Mol Cell Biochem 2017 Aug;432(1-2):41-54

Prevention of tumour cell apoptosis associated with sustained protein kinase B phosphorylation is more sensitive to regulation by insulin signalling than stimulation of proliferation and extracellular signal-regulated kinase 

Christoph Schmida, Claudia Ghirlandaa, Markus Niessena,b

aDivision of Endocrinology, Diabetology and Clinical Nutrition, University Hospital of Zurich, 8091 Zurich. bCompetence Centre for Systems Physiology and Metabolic Diseases, Swiss Federal Institute of Technology (ETH) Zurich, 8093 Zurich

Address correspondence and reprint requests to: Christoph Schmid, Raemistrasse 100, 8091 Zurich, Fax: +41-44-255’97’41; E-mail: christoph.schmid@usz.ch

 

Abstract

Insulin controls blood glucose while insulin-like growth factor (IGF) 1 is an important growth factor. Interestingly, both hormones have overlapping bioactivities and can activate the same intracellular signal transduction cascades. Growth control (mainly by IGF1) and metabolic function (predominantly by insulin) are believed to depend on activation of extracellular signal-regulated kinases (ERKs) 1/2 and protein kinase B (Akt/PKB), respectively. Therefore, insulin analogues that are used to normalize blood glucose are tested for their ability to preferentially activate Akt/PKB but not ERK1/2 and mitogenesis. Growth hormone, IGF1, and hyperinsulinaemia are associated with increased risk of growth progression of some cancer types. To test if continuous exposure to insulin can favour tumour growth, we studied insulin/IGF1-dependent activation of ERK1/2 and Akt/PKB by Western blotting, inhibition of apoptosis by ELISA, and induction of proliferation by [3H]-thymidine incorporation in Saos-2/B10 osteosarcoma cells. IGF1 and insulin both induced proliferation and prevented apoptosis effectively. Regulation of apoptosis was far more sensitive than regulation of proliferation. IGF1 and insulin activated PKB (Akt/PKB) rapidly and consistently maintained its phosphorylation. Activation of ERK1/2 was only observed in response to IGF1. Loss of p-Akt/PKB (but not of p-ERK1/2) was associated with increased apoptosis, and protection from apoptosis was lost when activation of Akt/PKB was inhibited. These findings in Saos-2/B10 cells were also replicated in the A549 cell line, originally derived from a human lung carcinoma. Therefore, IGF1 and insulin more likely (at lower concentrations) enhance tumour cell survival than proliferation, via activation and maintenance of phosphatidylinositol 3-kinase activity and p-Akt/PKB.

Keywords: Apoptosis, IGF, Insulin, Obesity, Osteosarcoma cells, PI3K.

 

Introduction

Insulin and IGF1 both promote growth. Conditions with high levels of insulin and/or IGF1, such as obesity or acromegaly, are associated with increased risk for certain types of cancer. Dietary restriction (DR), associated with lower levels of insulin/IGF1, is considered to increase life span and to be antitumourigenic [1]. Furthermore, epidemiological data, although difficult to interpret, suggest that not only obesity and type 2 diabetes but particularly the use of the long acting insulin analogue glargine may be linked to increased growth of cancer [2-4].

Insulin and IGF action is mediated via compounded intracellular signal transduction cascades [5-7]. Both hormones employ insulin receptor substrate proteins to activate phosphatidyl inositol 3 kinase (PI3K)-dependent or extracellular ligand-regulated kinases (ERK) 1/2-dependent downstream events. Metabolic insulin action depends on PI3K, and insulin typically is more potent than IGF in activating PI3K signalling in metabolic target tissues. IGF, on the other hand, potently activates ERK1/2 and there is ample evidence showing that this pathway links IGF to the regulation of mitogenesis [8].

Insulin, but not IGFs, lowers blood glucose by decreasing hepatic glucose production and increasing glucose uptake in skeletal muscle. It is secreted in a highly pulsatile fashion by pancreatic beta cells in response to rising plasma glucose levels [9-11]. The pulsatile pattern is disrupted in obesity and prediabetes, and hyperinsulinaemia is common in these patients. When endogenous insulin fails to maintain glucose homeostasis, as it is the case in diabetes, the hormone is replaced. In such cases, the doses of insulin required often exceed the amounts produced by healthy individuals, particularly in obese, insulin-resistant patients with type 2 diabetes. Current treatment modalities include injections of so-called long acting insulin analogues or continuous insulin infusion with pumps, with the goal to provide the patient with a constant baseline of circulating insulin. Although somewhat less effective than pulsatile insulin secretion in lowering blood glucose, this is done to increase predictive and reliable insulin delivery and to minimize the occurrence of hypoglycaemia, a condition that can result in unconsciousness and even death.

In order to optimize metabolic performance and safety of insulin analogues (insulin receptor-binding agonists) candidate molecules are tested for their ability to preferentially activate PI3K-dependent signalling but not ERK1/2. Our laboratory has performed a number of in vitro studies with IGFs, insulin and analogues thereof for more than 4 decades, more recently often using a Saos-2/B10 osteosarcoma cell line, as this particular cell line does not produce any IGF and is especially responsive to insulin/IGF [12-15].

Tissue (or tumour) size depends on cell number and cell size. The former depends on the balance between proliferation and apoptosis. A tissue will grow if proliferation rate exceeds apoptosis and if cell size remains the same or increases. A tissue will shrink if apoptosis rate exceeds proliferation and cell size remains the same or decreases. Thus far, cancer-related preclinical safety evaluation of insulin analogues has traditionally focused on assessing mitogenic potency only. Studies with xenografted tumours in mice have shown that growth of some (but not all) tumours is sensitive to DR [16]. The question could be asked whether cancer cells care if their host is hungry [17]. DR-resistant tumours carried mutations leading to constitutive activation of PI3K-dependent signalling, e.g. loss of PTEN. In culture, such tumour cells could proliferate independent of insulin or IGF. Importantly, apoptosis was much more affected by DR than proliferation in DR-sensitive tumours.

In our recently published study [18], we explored in more detail antiapoptotic effects of insulin and IGF1 in the same Saos-2/B10 osteosarcoma cell line. Our results showed that insulin and IGF1 are far more potent in regulating apoptosis than proliferation, and the concentrations required for apoptosis inhibition were in the high physiological or therapeutic dose range. Prevention of apoptosis required the continuous exposure to insulin or IGF1 and was dependent on sustained activation of the PI3K target Akt/PKB but not on ERK1/2. Our findings suggested that insulin/IGF1 more likely (at lower concentrations) enhance tumour growth via regulation of PI3K-Akt/PKB and apoptosis than via regulation of ERK1/2 and proliferation. Safety tests with insulin analogues should hence include assays to assess apoptosis and activation/maintenance of PI3K-dependent signalling.

In the following supplement we show complementary results obtained with insulin and the long acting analogue insulin glargine.

 

 

Figure 1: Time course of p-Akt/PKB and cleaved caspase-3 in Saos-2/B10 cells following serum deprivation, in the absence (control) and presence of insulin. Cells were serum-deprived and cultured for the indicated time periods with or without insulin (100 nmol/l). At the end of the incubations cells were washed and lysed. Lysates were analysed by Western blotting with antibodies against p-Akt/PKB (Ser473: Cell Signaling, Danvers, USA), cleaved caspase-3 (Asp175, Cell Signaling, Danvers, USA), and actin (clone C4, MAB1501, Millipore/Merck, Darmstadt, Germany).

 

 

 

Figure 2: Glargine stimulates phosphorylation of Akt/PKB and prevents apoptosis in Saos-2/B10 cells. Cells were serum-deprived for 4 hours and then lysed; glargine was added at the indicated time points prior to lysis. Lysates were analysed by immunoblotting (A) with antibodies against the non-phosphorylated and phosphorylated forms of Akt/PKB (BD Transduction Laboratories, San Jose,USA and Ser473: Cell Signaling, Danvers, USA) and ERK1/2 (Cell Signaling, Danvers, USA and Thr202/Tyr204: Cell Signaling, Danvers, USA), respectively. Actin (clone C4, MAB1501, Millipore/Merck, Darmstadt, Germany) was visualized as a control. Apoptosis (B) is shown relative to 4 hour control; apoptosis was assessed with the Cell Death Detection ELISAPLUS Kit from Boehringer Mannheim.

 

Supplement

Our study focused on experiments in a short time setting immediately following serum deprivation, when Saos-2/B10 cells undergo apoptosis and where effects of insulin and IGF1 could be tested. In this setting cleaved (activated) caspase-3 was detectable two hours after serum withdrawal but not if cells were cultured in the presence of insulin (Figure 1). In the control (without IGF1 or insulin), there is a time-dependent loss of p-Akt/PKB, concomitant with an increase in cleaved caspase-3. Insulin promotes rapid and sustained phosphorylation of Akt/PKB.

As shown in Figure 2, glargine (like insulin and IGF) potently phosphorylates/activates Akt/PKB and inhibits apoptosis. Importantly, minimal time period with low p-Akt/PKB is required to initiate apoptosis; late addition of IR binding agonists does not protect from apoptosis. In such experiments (Figures 1 and 2), insulin and IGF1 are at least as effective as serum in preventing apoptosis and in stimulating and maintaining p-Akt/PKB. However, they are less effective than serum in stimulating DNA synthesis.

EC50 values derived from dose-response curves are given for IGF1, insulin and insulin glargine in Table 1. For the latter two, EC50 values for mitogenesis lie well above levels typically observed in vivo. However, EC50 values for apoptosis inhibition are closer to levels observed in vivo. The EC50 values indicate increasing potency from insulin to glargine to IGF1. It is well recognized that insulin is far less mitogenic than IGF1, and that it is less antiapoptotic [13-15]. It should be kept in mind that IGFs (in contrast to insulin and its analogues) are tightly bound to IGFBPs in vivo.

Glargine dose-dependently decreases apoptosis. In the presence of wortmannin (inhibitor of PI3K) and thus prevention of Akt/PKB phosphorylation, glargine (like insulin or IGF1) no longer inhibits apoptosis (Table 2).

 

 

Table 1: EC50 values for induction of proliferation and inhibition of apoptosis in Saos-2/B10cellsby IR/IGFR agonists. Data are from at least 3 dose-response curves.

 

 

Table 2: Wortmannin inhibits glargine-dependent protection from apoptosis in Saos-2/B10 cells. Effects of insulin glargine on apoptosis, in the absence and presence of wortmannin (100 nmol/l). Condition without addition of substance was set to 100%. Saos-2/B10 cells were serum-deprived for 4 hours in the presence or absence of wortmannin plus/minus glargine. At the end of the incubation time, cells were lysed and apoptosis was determined with the Cell Death Detection ELISAPLUS Kit from Boehringer Mannheim. n=3 in triplicate.

 

References

[1]        Wei M, Brandhorst S, Shelehchi M, et al. (2017) Fasting-mimicking diet and markers/risk factors for aging, diabetes, cancer, and cardiovascular disease. Science translational medicine Feb 15; 9

[2]        Currie CJ, Johnson JA (2012) The safety profile of exogenous insulin in people with type 2 diabetes: justification for concern. Diabetes Obes Metab 14: 1-4

[3]        Smith U, Gale EA (2009) Does diabetes therapy influence the risk of cancer? Diabetologia 52: 1699-1708

[4]        Wu JW, Azoulay L, Majdan A, Boivin JF, Pollak M, Suissa S (2017) Long-Term Use of Long-Acting Insulin Analogs and Breast Cancer Incidence in Women With Type 2 Diabetes. J Clin Oncol 35: 3647-3653

[5]        Boller S, Joblin BA, Xu L, et al. (2012) From signal transduction to signal interpretation: an alternative model for the molecular function of insulin receptor substrates. Arch Physiol Biochem 118: 148-155

[6]        Schultze SM, Hemmings BA, Niessen M, Tschopp O (2012) PI3K/AKT, MAPK and AMPK signalling: protein kinases in glucose homeostasis. Expert Rev Mol Med 14: e1

[7]        Schultze SM, Jensen J, Hemmings BA, Tschopp O, Niessen M (2011) Promiscuous affairs of PKB/AKT isoforms in metabolism. Arch Physiol Biochem 117: 70-77

[8]        Czech MP (2017) Insulin action and resistance in obesity and type 2 diabetes. Nat Med 23: 804-814

[9]        Lefebvre PJ, Paolisso G, Scheen AJ, Henquin JC (1987) Pulsatility of insulin and glucagon release: physiological significance and pharmacological implications. Diabetologia 30: 443-452

[10]      Porksen N, Hollingdal M, Juhl C, Butler P, Veldhuis JD, Schmitz O (2002) Pulsatile insulin secretion: detection, regulation, and role in diabetes. Diabetes 51 Suppl 1: S245-254

[11]      Satin LS, Butler PC, Ha J, Sherman AS (2015) Pulsatile insulin secretion, impaired glucose tolerance and type 2 diabetes. Molecular aspects of medicine 42: 61-77

[12]      Froesch ER, Schmid C, Schwander J, Zapf J (1985) Actions of insulin-like growth factors. Annu Rev Physiol 47: 443-467

[13]      Kurtzhals P, Schaffer L, Sorensen A, et al. (2000) Correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use. Diabetes 49: 999-1005

[14]      Schmid C, Ghirlanda-Keller C, Zapf J (2001) Effects of IGF-I and -II, IGF binding protein-3 (IGFBP-3), and transforming growth factor-beta (TGF-beta) on growth and apoptosis of human osteosarcoma Saos-2/B-10 cells: lack of IGF-independent IGFBP-3 effects. Eur J Endocrinol 145: 213-221

[15]      Schmid C, Rutishauser J, Schlapfer I, Froesch ER, Zapf J (1991) Intact but not truncated insulin-like growth factor binding protein-3 (IGFBP-3) blocks IGF I-induced stimulation of osteoblasts: control of IGF signalling to bone cells by IGFBP-3-specific proteolysis? Biochem Biophys Res Commun 179: 579-585

[16]      Kalaany NY, Sabatini DM (2009) Tumours with PI3K activation are resistant to dietary restriction. Nature 458: 725-731

[17]      Pollak M (2009) Do cancer cells care if their host is hungry? Cell Metab 9: 401-403

[18]      Schmid C, Ghirlanda C, Niessen M (2017) Prevention of tumour cell apoptosis associated with sustained protein kinase B phosphorylation is more sensitive to regulation by insulin signalling than stimulation of proliferation and extracellular signal-regulated kinase. Mol Cell Biochem 432: 41-54