Overcoming retinoic acid resistance in HER2-enriched breast cancers: role of MYC.

HER2-enriched (HER2+) breast cancers express high levels of the growth-promoting HER2 protein. Although these cancers are treated with the HER2-targeted drug, trastuzumab, resistance to treatment is common. Retinoic acid (RA) is an anti-cancer agent that has been successfully used for the treatment of leukemia and holds promise for the treatment of solid cancers, including breast cancer. The HER2 gene is frequently co-amplified with RARA, a key determinant of RA sensitivity in breast cancers. It seems surprising, therefore, that HER2+ breast cancers are refractory to RA treatment. Here, we show that MYC mediates RA resistance by suppressing the expression of cellular retinoic acid binding protein 2 (CRABP2), resulting in RARα inactivation. CRABP2 is an intracellular RA transporter that delivers RA to the nuclear receptor RARα for its activation. Our results indicate that response to RA is enhanced by MYC depletion in HER2+ breast cancer cells and that RA treatment enhances trastuzumab responsiveness. Our findings support the use of RA and trastuzumab for the treatment of subsets of patients with breast cancers that are HER2-RARα co-amplified and have low levels of MYC.

A Novel Mechanism Underlying the Inhibitory Effects of Trastuzumab on the Growth of HER2-Positive Breast Cancer Cells.

To improve the efficacy of trastuzumab, it is essential to understand its mechanism of action. One of the significant issues that makes it difficult to determine the precise mechanism of trastuzumab action is the formation of various HER receptor dimers in HER2-positive breast cancer cells. So far, studies have focused on the role of HER2-HER3 heterodimers, and little is known regarding EGFR-HER2 heterodimers. Here, we study the role of trastuzumab on the cell signaling and cell proliferation mediated by EGFR-HER2 heterodimers in BT474 and SRBR3 cells. EGF stimulates the formation of both EGFR homodimer and EGFR-HER2 heterodimer. Trastuzumab only binds to HER2, not EGFR. Therefore, any effects of trastuzumab on EGF-induced activation of EGFR, HER2, and downstream signaling proteins, as well as cell proliferation, are through its effects on EGFR-HER2 heterodimers. We show that trastuzumab inhibits EGF-induced cell proliferation and cell cycle progression in BT474 and SKBR3 cells. Interestingly trastuzumab strongly inhibits EGF-induced Akt phosphorylation and slightly inhibits EGF-induced Erk activation, in both BT474 and SKBR3 cells. These data suggest the presence of a novel mechanism that allows trastuzumab to inhibit EGR-induced Akt activation and cell proliferation, without blocking EGF-induced EGFR-HER2 heterodimerization and activation. We show that trastuzumab inhibits EGF-induced lipid raft localization of the EGFR-HER2 heterodimer. Disruption of the lipid raft with MßCD blocks HER2-mediated AKT activation in a similar way to trastuzumab. MßCD and trastuzumab synergically inhibit AKT activation. We conclude that trastuzumab inhibits EGF-induced lipid raft localization of EGFR-HER2 heterodimer, which leads to the inhibition of Akt phosphorylation and cell proliferation, without blocking the formation and phosphorylation of the EGFR-HER2 heterodimer.

Analysis of Cell Cycle by Flow Cytometry.

The cell cycle of a cell is tightly controlled by several regulators. Dysregulation of cell cycle can lead to uncontrolled cell division which is one of the main characteristics of cancer cells. DNA content of a cell is changed during the cell cycle progression and can be measured by flow cytometry. In this chapter, we aim to provide a detailed protocol on how to analyze the cell cycle using flow cytometry.

Angioregulatory microRNAs in breast cancer: Molecular mechanistic basis and implications for therapeutic strategies.

Background: Cancer-associated angiogenesis is a fundamental process in tumor growth and metastasis. A variety of signaling regulators and pathways contribute to establish neovascularization, among them as small endogenous non-coding RNAs, microRNAs (miRNAs) play prominent dual regulatory function in breast cancer (BC) angiogenesis. Aim of Review: This review aims at describing the current state-of-the-art in BC angiogenesis-mediated by angioregulatory miRNAs, and an overview of miRNAs dysregulation association with the anti-angiogenic response in addition to potential clinical application of miRNAs-based therapeutics. Key Scientific Concepts of Review: Angioregulatory miRNA-target gene interaction is not only involved in sprouting vessels of breast tumors but also, trans-differentiation of BC cells to endothelial cells (ECs) in a process termed vasculogenic mimicry. Using canonical and non-canonical angiogenesis pathways, the tumor cell employs the oncogenic characteristics such as miRNAs dysregulation to increase survival, proliferation, oxygen and nutrient supply, and treatment resistance. Angioregulatory miRNAs in BC cells and their microenvironment have therapeutic potential in cancer treatment. Although, miRNAs dysregulation can serve as tumor biomarker nevertheless, due to the association of miRNAs dysregulation with anti-angiogenic resistant phenotype, clinical benefits of anti-angiogenic therapy might be challenging in BC. Hence, unveiling the molecular mechanism underlying angioregulatory miRNAs sparked a booming interest in finding new treatment strategies such as miRNA-based therapies in BC.

Trastuzumab Mechanism of Action; 20 Years of Research to Unravel a Dilemma.

Trastuzumab as a first HER2-targeted therapy for the treatment of HER2-positive breast cancer patients was introduced in 1998. Although trastuzumab has opened a new avenue to treat patients with HER2-positive breast cancer and other types of cancer, some patients are not responsive or become resistant to this treatment. So far, several mechanisms have been suggested for the mode of action of trastuzumab; however, the findings regarding these mechanisms are controversial. In this review, we aimed to provide a detailed insight into the various mechanisms of action of trastuzumab.

The role of Nrf2 in neural stem/progenitors cells: From maintaining stemness and self-renewal to promoting differentiation capability and facilitating therapeutic application in neurodegenerative disease.

Neurodegenerative diseases (NDs) cause progressive loss of neurons in nervous system. NDs are categorized as acute NDs such as stroke and head injury, besides chronic NDs including Alzheimer's, Parkinson's, Huntington's diseases, Friedreich's Ataxia, Multiple Sclerosis. The exact etiology of NDs is not understood but oxidative stress, inflammation and synaptic dysfunction are main hallmarks. Oxidative stress leads to free radical attack on neural cells which contributes to protein misfolding, glia cell activation, mitochondrial dysfunction, impairment of DNA repair system and subsequently cellular death. Neural stem cells (NSCs) support adult neurogenesis in nervous system during injuries which is limited to certain regions in brain. NSCs can differentiate into the neurons, astrocytes or oligodendrocytes. Impaired neurogenesis and inadequate induction of neurogenesis are the main obstacles in treatment of NDs. Protection of neural cells from oxidative damages and supporting neurogenesis are promising strategies to treat NDs. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional master regulator that maintains the redox homeostasis in cells by provoking expression of antioxidant, anti-inflammatory and cytoprotective genes. Nrf2 can strongly influence the NSCs function and fate determination by reducing levels of reactive oxygen species in benefit of NSC survival and neurogenesis. In this review we will summarize the role of Nrf2 in NSC function, and exogenous and endogenous therapeutic strategies in treatment of NDs.

DNA damage repair response in mesenchymal stromal cells: From cellular senescence and aging to apoptosis and differentiation ability.

Mesenchymal stromal cells (MSCs) are heterogeneous and contain several populations, including stem cells. MSCs' secretome has the ability to induce proliferation, differentiation, chemo-attraction, anti-apoptosis, and immunomodulation activities in stem cells. Moreover, these cells recognize tissue damage caused by drugs, radiation (e.g., Ultraviolet, infra-red) and oxidative stress, and respond in two ways: either MSCs differentiate into particular cell lineages to preserve tissue homeostasis, or they release a regenerative secretome to activate tissue repairing mechanisms. The maintenance of MSCs in quiescence can increase the incidence and accumulation of various forms of genomic modifications, particularly upon environmental insults. Thus, dysregulated DNA repair pathways can predispose MSCs to senescence or apoptosis, reducing their stemness and self-renewal properties. For instance, DNA damage can impair telomere replication, activating DNA damage checkpoints to maintain MSC function. In this review, we aim to summarize the role of DNA damage and associated repair responses in MSC senescence, differentiation and programmed cell death.

The Effects of Pertuzumab and Its Combination with Trastuzumab on HER2 Homodimerization and Phosphorylation.

Pertuzumab (Perjeta) is an anti-HER2 monoclonal antibody that is used for treatment of HER2-positive breast cancers in combination with trastuzumab (Herceptin) and docetaxel and showed promising clinical outcomes. Pertuzumab is suggested to block heterodimerization of HER2 with EGFR and HER3 that abolishes canonical function of HER2. However, evidence on the exact mode of action of pertuzumab in homodimerization of HER2 are limited. In this study, we investigated the effect of pertuzumab and its combination with trastuzumab on HER2 homodimerization, phosphorylation and whole gene expression profile in Chinese hamster ovary (CHO) cells stably overexpressing human HER2 (CHO-K6). CHO-K6 cells were treated with pertuzumab, trastuzumab, and their combination, and then HER2 homodimerization and phosphorylation at seven pY sites were investigated. The effects of the monoclonal antibodies on whole gene expression and the expression of cell cycle stages, apoptosis, autophagy, and necrosis were studied by cDNA microarray. Results showed that pertuzumab had no significant effect on HER2 homodimerization, however, trastuzumab increased HER2 homodimerization. Interestingly, pertuzumab increased HER2 phosphorylation at Y1127, Y1139, and Y1196 residues, while trastuzumab increased HER2 phosphorylation at Y1196. More surprisingly, combination of pertuzumab and trastuzumab blocked the phosphorylation of Y1005 and Y1127 of HER2. Our results also showed that pertuzumab, but not trastuzumab, abrogated the effect of HER2 overexpression on cell cycle in particular G1/S transition, G2/M transition, and M phase, whereas trastuzumab abolished the inhibitory effect of HER2 on apoptosis. Our findings confirm that pertuzumab is unable to inhibit HER2 homodimerization but induces HER2 phosphorylation at some pY sites that abolishes HER2 effects on cell cycle progress. These data suggest that the clinical effects of pertuzumab may mostly through the inhibition of HER2 heterodimers, rather than HER2 homodimers and that pertuzumab binding to HER2 may inhibit non-canonical HER2 activation and function in non-HER-mediated and dimerization-independent pathway(s).

Angioregulatory microRNAs in Colorectal Cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer mortality. Angiogenesis is a rate-determining step in CRC development and metastasis. The balance of angiogenic and antiangiogenic factors is crucial in this process. Angiogenesis-related genes can be regulated post-transcriptionally by microRNAs (miRNAs) and some miRNAs have been shown to shuttle between tumor cells and the tumor microenvironment (TME). MiRNAs have context-dependent actions and can promote or suppress angiogenesis dependent on the type of cancer. On the one hand, miRNAs downregulate anti-angiogenic targets and lead to angiogenesis induction. Tumor suppressor miRNAs, on the other hand, enhance anti-angiogenic response by targeting pro-angiogenic factors. Understanding the interaction between these miRNAs and their target mRNAs will help to unravel molecular mechanisms involved in CRC progression. The aim of this article is to review the current literature on angioregulatory miRNAs in CRC.

Mechanisms Underlying the Action and Synergism of Trastuzumab and Pertuzumab in Targeting HER2-Positive Breast Cancer.

Human epidermal growth factor receptor (HER) 2 (HER2) is overexpressed in 20⁻30% of breast cancers. HER2 is a preferred target for treating HER2-positive breast cancer. Trastuzumab and pertuzumab are two HER2-targeted monoclonal antibodies approved by the Food and Drug Administration (FDA) to use as adjuvant therapy in combination with docetaxel to treat metastatic HER2-positive breast cancer. Adding the monoclonal antibodies to treatment regimen has changed the paradigm for treatment of HER2-positive breast cancer. Despite improving outcomes, the percentage of the patients who benefit from the treatment is still low. Continued research and development of novel agents and strategies of drug combinations is needed. A thorough understanding of the molecular mechanisms underlying the action and synergism of trastuzumab and pertuzumab is essential for moving forward to achieve high efficacy in treating HER2-positive breast cancer. This review examined and analyzed findings and hypotheses regarding the action and synergism of trastuzumab and pertuzumab and proposed a model of synergism based on available information.

The effects of trastuzumab on HER2-mediated cell signaling in CHO cells expressing human HER2.

BACKGROUND: Targeted therapy with trastuzumab has become a mainstay for HER2-positive breast cancer without a clear understanding of the mechanism of its action. While many mechanisms have been suggested for the action of trastuzumab, most of them are not substantiated by experimental data. It has been suggested that trastuzumab functions by inhibiting intracellular signaling initiated by HER2, however, the data are very controversial. A major issue is the different cellular background of various breast cancer cells lines used in these studies. Each breast cancer cell line has a unique expression profile of various HER receptors, which could significantly affect the effects of trastuzumab. METHODS: To overcome this problem, in this research we adopted a cell model that allow us to specifically examine the effects of trastuzumab on a single HER receptor without the influence of other HER receptors. Three CHO cell lines stably expressing only human EGFR (CHO-EGFR), HER2 (CHO-K6), or HER3 (CHO-HER3) were used. Various methods including cytotoxicity assay, immunoblotting, indirect immunofluorescence, cross linking, and antibody-dependent cellular cytotoxicity (ADCC) were employed in this research. RESULTS: We showed that trastuzumab did not bind EGFR and HER3, and thus did not affect the homodimerization and phosphorylation of EGFR and HER3. However, overexpression of HER2 in CHO cells, in the absence of other HER receptors, resulted in the homodimerization of HER2 and the phosphorylation of HER2 at all major pY residues. Trastuzumab bound to HER2 specifically and with high affinity. Trastuzumab inhibited neither the homodimerization of HER2, nor the phosphorylation of HER2 at most phosphotyrosine residues. Moreover, trastuzumab did not inhibit the phosphorylation of ERK and AKT in CHO-K6 cells, and did not inhibit the proliferation of CHO-K6 cells. However, trastuzumab induced strong ADCC in CHO-K6 cells. CONCLUSION: We concluded that, in the absence of other HER receptors, trastuzumab exerts its antitumor activity through the induction of ADCC, rather than the inhibition of HER2-homodimerization and phosphorylation.

Dimerization Assessment of Epithelial Growth Factor Family of Receptor Tyrosine Kinases by Using Cross-Linking Reagent.

Dimerization of the epithelial growth factor (EGF) family of receptor tyrosine kinases is a crucial step for activation of these receptors. Different chemicals such as BS3 and DSS have been introduced to covalently bind the interacting receptors and fix the dimers. Unique properties of BS3 including higher water solubility and membrane impermeability make it suitable for assessing receptor-receptor interactions in live cells. In this protocol, we aim to explain a method to evaluate the dimerization of EGF receptors family using BS3 as a cross-linker reagent.

Multimodal tumor suppression by miR-302 cluster in melanoma and colon cancer.

The miR-302 family is one of the main groups of microRNAs, which are highly expressed in embryonic stem cells (ESCs). Previous reports have indicated that miR-302 can reduce the proliferation rate of some cancer cells while compromising on their oncogenic potential at the same time without having the same effect on normal somatic cells. In this study we aimed to further investigate the role of the miR-302 cluster in multiple cancer signaling pathways using A-375 melanoma and HT-29 colorectal cancer cells. Our results indicate that the miR-302 cluster has the potential to modulate oncogenic properties of cancer cells through inhibition of proliferation, angiogenesis and invasion, and through reversal of the epithelial-to-mesenchymal transition (EMT) in these cells. We showed for the first time that overexpression of miR-302 cluster sensitized A-375 and HT-29 cells to hypoxia and also to the selective BRAF inhibitor vemurafenib. MiR-302 is a pleiotropically acting miRNA family which may have significant implications in controlling cancer progression and invasion. It acts through a reprogramming process, which has a global effect on a multitude of cellular pathways and events. We propose that reprogramming of cancer cells by epigenetic factors, especially miRNAs might provide an efficient tool for controlling cancer and especially for those with more invasive nature.

In vivo toxicity of orally administrated silicon dioxide nanoparticles in healthy adult mice.

The increasing use of silica nanoparticles (SiNPs) in various applications including industrial, agriculture, and medicine has raised concerns about their potential risks to human health. Various nanotoxicity researches have been done on the assessment of SiNPs' toxic effects; however, a few in vivo investigations exist. In this investigation, an in vivo study was done in order to evaluate the oral toxicity of SiNPs. The biochemical levels of 19 different serum parameters were assessed. Moreover, the histopathological changes have been examined as well. We showed that SiNPs with diameters of 10-15 nm in size can cause significant changes in albumin, cholesterol, triglyceride, total protein, urea, HDL, and LDL as well as in alkaline phosphatase and aspartate aminotransferase activity. In addition, histopathological examinations demonstrated that SiNPs have toxic effects on various tissues including liver, kidney, lung, and testis.

Toxicity of ZnO nanoparticles in healthy adult mice.

In this study, we extended previous work to evaluate the oral toxicity of ZnO nanoparticles and their possible effects on different serum-elements and sexual hormones in the mouse. The histopathological changes have also been examined. Significant recorded increases in alanine aminotransferase and aspartate aminotransferase activity in all mice exposed to ZnO nanoparticles suggest that these nanoparticles can cause hepatic injury. Hepatocyte necrosis and other pathological observations also confirmed liver damage. Moreover, Glomeruli segmentation, hydropic degeneration in epithelial cells, necrosis of epithelial cells in tubules and swelling in epithelial cells of proximal tubules were found in all kidney tissues, which demonstrated that ZnO nanoparticles have severe toxicological effects on kidney. Serous inflammation, severe hyperemia in alveoli, and edema were observed as pathological findings in the lung which suggest that the lung is the third target tissue of the ZnO nanoparticles.