Eloise Ferreira

Novel patented therapeutic approaches targeting the 37/67 kDa laminin receptor for treatment of cancer and Alzheimer’s disease

Introduction: The 37/67 kDa high-affinity laminin receptor (laminin receptor precursor/laminin receptor, LRP/LR) is a multi-faceted cellular receptor. It plays a vital role in the malignancy of various cancer types where it is seen to contribute to invasion, adhesion, apoptosis evasion and angiogenesis. Furthermore, it has been found to play an important role in facilitating the processes leading to neurotoxicity in Alzheimer’s disease (AD). Various therapeutic options targeting this receptor have been patented with the outlook on application for the treatment/prevention of these diseases. Areas covered: The various roles that LRP/LR plays in cancer, AD and infectious diseases caused by viruses and bacteria have been examined in detail and an overview of the current patented therapeutic strategies targeting this receptor is given. Expert opinion: Molecular tools directed against LRP/LR, such as antibodies and small interfering RNA, could prove to be effective in the prevention of metastasis and angiogenesis while inducing apoptosis in cancers. Moreover, these strategies could also be applied to AD where LRP/LR is seen to facilitate the production and internalization of the neurotoxic Aβ peptide. This review provides a comprehensive overview of the mechanisms by which LRP/LR is involved in eliciting pathogenic events, while showing how the use of patented approaches targeting this receptor could be used to treat them.

Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity.

Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85–90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.

Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells.

Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment.

Anti-LRP/LR specific antibody IgG1-iS18 significantly impedes adhesion and invasion in early and late stage colorectal carcinoma cells.

Cancer is a highly complex disease that has become one of the leading causes of death globally. Metastasis, a major cause of cancer deaths, requires two crucial events, adhesion and invasion. The 37kDa/67kDa laminin receptor (laminin receptor precursor/high-affinity laminin receptor [LRP/LR]) enhances these two steps, consequently aiding in cancer progression. In this study, the role of LRP/LR in adhesion and invasion of early-stage (SW-480 and HT-29) and late-stage (DLD-1) colorectal cancer cells was investigated. Western blotting revealed that early- and late-stage colorectal cancer cells contained significantly higher total LRP/LR levels compared with poorly invasive MCF-7 breast cancer control cells. Flow cytometry revealed that both stages of colorectal cancer displayed significantly higher cell surface LRP/LR levels. Furthermore, upon treatment of colorectal cancer cells with the anti-LRP/LR-specific antibody IgG1-iS18, adhesion to laminin-1 was significantly reduced in both stages. Each stage’s invasive potential was determined using the Matrigel™ invasion assay, showing that invasion was significantly impeded in both colorectal cancer stages when the cells were incubated with IgG1-iS18. In addition, Pearson’s correlation coefficients propose that both total and cell surface LRP/LR levels are directly proportional to the adhesive and invasive potential of both stages of colorectal cancer. Hence, these findings indicate potential for use of the IgG1-iS18 antibody as a promising therapeutic tool for colorectal cancer patients at both stages.

Anti-LRP/LR-specific antibody IgG1-iS18 impedes adhesion and invasion of pancreatic cancer and neuroblastoma cells.

BackgroundCancer has become a global burden due to its high incidence and mortality rates, with an estimated 14.1 million cancer cases reported worldwide in 2012 particularly as a result of metastasis. Metastasis involves two crucial steps: adhesion and invasion, and the non-integrin receptor; the 37-kDa/67-kDa laminin receptor precursor/ high affinity laminin receptor (LRP/LR) has been shown to be overexpressed on the surface of tumorigenic cells, thus being implicated in the enhancement of these two crucial steps. The current study investigated the role of LRP/LR on the aggressiveness of pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells with respect to their adhesive and invasive potential.MethodsAsPC-1 and IMR-32 cells were utilized as the experimental cell lines for the study. Cell surface LRP/LR levels were visualised and quantified on the experimental and control (MCF-7) cell lines via confocal microscopy and flow cytometry, respectively. Total LRP/LR levels in the cell lines were assessed by Western blotting and the adhesive and invasive potential of the above-mentioned cell lines was determined before and after supplementation with the anti-LRP/LR specific antibody IgG1-iS18. Statistical significance of the data was confirmed via the use of the two-tailed student’s t-test and Pearson’s correlation coefficient.ResultsFlow cytometry revealed that AsPC-1 and IMR-32 cells displayed significantly higher cell surface LRP/LR levels in comparison to the MCF-7 control cell line. However, Western blotting and subsequent densitometric analysis revealed that all three tumorigenic cell lines displayed no significant difference in total LRP/LR levels. The treatment of AsPC-1 and IMR-32 cells with IgG1-iS18 caused a significant reduction in the adhesive and invasive potential of the cells to laminin-1 and through the ECM-like Matrigel™, respectively. Pearson’s correlation coefficients indicated a high correlation, thus suggesting a directly proportional relationship between cell surface LRP/LR levels and the adhesive and invasive potential of AsPC-1 and IMR-32 cells.ConclusionThese findings suggest that through the interference of the LRP/LR-laminin-1 interaction, the anti-LRP/LR specific antibody IgG1-iS18 may act as an alternative therapeutic tool for the treatment of metastatic pancreatic cancer and neuroblastoma.

37 kDa LRP::FLAG enhances telomerase activity and reduces senescent markers in vitro

One of the core regulators of cellular aging are telomeres, repetitive DNA sequences at the ends of chromosomes that are maintained by the ribonucleoprotein DNA polymerase complex, telomerase. Recently, we demonstrated that knockdown of the 37kDa/ 67kDa laminin receptor (LRP/LR), a protein that promotes cell viability in tumorigenic and normal cells, reduces telomerase activity. We therefore hypothesized that upregulating LRP/LR might increase telomerase activity and impede aging. Here we show that overexpression of LRP::FLAG resulted in significantly elevated hTERT levels, telomerase activity and telomere length, respectively, with concomitantly reduced levels of senescence markers. These data suggest a novel function of LRP/LR hampering the onset of senescence through elevating hTERT levels and telomerase activity, respectively. LRP::FLAG might therefore act as a potential novel anti-aging drug through the impediment of the cellular aging process.

IgG1-iS18 impedes the adhesive and invasive potential of early and late stage malignant melanoma cells.

Abstract The 37 kDa/67 kDa laminin receptor (LRP/LR) is a non‐integrin laminin receptor which is overexpressed in tumorigenic cells and supports progression of cancer via promoting metastasis, angiogenesis and telomerase activity and impediment of apoptosis. The present study investigates the role of LRP/LR on the metastatic potential of early (A375) and late (A375SM) stage malignant melanoma cells. Flow cytometry revealed that both early and late stage malignant melanoma cells display high levels of LRP/LR on their cell surface. Flow cytometry and western blot analysis showed that late stage malignant melanoma cells display significantly higher total and cell surface LRP/LR levels in comparison to early stage malignant melanoma cells and the poorly invasive breast cancer (MCF‐7) control cell line. Targeting LRP/LR using the LRP/LR specific antibody IgG1‐iS18 resulted in a significant reduction of the adhesive potential to laminin‐1 and the invasive potential through the ‘ECM‐simulating’ Matrigel™ of both early and late stage malignant melanoma cells. Furthermore, Pearsons correlation coefficient confirmed that increased LRP levels correlate with the increased invasive and adhesive potential in early and late stage melanoma cells. Thus, blocking LRP/LR using the IgG1‐iS18 antibody may therefore be a promising therapeutic strategy for early and late stage malignant melanoma treatment. Graphical abstract Figure. No Caption available. HighlightsA375SM cells have higher cell surface and total LRP/LR levels compared to A375 cells.A375 cells have lower adhesive and invasive potentials compared to A375SM cells.IgG1‐iS18 significantly decreases adhesion and invasion of both early (A375) and late stage malignant melanoma cells (A375SM).IgG1‐iS18 antibody was most effective on the metastatic late stage malignant melanoma cells.IgG1‐iS18 may act as a promising alternative therapeutic for treatment of early and late stage malignant melanoma cells.

Knockdown of LRP/LR induces apoptosis in pancreatic cancer and neuroblastoma cells through activation of caspases

Abstract The 37 kDa/67 kDa laminin receptor (LRP/LR) serves various physiological and pathological roles such as enhancing tumour‐related processes including metastasis, angiogenesis, cellular viability and telomerase activation in cancerous cell lines. The present study investigates the effect of siRNA mediated downregulation of LRP/LR on pancreatic cancer (AsPC‐1) and neuroblastoma (IMR‐32) cells. MTT and BrdU assays revealed that siRNA mediated downregulation of LRP resulted in a significant reduction in cell viability and cell proliferation. In addition, knock‐down of LRP resulted in phosphatidylserine externalization, diminished nuclear integrity and significantly enhanced caspase‐3 activity, which is indicative of apoptosis. LRP downregulation resulted in a significant increase in caspase‐8 activity in IMR‐32 cells and enhanced caspase‐8 and 9 activity in AsPC‐1 cells. These data recommend siRNA mediated knock‐down of LRP as a potential therapeutic avenue for the treatment of pancreatic cancer and neuroblastoma. HighlightssiRNA mediated knock‐down of LRP reduces cell viability of IMR‐32 and AsPC‐1 cells.siRNA mediated downregulation of LRP causes apoptosis in IMR‐32 and AsPC‐1 cells.knock‐down of LRP diminishes nuclear integrity and enhances caspase‐3 activity in IMR‐32 and AsPC‐1 cells.LRP downregulation resulted in an increase in caspase‐8 activity without increase in caspase‐9 activity in IMR‐32 cells.Downregulation of LRP enhances caspase‐8 and 9 activity in AsPC‐1 cells.

LRP/LR specific antibody IgG1-iS18 impedes neurodegeneration in Alzheimer's disease mice

Alzheimer’s disease (AD) is a neurodegenerative disease caused by accumulation of amyloid beta (Aβ) plaque and neurofibrillary tangle formation. We have shown in vitro, that knock-down and blockade of the 37 kDa/67 kDa Laminin Receptor (LRP/LR) resulted in reduced Aβ induced cytotoxicity and Aβ accumulation. In order to test the effect of blocking LRP/LR on Aβ formation and AD associated symptoms, AD transgenic mice received the anti-LRP/LR specific antibody, IgG1-iS18 through intranasal administration. We show that this treatment resulted in an improvement in memory, and decreased Aβ plaque formation. Moreover, a significant decrease in Aβ42 protein expression with a concomitant increase in amyloid precursor protein (APP) and telomerase reverse transcriptase (mTERT) levels was observed. These data recommend IgG1-iS18 as a potentially powerful therapeutic antibody for AD treatment.

siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases

ABSTRACT The 37 kDa/67 kDa laminin receptor (LRP/LR) is over‐expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell‐surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down‐regulated via siRNA technology. MTT assays revealed that LRP knock‐down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin‐V FITC/PI assays confirmed this observation. Additionally, caspase‐3 activity assays revealed that apoptosis was induced in both cell lines after siRNA‐mediated down‐regulation of LRP. Caspase‐8 and −9 activity assays suggested that post LRP knock‐down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. Implications: siRNAs mediated LRP knock‐down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis. HighlightsLate stage malignant A375SM melanoma cells exhibit higher cell surface and total LRP/LR levels in comparison to the early stage malignant A375 melanoma cells.siRNA technology lead to successful down‐regulation of LRP expression in early and late stage malignant melanoma cells.siRNA‐ mediated knock‐down of LRP expression results in significantly reduced cellular viability of A375 and A375SM cells.Knock‐down of LRP levels results in nuclear morphological changes indicating apoptotic induction in A375 and A375SM cells.Increased levels of apoptotic induction post siRNA‐mediated LRP knock‐down in A375 and A375SM melanoma cells.Significant increase in caspase‐3 activity in both early and late stage malignant melanoma cells post siRNA transfection.Apoptosis occurrs via the extrinsic pathway in A375 cells and via the intrinsic pathway inA375SM cells.High correlation between total LRP levels post siRNA transfection and induction of apoptosis in both melanoma cell lines.Apoptosis occurred via decreased laminin‐1/LRP/LR interaction and activation of survival pathways as well as activated caspase‐3.siRNAs targeting LRP expression may act ter as a alternative therapeutic tool for treatment of A375 and A375SM cells.