Up-regulation of acetyl-CoA carboxylase alpha and fatty acid synthase by human epidermal growth factor receptor 2 at the translational level in breast cancer cells

Expression of the HER2 oncogene is increased in approximately 30% of human breast carcinomas and is closely correlated with the expression of fatty acid synthase (FASN). In the present study, we determined the mechanism by which FASN and acetyl-CoA carboxylase alpha (ACCalpha) could be induced by HER2 overexpression. SK-BR-3 and BT-474 cells, breast cancer cells that overexpress HER2, expressed higher levels of FASN and ACCalpha compared with MCF-7 and MDA-MB-231 breast cancer cells in which HER2 expression is low. The induction of FASN and ACCalpha in BT474 cells were not mediated by the activation of SREBP-1. Exogenous HER2 expression in MDA-MB-231 cells induced the expression of FASN and ACCalpha, and the HER2-mediated increase in ACCalpha and FASN was inhibited by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. In addition, the activation of mTOR by the overexpression of RHEB in MDA-MB-231 cells increased the synthetic rates of both FASN and ACCalpha. On the other hand, FASN and ACCalpha were reduced in BT-474 cells by a blockade of the mTOR signaling pathway. These changes observed in their protein levels were not accompanied by changes in their mRNA levels. The 5'- and 3'-untranslated regions of both FASN and ACCalpha mRNAs were involved in selective translational induction that was mediated by mTOR signal transduction. These results strongly suggest that the major mechanism of HER2-mediated induction of FASN and ACCalpha in the breast cancer cells used in this study is translational regulation primarily through the mTOR signaling pathway.     

miR‐126 reduces trastuzumab resistance by targeting PIK3R2 and regulating AKT/mTOR pathway in breast cancer cells

MicroRNAs (miRNAs) have been found to play a key role in drug resistance. In the current study, we aimed to explore the potential role of miR‐126 in trastuzumab resistance in breast cancer cells. We found that the trastuzumab‐resistant cell lines SKBR3/TR and BT474/TR had low expression of miR‐126 and increased ability to migrate and invade. The resistance, invasion and mobilization abilities of the cells resistant to trastuzumab were reduced by ectopic expression of miR‐126 mimics. In comparison, inhibition of miR‐126 in SKBR3 parental cells had the opposite effect of an increased resistance to trastuzumab as well as invasion and migration. It was also found that miR‐126 directly targets PIK3R2 in breast cancer cells. PIK3R2‐knockdown cells showed decreased resistance to trastuzumab, while overexpression of PIK3R2 increased trastuzumab resistance. In addition, our finding showed that overexpression of miR‐126 reduced resistance to trastuzumab in the trastuzumab‐resistant cells and that inhibition of the PIK3R2/PI3K/AKT/mTOR signalling pathway was involved in this effect. SKBR3/TR cells also showed increased sensitivity to trastuzumab mediated by miR‐126 in vivo. In conclusion, the above findings demonstrated that overexpression of miR‐126 or down‐regulation of its target gene may be a potential approach to overcome trastuzumab resistance in breast cancer cells.     

Metformin inhibits breast cancer cell growth,colony formation and induces cell cycle arrest in vitro

The anti-diabetic drug metformin reduces human cancer incidence and improves the survival of cancer patients, including those with breast cancer. We studied the activity of metformin against diverse molecular subtypes of breast cancer cell lines in vitro. Metformin showed biological activity against all estrogen receptor (ER) positive and negative, erbB2 normal and abnormal breast cancer cell lines tested. It inhibited cellular proliferation, reduced colony formation and caused partial cell cycle arrest at the G1 checkpoint. Metformin did not induce apoptosis (as measured by DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 subtype breast cancer cell lines. At the molecular level, metformin treatment was associated with a reduction of cyclin D1 and E2F1 expression with no changes in p27kip1 or p21waf1. It inhibited mitogen activated protein kinase (MAPK) and Akt activity, as well as the mammalian target of rapamycin (mTOR) in both ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. In erbB2-overexpressing breast cancer cell lines, metformin reduced erbB2 expression at higher concentrations, and at lower concentrations within the therapeutic range, it inhibited erbB2 tyrosine kinase activity evidenced by a reduction of phosphorylated erbB2 (P-erbB2) at both auto- and Src- phosphorylation sites. These data suggest that metformin may have potential therapeutic utility against ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells.     

Protein-based identification of quantitative trait loci associated with malignant transformation in two HER2+ cellular models of breast cancer

Background

A contemporary view of the cancer genome reveals extensive rearrangement compared to normal cells. Yet how these genetic alterations translate into specific proteomic changes that underpin acquiring the hallmarks of cancer remains unresolved. The objectives of this study were to quantify alterations in protein expression in two HER2+ cellular models of breast cancer and to infer differentially regulated signaling pathways in these models associated with the hallmarks of cancer.

Results

A proteomic workflow was used to identify proteins in two HER2 positive tumorigenic cell lines (BT474 and SKBR3) that were differentially expressed relative to a normal human mammary epithelial cell line (184A1). A total of 64 (BT474-184A1) and 69 (SKBR3-184A1) proteins were uniquely identified that were differentially expressed by at least 1.5-fold. Pathway inference tools were used to interpret these proteins in terms of functionally enriched pathways in the tumor cell lines. We observed "protein ubiquitination" and "apoptosis signaling" pathways were both enriched in the two breast cancer models while "IGF signaling" and "cell motility" pathways were enriched in BT474 and "amino acid metabolism" were enriched in the SKBR3 cell line.

Conclusion

While "protein ubiquitination" and "apoptosis signaling" pathways were common to both the cell lines, the observed patterns of protein expression suggest that the evasion of apoptosis in each tumorigenic cell line occurs via different mechanisms. Evidently, apoptosis is regulated in BT474 via down regulation of Bid and in SKBR3 via up regulation of Calpain-11 as compared to 184A1.     

Distinct apoptotic blocks mediate resistance to panHER inhibitors in HER2+ breast cancer cells

Despite the development of novel targeted therapies, de novo or acquired chemoresistance remains a significant factor for treatment failure in breast cancer therapeutics. Neratinib and dacomitinib are irreversible panHER inhibitors, which block their autophosphorylation and downstream signaling. Moreover, neratinib and dacomitinib have been shown to activate cell death in HER2-overexpressing cell lines. Here we showed that increased MCL1 and decreased BIM and PUMA mediated resistance to neratinib in ZR-75-30 and SKBR3 cells while increased BCL-XL and BCL-2 and decreased BIM and PUMA promoted neratinib resistance in BT474 cells. Cells were also cross-resistant to dacomitinib. BH3 profiles of HER2+ breast cancer cells efficiently predicted antiapoptotic protein dependence and development of resistance to panHER inhibitors. Reactivation of ERK1/2 was primarily responsible for acquired resistance in SKBR3 and ZR-75-30 cells. Adding specific ERK1/2 inhibitor SCH772984 to neratinib or dacomitinib led to increased apoptotic response in neratinib-resistant SKBR3 and ZR-75-30 cells, but we did not detect a similar response in neratinib-resistant BT474 cells. Accordingly, suppression of BCL-2/BCL-XL by ABT-737 was required in addition to ERK1/2 inhibition for neratinib- or dacomitinib-induced apoptosis in neratinib-resistant BT474 cells. Our results showed that different mitochondrial apoptotic blocks mediated acquired panHER inhibitor resistance in HER2+ breast cancer cell lines as well as highlighted the potential of BH3 profiling assay in prediction of panHER inhibitor resistance in breast cancer cells.     

Mechanisms by Which Low Glucose Enhances the Cytotoxicity of Metformin to Cancer Cells Both In Vitro and In Vivo

Different cancer cells exhibit altered sensitivity to metformin treatment. Recent studies suggest these findings may be due in part to the common cell culture practice of utilizing high glucose, and when glucose is lowered, metformin becomes increasingly cytotoxic to cancer cells. In low glucose conditions ranging from 0 to 5 mM, metformin was cytotoxic to breast cancer cell lines MCF7, MDAMB231 and SKBR3, and ovarian cancer cell lines OVCAR3, and PA-1. MDAMB231 and SKBR3 were previously shown to be resistant to metformin in normal high glucose medium. When glucose was increased to 10 mM or above, all of these cell lines become less responsive to metformin treatment. Metformin treatment significantly reduced ATP levels in cells incubated in media with low glucose (2.5 mM), high fructose (25 mM) or galactose (25 mM). Reductions in ATP levels were not observed with high glucose (25 mM). This was compensated by enhanced glycolysis through activation of AMPK when oxidative phosphorylation was inhibited by metformin. However, enhanced glycolysis was either diminished or abolished by replacing 25 mM glucose with 2.5 mM glucose, 25 mM fructose or 25 mM galactose. These findings suggest that lowering glucose potentiates metformin induced cell death by reducing metformin stimulated glycolysis. Additionally, under low glucose conditions metformin significantly decreased phosphorylation of AKT and various targets of mTOR, while phospho-AMPK was not significantly altered. Thus inhibition of mTOR signaling appears to be independent of AMPK activation. Further in vivo studies using the 4T1 breast cancer mouse model confirmed that metformin inhibition of tumor growth was enhanced when serum glucose levels were reduced via low carbohydrate ketogenic diets. The data support a model in which metformin treatment of cancer cells in low glucose medium leads to cell death by decreasing ATP production and inhibition of survival signaling pathways. The enhanced cytotoxicity of metformin against cancer cells was observed both in vitro and in vivo.     

Metformin targets Stat3 to inhibit cell growth and induce apoptosis in triple-negative breast cancers

A distinct group of breast cancers, called “basal” or “triple-negative” (TN) cancers express both basal cytokeratins and the epidermal growth factor receptor, but fail to express estrogen receptors, progesterone receptors or HER2 and have stem-like or mesenchymal features. They are particularly aggressive, are frequently chemo-resistant, with p53 mutation, up-regulation of IL-6 and Stat3. Because TN cells are particularly sensitive to the anti-diabetic agent metformin, we hypothesized that it may target JAK2/Stat3 signaling. The effects of metformin upon Stat3 expression and activation were examined in four human TN cell lines. Metformin’s effects were also studied in sublines with forced over-expression of constitutively active (CA) Stat3, as well as lines with stable knockdown of Stat3. Metformin inhibited Stat3 activation (P-Stat3) at Tyr705 and Ser727 and downstream signaling in each of the four parental cell lines. CA-Stat3 transfection attenuated, whereas Stat3 knockdown enhanced, the effects of metformin upon growth inhibition and apoptosis induction. A Stat3 specific inhibitor acted synergistically with metformin in reducing cell growth and inducing apoptosis. An mTOR inhibitor showed no significant interaction with metformin. In summary, Stat3 is a critical regulator of metformin action in TN cancer cells, providing the potential for enhancing metformin’s efficacy in the clinical setting.     

Phosphorylated ribosomal S6 (p-rpS6) as a post-treatment indicator of HER2 signalling targeted drug resistance

Abstract

Objective: To identify clinically relevant predictive biomarkers of trastuzumab resistance.

Material and methods: MTT, FACS assays, immunoblotting and immunocytochemistry were used to phenotypically characterize drug responses of two cell models BT474R and SKBR3R. Student's t-test and Spearman's correlation were applied for statistic analysis.

Results: The activity of a downstream effector of the HER2 pathway phosphorylated ribosomal protein S6 (p-rpS6), was suppressed by trastuzumab in the parental cell lines yet remained unchanged in the resistant cells following treatment. The level of p-rpS6 was inversely correlated to the drug induced growth inhibition of trastuzumab-resistant cells when they are treated with selected HER2 targeting drugs.

Conclusion: p-rpS6 is a robust post-treatment indicator of HER2 pathway-targeted therapy resistance.     

Activation of Akt by Advanced Glycation End Products (AGEs): Involvement of IGF-1 Receptor and Caveolin-1

Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ) on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3-Kinase and Akt are involved in the facilitation of adipogenesis by AGEs.     

Targeting of HER3 with Functional Cooperative miRNAs Enhances Therapeutic Activity in HER2-Overexpressing Breast Cancer Cells

Background

The HER3 receptor functions as a major cause of drug resistance in cancer treatment. It is believed that therapeutic targeting of HER3 is required to improve patient outcomes. It is not clear whether a novel strategy with two functional cooperative miRNAs would effectively inhibit erbB3 expression and potentiate the anti-proliferative/anti-survival effects of a HER2-targeted therapy (trastuzumab) and chemotherapy (paclitaxel) on HER2-overexpressing breast cancer cells.

Results

Combination of miR-125a and miR-205, as compared to either miRNA alone, potently inhibited expression of HER3 in HER2-overexpressing breast cancer BT474 cells. Co-expression of the two miRNAs not only reduced the levels of phosphorylated erbB3 (P-erbB3), Akt (P-Akt), and Src (P-Src), it also inhibited cell proliferation and increased cells at G1 phase. A multi-miRNA lentiviral vector - the cluster of miR-125a and miR-205 - was constructed to simultaneously express the two miRNAs in HER2-overexpressing breast cancer cells. Concurrent expression of miR-125a and miR-205 via the miRNA cluster transfection significantly enhanced trastuzumab-mediated growth inhibition and cell cycle G1 arrest in BT474 cells and markedly increased paclitaxel-induced apoptosis in another HER2-overexpressing breast cancer cell line HCC1954.

Conclusions

Here, we showed that functional cooperative miRNAs effectively suppressed erbB3 expression. This novel approach targeting of HER3 was able to enhance the therapeutic efficacy of trastuzumab and paclitaxel against HER2-overexpressing breast cancer.

     

Rapamycin Enhances the Anti-Cancer Effect of Dasatinib by Suppressing Src/PI3K/mTOR Pathway in NSCLC Cells

Src and the mammalian target of rapamycin (mTOR) signaling are commonly activated in non-small cell lung cancer (NSCLC) and hence potential targets for chemotherapy. Although the combined use of Src inhibitor Dasatinib with other chemotherapeutic agents has shown superior efficacy for cancer treatment, the mechanisms that lead to enhanced sensitivity of Dasatinib are not completely understood. In this study, we found that Rapamycin dramatically enhanced Dasatinib-induced cell growth inhibition and cell cycle G1 arrest in human lung adenocarcinoma A549 cells without affecting apoptosis. The synergistic effects were consistently correlated with the up-regulation of cyclin-dependent kinases inhibitor proteins, including p16, p19, p21, and p27, as well as the repression of Cdk4 expression and nuclear translocation. Mechanistic investigations demonstrated that FoxO1/FoxO3a and p70S6K/4E-BP1, the molecules at downstream of Src-PI3K-Akt and mTOR signaling, were significantly suppressed by the combined use of Dasatinib and Rapamycin. Restraining Src and mTOR with small interfering RNA in A549 cells further confirmed that the Src/PI3K/mTOR Pathway played a crucial role in enhancing the anticancer effect of Dasatinib. In addition, this finding was also validated by a series of assays using another two NSCLC cell lines, NCI-H1706 and NCI-H460. Conclusively, our results suggested that the combinatory application of Src and mTOR inhibitors might be a promising therapeutic strategy for NSCLC treatment.     

Induction of Autophagy Is an Early Response to Gefitinib and a Potential Therapeutic Target in Breast Cancer

Gefitinib (Iressa^®, ZD1839) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. We report on an early cellular response to gefitinib that involves induction of functional autophagic flux in phenotypically diverse breast cancer cells that were sensitive (BT474 and SKBR3) or insensitive (MCF7-GFPLC3 and JIMT-1) to gefitinib. Our data show that elevation of autophagy in gefitinib-treated breast cancer cells correlated with downregulation of AKT and ERK1/2 signaling early in the course of treatment. Inhibition of autophagosome formation by BECLIN-1 or ATG7 siRNA in combination with gefitinib reduced the abundance of autophagic organelles and sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death. However, inhibition of the late stage of gefitinib-induced autophagy with hydroxychloroquine (HCQ) or bafilomycin A1 significantly increased (p<0.05) cell death in gefitinib-sensitive SKBR3 and BT474 cells, as well as in gefitinib-insensitive JIMT-1 and MCF7-GFPLC3 cells, relative to the effects observed with the respective single agents. Treatment with the combination of gefitinib and HCQ was more effective (p<0.05) in delaying tumor growth than either monotherapy (p>0.05), when compared to vehicle-treated controls. Our results also show that elevated autophagosome content following short-term treatment with gefitinib is a reversible response that ceases upon removal of the drug. In aggregate, these data demonstrate that elevated autophagic flux is an early response to gefitinib and that targeting EGFR and autophagy should be considered when developing new therapeutic strategies for EGFR expressing breast cancers.     

Metformin sensitizes insulin signaling through AMPK-mediated PTEN down-regulation in preadipocyte 3T3-L1 cells

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.     

IGF-1 Regulates Cyr61 Induced Breast Cancer Cell Proliferation and Invasion

Background

Studies from our laboratory and others have shown that cysteine-rich 61 (Cyr61) may be involved in tumor proliferation and invasion. In earlier studies, we demonstrated increased insulin-like growth factor-I (IGF-1) is associated with breast tumor formation and poor clinical outcomes. In our current study we have investigated IGF-1 regulation of Cyr61 and whether targeting IGF-1 could inhibit Cyr61 induced tumor growth and proliferation.

Methods

Several ATCC derived normal and breast cancer cell lines were used in this study: MDA-MB231, BT474, MCF-7, and SKBR3. We also tested cells stably transfected in our laboratory with active Akt1 (pAkt; SKBR3/AA and MCF-7/AA) and dominant negative Akt1 (SKBR3/DN and MCF-7/DN). In addition, we used MCF-7 cells transfected with full length Cyr61 (CYA). Monolayer cultures treated with IGF-1 were analyzed for Cyr61 expression by RT-PCR and immunohistochemical staining. Migration assays and MTT based proliferation assays were used to determine invasive characteristics in response to IGF-1/Cyr61 activation.

Results

Cells with activated Akt have increased levels of Cyr61. Conversely, cells with inactive Akt have decreased levels of Cyr61. IGF-1 treatment increased Cyr61 expression significantly and cells with high level of Cyr61 demonstrate increased invasiveness and proliferation. Cyr61 overexpression and activation led to decrease in E-cadherin and decrease in FOXO1. Inhibition of the PI3K and MAPK pathways resulted in significant decrease in invasiveness and proliferation, most notably in the PI3K pathway inhibited cells.

Conclusion

The findings of this study show that IGF-1 upregulates Cyr61 primarily through activation of the Akt-PI3K pathway. IGF-1 induced MAPK plays a partial role. Increase in Cyr61 leads to increase in breast cancer cell growth and invasion. Hence, targeting Cyr61 and associated pathways may offer an opportunity to inhibit IGF-1 mediated Cyr61 induced breast cancer growth and invasion.     

Glucose promotes breast cancer aggression and reduces metformin efficacy

Metformin treatment has been associated with a decrease in breast cancer risk and improved survival. Metformin induces complex cellular changes, resulting in decreased tumor cell proliferation, reduction of stem cells, and apoptosis. Using a carcinogen-induced rodent model of mammary tumorigenesis, we recently demonstrated that overfeeding in obese animals is associated with a 50% increase in tumor glucose uptake, increased proliferation, and tumor cell reprogramming to an “aggressive” metabolic state. Metformin significantly inhibited these pro-tumorigenic effects. We hypothesized that a dynamic relationship exists between chronic energy excess (glucose by dose) and metformin efficacy/action.

Media glucose concentrations above 5 mmol/L was associated with significant increase in breast cancer cell proliferation, clonogenicity, motility, upregulation/activation of pro-oncogenic signaling, and reduction in apoptosis. These effects were most significant in triple-negative breast cancer (TNBC) cell lines. High-glucose conditions (10 mmol/L or above) significantly abrogated the effects of metformin. Mechanisms of metformin action at normal vs. high glucose overlapped but were not identical; for example, metformin reduced IGF-1R expression in both the HER2+ SK-BR-3 and TNBC MDA-MB-468 cell lines more significantly at 5, as compared with 10 mmol/L glucose. Significant changes in gene profiles related to apoptosis, cellular processes, metabolic processes, and cell proliferation occurred with metformin treatment in cells grown at 5 mmol/L glucose, whereas under high-glucose conditions, metformin did not significantly increase apoptotic/cellular death genes. These data indicate that failure to maintain glucose homeostasis may promote a more aggressive breast cancer phenotype and alter metformin efficacy and mechanisms of action.     

Metformin Induces Apoptosis and Downregulates Pyruvate Kinase M2 in Breast Cancer Cells Only When Grown in Nutrient-Poor Conditions

Introduction

Metformin is proposed as adjuvant therapy in cancer treatment because of its ability to limit cancer incidence by negatively modulating the PI3K/AKT/mTOR pathway. In vitro, in addition to inhibiting cancer cell proliferation, metformin can also induce apoptosis. The molecular mechanism underlying this second effect is still poorly characterized and published data are often contrasting. We investigated how nutrient availability can modulate metformin-induced apoptosis in three breast cancer cell lines.

Material and Methods

MCF7, SKBR3 and MDA-MB-231 cells were plated in MEM medium supplemented with increasing glucose concentrations or in DMEM medium and treated with 10 mM metformin. Cell viability was monitored by Trypan Blue assay and treatment effects on Akt/mTOR pathway and on apoptosis were analysed by Western Blot. Moreover, we determined the level of expression of pyruvate kinase M2 (PKM2), a well-known glycolytic enzyme expressed in cancer cells.

Results

Our results showed that metformin can induce apoptosis in breast cancer cells when cultured at physiological glucose concentrations and that the pro-apoptotic effect was completely abolished when cells were grown in high glucose/high amino acid medium. Induction of apoptosis was found to be dependent on AMPK activation but, at least partially, independent of TORC1 inactivation. Finally, we showed that, in nutrient-poor conditions, metformin was able to modulate the intracellular glycolytic equilibrium by downregulating PKM2 expression and that this mechanism was mediated by AMPK activation.

Conclusion

We demonstrated that metformin induces breast cancer cell apoptosis and PKM2 downregulation only in nutrient-poor conditions. Not only glucose levels but also amino acid concentration can influence the observed metformin inhibitory effect on the mTOR pathway as well as its pro-apoptotic effect. These data demonstrate that the reduction of nutrient supply in tumors can increase metformin efficacy and that modulation of PKM2 expression/activity could be a promising strategy to boost metformin anti-cancer effect.     

Survivin expression in tuberous sclerosis complex cells

Tuberous Sclerosis Complex (TSC) is a tumor suppressor gene disorder with mutations of TSC1/TSC2 genes. This leads to the development of hamartomas that most frequently affect central nervous system, kidney, and skin. Angiomyolipomas are abdominal masses made up of muscle vessels and adipose tissues that grow mostly in proximity to kidneys and liver. Bleeding and kidney failure are the major justification for surgery. This study shows that angiomyolipoma-derived human smooth muscle TSC2-/- cells express the apoptosis inhibitor protein survivin when exposed to IGF-1. Survivin expression is also triggered whenever culture conditions perturb normal TSC2-/- cell function, such as the omission of EGF from the growth medium, the supplementation of anti-EGFR, blockade of PI3K and ERK, or inhibition of mTOR. Interestingly, single or simultaneous inhibition of PI3K by LY294002 and ERK by PD98059 does not prevent IGF-1-mediated survivin expression. Apoptogenic Smac/DIABLO, which is constitutively expressed by TSC2-/- A+ cells, is down-regulated by IGF-1 even in the presence of LY294002 and PD98059. These cells release IGF-1 by means of a negative feedback-regulated mechanism that is overrun when they are exposed to antibodies to IGF-1R, which increases the released amount by more than 400%. The autocrine release of IGF-1 may therefore be a powerful mechanism of survival of the tightly packed cells in the thick-walled vessels of TSC angiomyolipoma and in lymphangioleiomyomatosis (LAM) nodules. Future experimental therapies for TSC and LAM may result from the targeted inhibition of survivin, which may enhance sensitivity to TSC2 therapy.     

89Zr-mAb3481 PET for HER3 tumor status assessment during lapatinib treatment

Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with tyrosine kinase inhibitor lapatinib can induce a compensatory HER3 increase, which may attenuate antitumor efficacy. Therefore, we explored in vivo HER3 tumor status assessment after lapatinib treatment with zirconium-89 (⁸⁹Zr)-labeled anti-HER3 antibody mAb3481 positron emission tomography (PET). Lapatinib effects on HER3 cell surface expression and mAb3481 internalization were evaluated in human breast (BT474, SKBR3) and gastric (N87) cancer cell lines using flow cytometry. Next, in vivo effects of daily lapatinib treatment on⁸⁹Zr-mAb3481 BT474 and N87 xenograft tumor uptake were studied. PET-scans (BT474 only) were made after daily lapatinib treatment for 9 days, starting 3 days prior to ⁸⁹Zr-mAb3481 administration. Subsequently, ex vivo ⁸⁹Zr-mAb3481 organ distribution analysis was performed and HER3 tumor levels were measured with Western blot and immunohistochemistry. In vitro, lapatinib increased membranous HER3 in BT474, SKBR3 and N87 cells, and consequently mAb3481 internalization 1.7-fold (BT474), 1.4-fold (SKBR3) and 1.4-fold (N87). ⁸⁹Zr-mAb3481 BT474 tumor uptake was remarkably high at SUV_mean 5.6±0.6 (51.8±7.7%ID/g) using a 10 μg ⁸⁹Zr-mAb3481 protein dose in vehicle-treated mice. However, compared to vehicle, lapatinib did not affect ⁸⁹Zr-mAb3481 ex vivo uptake in BT474 and N87 tumors, while HER3 tumor expression remained unchanged. In conclusion, lapatinib increased in vitro HER3 tumor cell expression, but not when these cells were xenografted. ⁸⁹Zr-mAb3481 PET accurately reflected HER3 tumor status. ⁸⁹Zr-mAb3481 PET showed high, HER3-specific tumor uptake, and such an approach might sensitively assess HER3 tumor heterogeneity and treatment response in patients.