al. 2007;
Orita et al. 2007; Orita et al. 2008; Migita et al. 2009). SREBP, the transcriptional
factor that controls FASN expression, becomes dysregulated in prostate cancer
progression. During progression to castrate resistance it responds to androgen and
growth factor signalling, rather than traditional regulatory pathways, ultimately
resulting in increased FASN expression (Swinnen et al. 1996; Swinnen et al.
1997b; Baron et al. 2004; Ettinger et al. 2004).
Insulin stimulation of FASN was first investigated by Monaco in 1977 in MCF7
breast cancer cell lines because these cells responded similarly to normal mammary
gland tissue in response to insulin, androgens, estrogens, and glucocorticoids
(Monaco et al. 1977). Measuring a surrogate for total fatty acid synthesis and 14C-
acetate for de novo fatty acid synthesis, the authors showed that insulin at
134
physiological concentrations increased fatty acid synthesis. In this study, total
cellular mRNA and protein production did not seem to correlate to the increase in 14C-acetate labelled lipids; however, the authors did not examine specific enzymes.
Interestingly, FASN, also called oncogenic antigen-519 or OA-519, was not
suggested as an oncogene until the 1990s (Epstein et al. 1995).
Lipids are a well-known major source of energy for prostate cancer cells, but they
also have a multitude of other functionalities (Liu 2006). It is thought that
increased lipid content is necessary for processes such as cell growth, cell
proliferation, and membrane expansion, as well as FASN-mediated supply of
palmitate and myristate for intracellular trafficking and changes in phospholipid
content in the cell membranes, which alters signalling pathways (Baron et al. 2004;
Swinnen et al. 2006). In CaP, it has also been shown that fatty acid activation is
important for initiation of steroidogenesis, through the action of hormone sensitive
FASN Santa Cruz 200 180 Rabbit sc-20140 Human, mouse
HMGR Millipore 500 63 Rabbit O7572
Human, mouse, rabbit, rat
Secondary Antibodies
242
Anti-Rabbit (IRDye 680CW) Licor 10 000 N/A Donkey
926-68023 Rabbit
Anti-Goat (IRDye 800CW) Licor 11 000 N/A Donkey
926-32214 Goat
Anti-Mouse (IRDye 800CW) Licor 12 000 N/A Donkey
926-32212 Mouse
243
References
244
245
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