GABA(A) Receptor Pi (GABRP) Stimulates Basal-like Breast Cancer Cell Migration through Activation of Extracellular-regulated Kinase 1/2 (ERK1/2)

Breast cancer is a heterogeneous disease comprised of distinct subtypes predictive of patient outcome. Tumors of the basal-like subtype have a poor prognosis due to inherent aggressiveness and the lack of targeted therapeutics. Basal-like tumors typically lack estrogen receptor-α, progesterone receptor and HER2/ERBB2, or in other words they are triple negative (TN). Continued evaluation of basal-like breast cancer (BLBC) biology is essential to identify novel therapeutic targets. Expression of the pi subunit of the GABA(A) receptor (GABRP) is associated with the BLBC/TN subtype, and herein, we reveal its expression also correlates with metastases to the brain and poorer patient outcome. GABRP expression in breast cancer cell lines also demonstrates a significant correlation with the basal-like subtype suggesting that GABRP functions in the initiation and/or progression of basal-like tumors. To address this postulate, we stably silenced GABRP in two BLBC cell lines, HCC1187 and HCC70 cells. Decreased GABRP reduces in vitro tumorigenic potential and migration concurrent with alterations in the cytoskeleton, specifically diminished cellular protrusions and expression of the BLBC-associated cytokeratins, KRT5, KRT6B, KRT14, and KRT17. Silencing GABRP also decreases phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) in both cell lines and selective inhibition of ERK1/2 similarly decreases the basal-like cytokeratins as well as migration. Combined, these data reveal a GABRP-ERK1/2-cytokeratin axis that maintains the migratory phenotype of basal-like breast cancer. GABRP is a component of a cell surface receptor, thus, these findings suggest that targeting this new signaling axis may have therapeutic potential in BLBC.     

Nuclear localization of GLI1 and elevated expression of FOXC2 in breast cancer is associated with the basal-like phenotype

Aberrant sonic hedgehog (SHH)/glioma-associated oncogene (GLI) signaling has been shown in the development of many tumors. The aims of the present study are to determine the expression of two SHH signaling molecules, the glioma-associated oncogene homolog 1 (GLI1) and forkhead box C2 (FOXC2), in invasive breast cancers (IBC), to evaluate their association with clinicopathological parameters, and to determine their prognostic significance in breast cancer patients. Expression of GLI1 and FOXC2 were assessed by immunohistochemical analysis of a tissue microarray containing 262 unselected IBC cases. A statistical analysis was performed to assess the correlation of GLI1 and FOXC2 expression with the patients' clinicopathological parameters, postoperative survival rate, and molecular subtypes. Immunoreactivity of GLI1 and FOXC2 was observed in 84% and 75% of all breast cancer tissues, respectively. There was a significant correlation between nuclear FOXC2 and GLI1 expressions in these breast cancers, which was associated with estrogen receptor (ER) negativity. Furthermore, there was a significant association between nuclear expression of GLI1 and FOXC2 and a basal-like breast cancer phenotype. Patients with nuclear GLI1 or FOXC2-expressing tumors had a significantly shorter survival time than those without nuclear FOXC2 or GLI1 expression. Multivariate analysis showed that nuclear GLI1 or FOXC2 expression was an independent factor for predicting the prognosis of basal-like breast cancer. In conclusion, there was a significant correlation between expression of nuclear GLI1 or FOXC2 and human breast cancer. More specifically, elevated levels of these proteins were associated with the basal-like breast cancer phenotype and with a poor rate of disease-free survival. These data suggest that GLI1 and FOXC2 are involved in tumorigenesis and that they may be useful as diagnostic and therapeutic targets for human basal-like breast cancers. Additional studies are warranted to better understand the biological significance of GLI1 and FOXC2, to further refine statistics related to patient prognosis, and to optimize treatment of patients with basal-like breast cancer.     

Analysis of the MMP-dependent and independent functions of tissue inhibitor of metalloproteinase-2 on the invasiveness of breast cancer cells

Matrix metalloproteinases (MMPs) are secreted endopeptidases that play an essential role in remodeling the extracellular matrix (ECM). MMPs are primarily active during development, when the majority of ECM remodeling events occurs. In adults, elevated MMP activity has been observed in many pathological conditions such as cancer and osteoarthritis. The proteolytic activity of MMPs is controlled by their natural inhibitors - the tissue inhibitor of metalloproteinases (TIMPs). In addition to blocking MMP-mediated proteolysis, TIMPs have a number of MMP-independent functions including binding to cell surface proteins thereby stimulating signaling cascades. TIMP-2, the most studied member of the family, can both inhibit and activate MMPs directly, as well as inhibit MMP activity indirectly by upregulating expression of RECK, a membrane anchored MMP regulator. While TIMP-2 has been shown to play important roles in breast cancer, we describe how the MMP-independent effects of TIMP-2 can modulate the invasiveness of MCF-7, T47D and MDA-MB-231 breast cancer cells. Using an ALA + TIMP-2 mutant which is devoid of MMP inhibition, but still capable of initiating specific cell signaling cascades, we show that TIMP-2 can differentially affect MMP activity and cellular invasiveness in both an MMP dependent and independent manner. More specifically, MMP activity and invasiveness is increased with the addition of exogenous TIMP-2 in poorly invasive cell lines whereas it is decreased in highly invasive cells lines (MDA-MB-231). Conversely, the addition of ALA + TIMP-2 resulted in decreased invasiveness regardless of cell line.     

Interactions with fibroblasts are distinct in Basal-like and luminal breast cancers

Basal-like breast cancers have several well-characterized distinguishing molecular features, but most of these are features of the cancer cells themselves. The unique stromal-epithelial interactions, and more generally, microenvironmental features of basal-like breast cancers have not been well characterized. To identify characteristic microenvironment features of basal-like breast cancer, we performed cocultures of several basal-like breast cancer cell lines with fibroblasts and compared these with cocultures of luminal breast cancer cell lines with fibroblasts. Interactions between basal-like cancer cells and fibroblasts induced expression of numerous interleukins and chemokines, including IL-6, IL-8, CXCL1, CXCL3, and TGFβ. Under the influence of fibroblasts, basal-like breast cancer cell lines also showed increased migration in vitro. Migration was less pronounced for luminal lines; but, these lines were more likely to have altered proliferation. These differences were relevant to tumor biology in vivo, as the gene set that distinguished luminal and basal-like stromal interactions in coculture also distinguishes basal-like from luminal tumors with 98% accuracy in 10-fold cross-validation and 100% accuracy in an independent test set. However, comparisons between cocultures where cells were in direct contact and cocultures where interaction was solely through soluble factors suggest that there is an important impact of direct cell-to-cell contact. The phenotypes and gene expression changes invoked by cancer cell interactions with fibroblasts support the microenvironment and cell-cell interactions as intrinsic features of breast cancer subtypes.     

MT1‐MMP modulates melanoma cell dissemination and metastasis through activation of MMP2 and RAC1

Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. MT1‐MMP is a membrane‐associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion‐promoting, pro‐tumorigenic MMP in cancer. We show that deregulation of MT1‐MMP expression happens as early as the transition from nevus to primary melanoma and continues to increase during melanoma progression. Furthermore, MT1‐MMP expression is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1‐MMP in melanoma pathogenesis. We demonstrate that MT1‐MMP is directly required for melanoma cells to metastasize, as cells deprived of MT1‐MMP fail to form distant metastasis in an orthotopic mouse melanoma model. We show that MT1‐MMP affects cell invasion by activating its target MMP2. Importantly, we demonstrate, for the first time, that activation of MMP2 by MT1‐MMP is required to sustain RAC1 activity and promote MT1‐MMP‐dependent cell motility. These data highlight a novel MT1‐MMP/MMP2/RAC1 signaling axis in melanoma that may represent an intriguing molecular target for the treatment of invasive melanoma.     

Roles of the Cyclooxygenase 2 Matrix Metalloproteinase 1 Pathway in Brain Metastasis of Breast Cancer

Brain is one of the major sites of metastasis in breast cancer; however, the pathological mechanism of brain metastasis is poorly understood. One of the critical rate-limiting steps of brain metastasis is the breaching of blood-brain barrier, which acts as a selective interface between the circulation and the central nervous system, and this process is considered to involve tumor-secreted proteinases. We analyzed clinical significance of 21 matrix metalloproteinases on brain metastasis-free survival of breast cancer followed by verification in brain metastatic cell lines and found that only matrix metalloproteinase 1 (MMP1) is significantly correlated with brain metastasis. We have shown that MMP1 is highly expressed in brain metastatic cells and is capable of degrading Claudin and Occludin but not Zo-1, which are key components of blood-brain barrier. Knockdown of MMP1 in brain metastatic cells significantly suppressed their ability of brain metastasis in vivo, whereas ectopic expression of MMP1 significantly increased the brain metastatic ability of the cells that are not brain metastatic. We also found that COX2 was highly up-regulated in brain metastatic cells and that COX2-induced prostaglandins were directly able to promote the expression of MMP1 followed by augmenting brain metastasis. Furthermore, we found that COX2 and prostaglandin were able to activate astrocytes to release chemokine (C-C motif) ligand 7 (CCL7), which in turn promoted self-renewal of tumor-initiating cells in the brain and that knockdown of COX2 significantly reduced the brain metastatic ability of tumor cells. Our results suggest the COX2-MMP1/CCL7 axis as a novel therapeutic target for brain metastasis.     

蛋白酪氨酸磷酸酶SHP-2在乳腺癌细胞移动及粘附中的作用

探讨蛋白酪氨酸磷酸酶SHP 2在乳腺癌细胞MCF 7的移动及粘附中的作用 .利用基因重组技术分别将野生型SHP 2与突变型SHP 2与绿色荧光蛋白GFP的基因片段构成重组质粒 (SHP 2 GFP、SHP 2C >S GFP) .脂质体转染法分别转入MCF 7中 ,表达成功后筛选并建立SHP 2 GFP和SHP 2C >S GFP细胞株 .荧光显微镜观察细胞移动情况 ,免疫印迹法检测粘附分子E 钙粘蛋白和金属蛋白酶MMP 1及MMP 9的表达 .实验后建立SHP 2 GFP及SHP 2C >S GFP细胞株 ,同时观察到SHP 2C >S GFP细胞的形态发生明显改变 :从梭形状态变成圆形状态 .荧光显微镜发现 ,MCF 7细胞和SHP 2 GFP、SHP 2C >S GFP转染的细胞在 3h、6h、9h的移动情况分别是MCF 7为 10 %、2 3%、5 4% ,SHP 2 GFP为 15 %、4 9%、98% ,SHP 2C >S GFP为 4 %、11%、30 % .免疫印迹结果表明 ,SHP 2C >S GFP细胞的E 钙粘蛋白表达比SHP 2 GFP细胞明显升高 (P <0 0 5 ) .MMP 1及MMP 9的表达量在SHP 2 GFP细胞中有所增强 (P <0 0 5 ) .实验表明 ,SHP 2可能通过调节粘附分子和基质金属磷酸酶而在细胞移动、粘附中发挥重要作用     

Golgi Calcium Pump Secretory Pathway Calcium ATPase 1 (SPCA1) Is a Key Regulator of Insulin-like Growth Factor Receptor (IGF1R) Processing in the Basal-like Breast Cancer Cell Line MDA-MB-231

Calcium signaling is a key regulator of pathways important in tumor progression, such as proliferation and apoptosis. Most studies assessing altered calcium homeostasis in cancer cells have focused on alterations mediated through changes in cytoplasmic free calcium levels. Here, we show that basal-like breast cancers are characterized by an alteration in the secretory pathway calcium ATPase 1 (SPCA1), a calcium pump localized to the Golgi. Inhibition of SPCA1 in MDA-MB-231 cells produced pronounced changes in cell proliferation and morphology in three-dimensional culture, without alterations in sensitivity to endoplasmic reticulum stress induction or changes in global calcium signaling. Instead, the effects of SPCA1 inhibition in MDA-MB-231 cells reside in altered regulation of calcium-dependent enzymes located in the secretory pathway, such as proprotein convertases. Inhibition of SPCA1 produced a pronounced alteration in the processing of insulin-like growth factor receptor (IGF1R), with significantly reduced levels of functional IGF1Rβ and accumulation of the inactive trans-Golgi network pro-IGF1R form. These studies identify for the first time a calcium transporter associated with the basal-like breast cancer subtype. The pronounced effects of SPCA1 inhibition on the processing of IGF1R in MDA-MB-231 cells independent of alterations in global calcium signaling also demonstrate that some calcium transporters can regulate the processing of proteins important in tumor progression without major alterations in cytosolic calcium signaling. Inhibitors of SPCA1 may offer an alternative strategy to direct inhibitors of IGF1R and attenuate the processing of other proprotein convertase substrates important in basal breast cancers.     

Identification of nuclear export inhibitor-based combination therapies in preclinical models of triple-negative breast cancer

An estimated 284,000 Americans will be diagnosed with breast cancer in 2021. Of these individuals, 15–20% have basal-like triple-negative breast cancer (TNBC), which is known to be highly metastatic. Chemotherapy is standard of care for TNBC patients, but chemoresistance is a common clinical problem. There is currently a lack of alternative, targeted treatment strategies for TNBC; this study sought to identify novel therapeutic combinations to treat basal-like TNBCs. For these studies, four human basal-like TNBC cell lines were utilized to determine the cytotoxicity profile of 1363 clinically-used drugs. Ten promising therapeutic candidates were identified, and synergism studies were performed in vitro. Two drug combinations that included KPT-330, an XPO1 inhibitor, were synergistic in all four cell lines. In vivo testing of four basal-like patient-derived xenografts (PDX) identified one combination, KPT-330 and GSK2126458 (a PI3K/mTOR inhibitor), that decreased tumor burden in mice significantly more than monotherapy with either single agent. Bulk and single-cell RNA-sequencing, immunohistochemistry, and analysis of published genomic datasets found that XPO1 was abundantly expressed in human basal-like TNBC cell lines, PDXs, and patient tumor samples. Within basal-like PDXs, XPO1 overexpression was associated with increased proliferation at the cellular level. Within patient datasets, XPO1 overexpression was correlated with greater rates of metastasis in patients with basal-like tumors. These studies identify a promising potential new combination therapy for patients with basal-like breast cancer.     

Identification of nuclear export inhibitor-based combination therapies in preclinical models of triple-negative breast cancer

An estimated 284,000 Americans will be diagnosed with breast cancer in 2021. Of these individuals, 15–20% have basal-like triple-negative breast cancer (TNBC), which is known to be highly metastatic. Chemotherapy is standard of care for TNBC patients, but chemoresistance is a common clinical problem. There is currently a lack of alternative, targeted treatment strategies for TNBC; this study sought to identify novel therapeutic combinations to treat basal-like TNBCs. For these studies, four human basal-like TNBC cell lines were utilized to determine the cytotoxicity profile of 1363 clinically-used drugs. Ten promising therapeutic candidates were identified, and synergism studies were performed in vitro. Two drug combinations that included KPT-330, an XPO1 inhibitor, were synergistic in all four cell lines. In vivo testing of four basal-like patient-derived xenografts (PDX) identified one combination, KPT-330 and GSK2126458 (a PI3K/mTOR inhibitor), that decreased tumor burden in mice significantly more than monotherapy with either single agent. Bulk and single-cell RNA-sequencing, immunohistochemistry, and analysis of published genomic datasets found that XPO1 was abundantly expressed in human basal-like TNBC cell lines, PDXs, and patient tumor samples. Within basal-like PDXs, XPO1 overexpression was associated with increased proliferation at the cellular level. Within patient datasets, XPO1 overexpression was correlated with greater rates of metastasis in patients with basal-like tumors. These studies identify a promising potential new combination therapy for patients with basal-like breast cancer.     

Toll-Like Receptor 4 Prompts Human Breast Cancer Cells Invasiveness via Lipopolysaccharide Stimulation and Is Overexpressed in Patients with Lymph Node Metastasis

Toll-like receptor (TLR)4-mediated signaling has been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study investigated the expression and biological role of TLR4 in human breast cancer metastasis. MCF-7 and MDA-MB-231 are human breast cancer cell lines with low and high metastatic potential, respectively. Using lipopolysaccharide (LPS) to stimulate MCF-7 and MDA-MB-231 cells, expression of TLR4 mRNA and protein increased compared with that in control cells. TLR4 activation notably up-regulated expression of matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor(VEGF) mRNA and their secretion in the supernatants of both cell lines. LPS enhanced invasion of MDA-MB-231 cells by transwell assay and MCF-7 cells by wound healing assay. LPS triggered increased expression of TLR4 downstream signaling pathway protein myeloid differentiation factor 88(MyD88) and resulted in interleukin (IL)-6 and IL-10 higher production by human breast cancer cells. Stimulation of TLR4 with LPS promoted tumorigenesis and formed metastatic lesions in liver of nude mice. Moreover, expression of TLR4 and MyD88 as well as invasiveness and migration of the cells could be blocked by TLR4 antagonist. Combined with clinicopathological parameters, TLR4 was overexpressed in human breast cancer tissue and correlated with lymph node metastasis. These findings indicated that TLR4 may participate in the progression and metastasis of human breast cancer and provide a new therapeutic target.     

PTK 7 Is a Transforming Gene and Prognostic Marker for Breast Cancer and Nodal Metastasis Involvement

Protein Tyrosin Kinase 7 (PTK7) is upregulated in several human cancers; however, its clinical implication in breast cancer (BC) and lymph node (LN) is still unclear. In order to investigate the function of PTK7 in mediating BC cell motility and invasivity, PTK7 expression in BC cell lines was determined. PTK7 signaling in highly invasive breast cancer cells was inhibited by a dominant-negative PTK7 mutant, an antibody against the extracellular domain of PTK7, and siRNA knockdown of PTK7. This resulted in decreased motility and invasivity of BC cells. We further examined PTK7 expression in BC and LN tissue of 128 BC patients by RT-PCR and its correlation with BC related genes like HER2, HER3, PAI1, MMP1, K19, and CD44. Expression profiling in BC cell lines and primary tumors showed association of PTK7 with ER/PR/HER2-negative (TNBC-triple negative BC) cancer. Oncomine data analysis confirmed this observation and classified PTK7 in a cluster with genes associated with agressive behavior of primary BC. Furthermore PTK7 expression was significantly different with respect to tumor size (ANOVA, p = 0.033) in BC and nodal involvement (ANOVA, p = 0.007) in LN. PTK7 expression in metastatic LN was related to shorter DFS (Cox Regression, p = 0.041). Our observations confirmed the transforming potential of PTK7, as well as its involvement in motility and invasivity of BC cells. PTK7 is highly expressed in TNBC cell lines. It represents a novel prognostic marker for BC patients and has potential therapeutic significance.     

High-density array analysis of DNA methylation in Tamoxifen-resistant breast cancer cell lines

Roughly two-thirds of all breast cancers are ERα-positive and can be treated with the antiestrogen, Tamoxifen, however resistance occurs in 33% of women who take the drug for more than 5 y. Aberrant DNA methylation, an epigenetic mechanism that alters gene expression in cancer, is thought to play a role in this resistance. To develop an understanding of Tamoxifen-resistance and identify novel pathways and targets of aberrant methylation, DNA from MCF-7 breast cancer cells and Tamoxifen-resistant derivatives, TMX2–11 and TMX2–28, were analyzed using the Illumina HumanMethylation450 BeadChip. Normalizing against MCF-7 values, ERα-positive TMX2–11 had 4000 hypermethylated sites and ERα-negative TMX2–28 had over 33?000. Analysis of CpG sites altered in both TMX2–11 and TMX2–28 revealed that the Tamoxifen-resistant cell lines share 3000 hypermethylated and 200 hypomethylated CpGs. ZNF350 and MAGED1, two genes hypermethylated in both cell lines, were examined in greater detail. Treatment with 5-aza-2′deoxycitidine caused a significant reduction in promoter methylation of both ZNF350 and MAGED1 and a corresponding increase in expression in TMX2–28. A similar relationship between methylation and expression was not detected in TMX2–11. Our findings are indicative of the variable responses to methylation-targeted breast cancer therapy and highlight the need for biomarkers that accurately predict treatment outcome.     

TrkB Promotes Breast Cancer Metastasis via Suppression of Runx3 and Keap1 Expression

In metastatic breast cancer, the acquisition of malignant traits has been associated with the increased rate of cell growth and division, mobility, resistance to chemotherapy, and invasiveness. While screening for the key regulators of cancer metastasis, we observed that neurotrophin receptor TrkB is frequently overexpressed in breast cancer patients and breast cancer cell lines. Additionally, we demonstrate that TrkB expression and clinical breast tumor pathological phenotypes show significant correlation. Moreover, TrkB expression was significantly upregulated in basal-like, claudin-low, and metaplastic breast cancers from a published microarray database and in patients with triple-negative breast cancer, which is associated with a higher risk of invasive recurrence. Interestingly, we identified a new TrkB-regulated functional network that is important for the tumorigenicity and metastasis of breast cancer. We demonstrated that TrkB plays a key role in regulation of the tumor suppressors Runx3 and Keap1. A markedly increased expression of Runx3 and Keap1 was observed upon knockdown of TrkB, treatment with a TrkB inhibitor, and in TrkB kinase dead mutants. Additionally, the inhibition of PI3K/AKT activation significantly induced Runx3 and Keap1 expression. Furthermore, we showed that TrkB enhances metastatic potential and induces proliferation. These observations suggest that TrkB plays a key role in tumorigenicity and metastasis of breast cancer cells through suppression of Runx3 or Keap1 and that it is a promising target for future intervention strategies for preventing tumor metastasis and cancer chemoprevention.     

The effect of a DNA repair gene on cellular invasiveness: XRCC3 over-expression in breast cancer cells

Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3) and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion.