Parity and Short-Term Estradiol Treatment Utilizes Similar Cellular Mechanisms to Confer Protection Against Breast Cancer

Background: Protective effect of early pregnancy and short-term estrogen treatment (STET), against breast cancer is well established. The underlying mechanisms are not well understood. In this study, we compared the mammary gland cellular microenvironment influenced/induced by parity and STET alongside age-matched controls. Methods: Parous, STET, and control rats were injected with N-methyl-N-nitrosourea at 15 weeks and monitored for the development of mammary cancer. A subset of 4 rats were killed five weeks post carcinogen treatment and mammary gland samples were isolated and subjected to molecular analysis. Results: Our results demonstrated a reduction in cell survival, extracellular matrix associated proliferation, hormonal and growth factor receptor pathways in the experimental groups compared to control rats. Moreover, concomitant reductions in the EMT markers along with cell migration regulators were also observed in parous and STET groups. Hormonal receptor such as GHR, PR, ERα and growth factor receptors IGFR, EGFR and erbB2 were down regulated in the treatment groups. Further analysis revealed that parity and STET drastically reduced the expression, activation of JAK2 and nuclear localization of STATs. Conclusion: Parity and STET by targeting major cell signaling pathways involved in cell survival, cell migration and cell death reduces the mammary tumor promoting environment

Targeting insulin-like growth factor 1 receptor inhibits pancreatic cancer growth and metastasis.

Pancreatic cancer is one of the most lethal cancers. Increasing incidence and mortality indicates that there is still much lacking in detection and management of the disease. This is partly due to a lack of specific symptoms during early stages of the disease. Several growth factor receptors have been associated with pancreatic cancer. Here, we have investigated if an RNA interference approach targeted to IGF-IR could be effective and efficient against pancreatic cancer growth and metastasis. For that, we evaluated the effects of IGF-1R inhibition using small interfering RNA (siRNAs) on tumor growth and metastasis in HPAC and PANC-1 pancreatic cancer cell lines. We found that silencing IGF-1R inhibits pancreatic cancer growth and metastasis by blocking key signaling pathways such AKT/PI3K, MAPK, JAK/STAT and EMT. Silencing IGF-1R resulted in an anti-proliferative effect in PANC-1 and HPAC pancreatic cancer cell lines. Matrigel invasion, transwell migration and wound healing assays also revealed a role for IGF-1R in metastatic properties of pancreatic cancer. These results were further confirmed using Western blotting analysis of key intermediates involved in proliferation, epithelial mesenchymal transition, migration, and invasion. In addition, soft agar assays showed that silencing IGF-1R also blocks the colony forming capabilities of pancreatic cancer cells in vitro. Western blots, as well as, flow cytometric analysis revealed the induction of apoptosis in IGF-1R silenced cells. Interestingly, silencing IGF-1R also suppressed the expression of insulin receptor β. All these effects together significantly control pancreatic cancer cell growth and metastasis. To conclude, our results demonstrate the significance of IGF-1R in pancreatic cancer

Effect of silencing IGF-1R on proliferation and colony formation in pancreatic cancer cell lines.

<p>(A) Expression levels of IGF-1R in PANC-1, HPAC and MIA PaCa-2 were compared with normal pancreatic cells from rat using western blot. (B) Representative immunohistochemical analysis of IGF-1R by tumor stage in pancreatic adenocarcinoma tissues and normal pancreas tissue. (C) PANC-1 and HPAC cells were transfected with three predesigned IGF-1R siRNAs (A, B and C) at three different concentrations (10, 30 and 50 nM) along with scrambled control siRNA. Silencing efficacy of IGF-1R siRNA was determined using western blot in PANC-1 and HPAC cells. (D) Effect of IGF-1R siRNA on cell viability of PANC-1 and HPAC. Cells were transfected with 30 nM and 50 nM of IGF-1R siRNA in PANC-1 and HPAC cells respectively. Cell viability was assayed at 48 h post transfection using MTS assay kit. Results represented as mean ± standard deviation (n = 3). (E) Inhibition of IGF1R expression blocks colony forming capabilities of pancreatic cancer cells, PANC-1 and HPAC. A soft agar assay was used to study the colony formation ability of PANC-1 and HPAC cells. Forty eight hours after the siRNA transfection, PANC-1 and HPAC cells were allowed to grow in 0.7% agarose in RPMI-1640-supplemented with 10% FBS for 16 and 22 days, respectively. Shown here are representative pictures of colony formation from two independent experiments done in triplicate. (F) Percentage colonies in both PANC-1 and HPAC cells were calculated with scrambled control (SCR) serving as the baseline.</p

IGF-1R silencing inhibits invading ability and epithelial-mesenchymal transition of pancreatic cancer cells.

<p>(A) PANC-1 and HPAC cell invasion was assessed in transwell chambers coated with matrigel. Cells that invaded the matrigel-coated insert were fixed, stained and captured at 20× magnification. (B) Number of invaded cells were counted and expressed as percentage invasion. Experiments were done in triplicate (C) IGF-1R silencing inhibits expression of several epithelial-mesenchymal transition markers. Total protein lysates from scrambled control and IGF-1R silenced PANC-1 and HPAC cells were analyzed for expression of Notch-2, Snail, E-cadherin, N-Cadherin, Zeb, Vimentin, and Slug along with internal control β-actin. (D) Densitometic values of EMT markers are shown as % expression. PS-PANC-1 Scrambled, PI-PANC-1 IGF-1R silenced, HS-HPAC Scrambled, HI-HPAC IGF-1R silenced.</p

Silencing IGF-1R alters ERK and STAT signaling in PANC-1 and HPAC cells.

<p>(A & C): The effect of IGF-1R suppression on ERK and STAT signaling was examined in pancreatic cancer cells. Whole cell lysates were separated by SDS-PAGE and analyzed by Western blot for expression levels of phospho-ERK, ERK, IR-β, phospho-IRS-1, IRS, phospho-STAT3, STAT3, COX-2 and β-actin. (B & D): Representative blots are presented and corresponding densitometric analysis is shown to the right of each image. PS-PANC-1 Scrambled, PI-PANC-1 IGF-1R silenced, HS-HPAC Scrambled, HI-HPAC IGF-1R silenced.</p

The effect of IGF-1R siRNA on apoptosis of pancreatic cancer cells.

<p>(A) IGF1R inhibition induces apoptosis in PANC1 & HPAC cells. Post transfection cells were stained with Annexin-V-FITC and propidium iodide followed by flow cytometry. The percentage of early apoptotic (bottom right quadrant), apoptotic (top right quadrant), late apoptotic and necrotic cells (top left quadrant), and live healthy cells (bottom left quadrant) are shown. (B) Percentage apoptosis and cell death is summarized for three independent experiments in PANC-1 and HPAC cells. (C) IGF-1R inhibition induces death receptor and mitochondrial mediated apoptosis in PANC1 & HPAC cells. Bax, Bcl-2, caspase 8, caspase 3 and cleaved PARP and β-actin expression was assayed using Western blot in IGF-1R siRNA-transfected PANC-1 and HPAC cells. (D) Representative densitometry analysis shows significant potentiation of apoptosis via intrinsic and extrinsic pathways. PS-PANC-1 Scrambled, PI-PANC-1 IGF-1R silenced, HS-HPAC Scrambled, HI-HPAC IGF-1R silenced.</p

IGF1R silencing suppressed cell migration in pancreatic cancer cell lines.

<p>(A) Wound healing assay was performed to evaluate the migration of PANC-1 and HPAC cells after silencing IGF-1R. Forty eight hours after siRNA transfection, wound healing capacity of cells were monitored with automated Nikon Biostation CT at 2 h intervals up to 96 h. (B & C): Cell migration was determined by the rate of cells moving towards the scratched area. The percentage migration was calculated by the NIS-Element AR software. Similar results were obtained in three independent experiments. (D) Silencing IGF-1R expression inhibits migration of PANC-1 and HPAC cells. Cell migratory abilities were determined using uncoated transwell Boyden chambers. Post transfection PANC-1 and HPAC cells were allowed to migrate through pores to the bottom surface of transwell. Migrated cells were fixed and stained with 0.2% crystal violet in 5% formalin. Data are representative of five random microscopic field images taken at 20X magnification (E) Percentage migration for transwell assays is shown for IGF-1R silenced PANC-1 and HPAC cells from the results of three independent experiments.</p

Suppression of IGF-1R alters key signaling molecules in PANC-1 and HPAC cells.

<p>(A, C & E): The effect of IGF-1R suppression on AKT/PI3K signaling was examined in pancreatic cancer cells. PANC-1 and HPAC cells were treated with IGF-1R siRNA for 48 h. The cells were harvested and the expression of phospho-AKT, AKT, phospho-PI3K, PI3K, phospho-PTEN, phospho-mTOR, mTOR, phospho-p70s6kinase, p70s6kinase and the internal control β-actin was measured by Western blotting. (B, D & F): Densitometric analysis is also shown to the right of each representative image.</p

Hyperglycemia enhances the proliferation of non-tumorigenic and malignant mammary epithelial cells through increased leptin/IGF1R signaling and activation of AKT/mTOR.

Obesity and diabetes are associated with increased breast cancer risk and worse disease progression once cancer is diagnosed; however, the exact etiology behind these observations remains to be fully elucidated. Due to the global obesity/diabetes pandemic, it is imperative to understand how these diseases promote and enhance breast cancer and other common cancers. In this study we demonstrate that hyperglycemia promotes breast cancer by altering leptin/IGF1R and AKT/mTOR signaling. To our knowledge, we show for the first time that in breast epithelial cells, hyperglycemia alone directly impacts leptin signaling. Hyperglycemia increased proliferation of both non-tumorigenic and malignant mammary epithelial cells. These observations coincided with increased leptin receptor and IGF1R receptor, as well as, increased levels of GRB2, pJAK2, pSTAT3, pIRS1/2, pAKT, and p-mTOR. Moreover, pJAK2 was almost completely colocalized with leptin receptor under high glucose conditions. These results demonstrate how hyperglycemia can potentially increase the risk of breast cancer in premalignant lesions and enhance cancer progression in malignant cells

Leptin receptor expression.

<p>Cells were cultured in normal (N) and high glucose (HG) for 24 and 72 hr. Changes in leptin receptor expression were assessed via Western blot for estrogen-receptor positive breast cancer cells (A), triple-negative breast cancer cells (B), and non-tumorigenic breast epithelial cells (C). A total of 10 µg of protein was loaded for each sample. Results are representative of at least three separate experiments. ‘OB-Rs’ is the short isoform of leptin receptor, ‘OB-Rl’ is the long isoform of leptin receptor.</p