Mitochondrial DNA mutations regulate metastasis of human breast cancer cells

Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously expressed a highly metastatic potential, mitochondrial respiration defects, and ROS overproduction. Since mitochondrial respiratory function is controlled by both mtDNA and nuclear DNA, it is possible that nuclear DNA mutations contribute to the mitochondrial respiration defects and the highly metastatic potential found in MDA-MB-231 cells. To examine this possibility, we carried out mtDNA replacement of MDA-MB-231 cells by normal human mtDNA. For the complete mtDNA replacement, first we isolated mtDNA-less (ρ(0)) MDA-MB-231 cells, and then introduced normal human mtDNA into the ρ(0) MDA-MB-231 cells, and isolated trans-mitochondrial cells (cybrids) carrying nuclear DNA from MDA-MB-231 cells and mtDNA from a normal subject. The normal mtDNA transfer simultaneously induced restoration of mitochondrial respiratory function and suppression of the highly metastatic potential expressed in MDA-MB-231 cells, but did not suppress ROS overproduction. These observations suggest that mitochondrial respiration defects observed in MDA-MB-231 cells are caused by mutations in mtDNA but not in nuclear DNA, and are responsible for expression of the high metastatic potential without using ROS-mediated pathways. Thus, human tumor cells possess an mtDNA-mediated metastatic pathway that is required for expression of the highly metastatic potential in the absence of ROS production.     

Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis.     

Heparin-like polysaccharides reduce osteolytic bone destruction and tumor growth in a mouse model of breast cancer bone metastasis

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.     

The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption

Metastasis accounts for most deaths from breast cancer, driving the need for new therapeutics that can impede disease progression. Rationally designed peptides that take advantage of cancer-specific differences in cellular physiology are an emerging technology that offer promise as a treatment for metastatic breast cancer. We developed CT20p, a hydrophobic peptide based on the C terminus of Bax that exhibits similarities with antimicrobial peptides, and previously reported that CT20p has unique cytotoxic actions independent of full-length Bax. In this study, we identified the intracellular actions of CT20p which precede cancer cell-specific detachment and death. Previously, we found that CT20p migrated in the heavy membrane fractions of cancer cell lysates. Here, using MDA-MB-231 breast cancer cells, we demonstrated that CT20p localizes to the mitochondria, leading to fusion-like aggregation and mitochondrial membrane hyperpolarization. As a result, the distribution and movement of mitochondria in CT20p-treated MDA-MB-231 cells was markedly impaired, particularly in cell protrusions. In contrast, CT20p did not associate with the mitochondria of normal breast epithelial MCF-10A cells, causing little change in the mitochondrial membrane potential, morphology or localization. In MDA-MB-231 cells, CT20p triggered cell detachment that was preceded by decreased levels of α5β1 integrins and reduced F-actin polymerization. Using folate-targeted nanoparticles to encapsulate and deliver CT20p to murine tumors, we achieved significant tumor regression within days of peptide treatment. These results suggest that CT20p has application in the treatment of metastatic disease as a cancer-specific therapeutic peptide that perturbs mitochondrial morphology and movement ultimately culminating in disruption of the actin cytoskeleton, cell detachment, and loss of cell viability.     

Significance of PELP1 in ER-negative breast cancer metastasis

Breast cancer metastasis is a major clinical problem. The molecular basis of breast cancer progression to metastasis remains poorly understood. PELP1 is an estrogen receptor (ER) coregulator that has been implicated as a proto-oncogene whose expression is deregulated in metastatic breast tumors and whose expression is retained in ER-negative tumors. We examined the mechanism and significance of PELP1-mediated signaling in ER-negative breast cancer progression using two ER-negative model cells (MDA-MB-231 and 4T1 cells) that stably express PELP1-shRNA. These model cells had reduced PELP1 expression (75% of endogenous levels) and exhibited less propensity to proliferate in growth assays in vitro. PELP1 downregulation substantially affected migration of ER-negative cells in Boyden chamber and invasion assays. Using mechanistic studies, we found that PELP1 modulated expression of several genes involved in the epithelial mesenchymal transition (EMT), including MMPs, SNAIL, TWIST, and ZEB. In addition, PELP1 knockdown reduced the in vivo metastatic potential of ER-negative breast cancer cells and significantly reduced lung metastatic nodules in a xenograft assay. These results implicate PELP1 as having a role in ER-negative breast cancer metastasis, reveal novel mechanism of coregulator regulation of metastasis via promoting cell motility/EMT by modulating expression of genes, and suggest PELP1 may be a potential therapeutic target for metastatic ER-negative breast cancer.     

BMP9对人乳腺癌细胞MDA-MB-231骨转移的影响及可能机制

探讨BMP9对人乳腺癌MDA-MB-231细胞骨转移能力的影响及其可能的机制。扩增高滴度的BMP9表达腺病毒,感染MDA-MB-231细胞,制备表达BMP9的重组MDA-MB-231/ BMP9细胞,以此作为实验组;同时以含GFP的空载腺病毒感染该细胞为MDA-MB-231/GFP,联合MDA-MB-231共同作为对照组;RT-PCR及Western blot检测重组MDA-MB-231/ BMP9细胞中BMP9以及磷酸化Smad1(PSmad1)的表达;定量PCR及Western blot检测三组细胞中CTGFmRNA和蛋白水平的表达情况,最后结合X片运用免疫组织化学染色的方法检测三组标本中CTGF的表达。结果发现重组MDA-MB-231/ BMP9细胞中存在BMP9的表达;与对照组细胞相比,MDA-MB-231/ BMP9细胞中存在PSmad1的活化增强及CTGF的表达下调;X片发现实验组裸鼠胫骨溶骨性缺损减少;瘤体组织免疫组化发现实验组CTGF表达下调。所以BMP9可以在体内抑制乳腺癌MDA-MB-231细胞的骨转移并且这种抑制作用有可能是通过下调CTGF来实现的。     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.     

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaP_M cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis.     

Clinical-translational strategies for the elevation of Nm23-H1 metastasis suppressor gene expression

Interruption of the tumor metastatic process is a new, thought provoking molecular target for the treatment of cancer. The Nm23-H1 metastasis suppressor gene stands as a validated molecular target owing to its reduced expression in many aggressive human tumors, and the reduction in metastatic potential in vivo upon re-expression in multiple cell lines. Several compounds have been identified which elevate Nm23-H1 expression in vitro including indomethacin, γ Linolenic Acid, trichostatin A, 5-aza-deoxycytidine, and high dose medroxyprogesterone acetate. Using a model of lung metastatic colonization by MDA-MB-231 human breast carcinoma cells, we demonstrated that high dose MPA reduced the formation of overt lung metastases by 37–46% and those metastases that formed were statistically smaller. A Phase II clinical trial of high dose MPA, alone or in combination with metronomic chemotherapy has recently opened.     

DC Electric Fields Direct Breast Cancer Cell Migration, Induce EGFR Polarization, and Increase the Intracellular Level of Calcium Ions

Migration of cancer cells leads to invasion of primary tumors to distant organs (i.e., metastasis). Growing number of studies have demonstrated the migration of various cancer cell types directed by applied direct current electric fields (dcEF), i.e., electrotaxis, and suggested its potential implications in metastasis. MDA-MB-231 cell, a human metastatic breast cancer cell line, has been shown to migrate toward the anode of dcEF. Further characterizations of MDA-MB-231 cell electrotaxis and investigation of its underlying signaling mechanisms will lead to a better understanding of electrically guided cancer cell migration and metastasis. Therefore, we quantitatively characterized MDA-MB-231 cell electrotaxis and a few associated signaling events. Using a microfluidic device that can create well-controlled dcEF, we showed the anode-directing migration of MDA-MB-231 cells. In addition, surface staining of epidermal growth factor receptor (EGFR) and confocal microscopy showed the dcEF-induced anodal EGFR polarization in MDA-MB-231 cells. Furthermore, we showed an increase of intracellular calcium ions in MDA-MB-231 cells upon dcEF stimulation. Altogether, our study provided quantitative measurements of electrotactic migration of MDA-MB-231 cells, and demonstrated the electric field-mediated EGFR and calcium signaling events, suggesting their involvement in breast cancer cell electrotaxis.     

Ketone body utilization drives tumor growth and metastasis

We have previously proposed that catabolic fibroblasts generate mitochondrial fuels (such as ketone bodies) to promote the anabolic growth of human cancer cells and their metastasic dissemination. We have termed this new paradigm “two-compartment tumor metabolism.” Here, we further tested this hypothesis by using a genetic approach. For this purpose, we generated hTERT-immortalized fibroblasts overexpressing the rate-limiting enzymes that promote ketone body production, namely BDH1 and HMGCS2. Similarly, we generated MDA-MB-231 human breast cancer cells overexpressing the key enzyme(s) that allow ketone body re-utilization, OXCT1/2 and ACAT1/2. Interestingly, our results directly show that ketogenic fibroblasts are catabolic and undergo autophagy, with a loss of caveolin-1 (Cav-1) protein expression. Moreover, ketogenic fibroblasts increase the mitochondrial mass and growth of adjacent breast cancer cells. However, most importantly, ketogenic fibroblasts also effectively promote tumor growth, without a significant increase in tumor angiogenesis. Finally, MDA-MB-231 cells overexpressing the enzyme(s) required for ketone re-utilization show dramatic increases in tumor growth and metastatic capacity. Our data provide the necessary genetic evidence that ketone body production and re-utilization drive tumor progression and metastasis. As such, ketone inhibitors should be designed as novel therapeutics to effectively treat advanced cancer patients, with tumor recurrence and metastatic disease. In summary, ketone bodies behave as onco-metabolites, and we directly show that the enzymes HMGCS2, ACAT1/2 and OXCT1/2 are bona fide metabolic oncogenes.     

cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-β. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.     

NHE1 mediates MDA-MB-231 cells invasion through the regulation of MT1-MMP

Na⁺/H⁺ exchanger 1 (NHE1), an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been shown to play a key role in breast cancer metastasis. However, the exact mechanism by which NHE1 mediates breast cancer metastasis is not yet well known. We showed here that inhibition of NHE1 activity, with specific inhibitor Cariporide, could suppress MDA-MB-231 cells invasion as well as the activity and expression of MT1-MMP. Overexpression of MT1-MMP resulted in a distinguished increase in MDA-MB-231 cells invasiveness, but treatment with Cariporide reversed the MT1-MMP-mediated enhanced invasiveness. To explore the role of MAPK signaling pathways in NHE1-mediated breast cancer metastasis, we compared the difference of constitutively phosphorylated ERK1/2, p38 MAPK and JNK in non-invasive MCF-7 cells and invasive MDA-MB-231cells. Interestingly, we found that the phosphorylation levels of ERK1/2 and p38 MAPK in MDA-MB-231 cells were higher than in MCF-7 cells, but both MCF-7 cells and MDA-MB-231 cells expressed similar constitutively phosphorylated JNK. Treating MDA-MB-231 cells with Cariporide led to decreased phosphorylation level of both p38 MAPK and ERK1/2 in a time-dependent manner, but JNK activity was not influenced. Supplementation with MAPK inhibitor (MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125) or Cariporide all exhibited significant depression of MDA-MB-231 cells invasion and MT1-MMP expression. Furthermore, we co-treated MDA-MB-231 cells with MAPK inhibitor and Cariporide. The result showed that Cariporide synergistically suppressed invasion and MT1-MMP expression with MEK inhibitor and p38 MAPK inhibitor, but not be synergistic with the JNK inhibitor. These findings suggest that NHE1 mediates MDA-MB-231 cells invasion partly through regulating MT1-MMP in ERK1/2 and p38 MAPK signaling pathways dependent manner.     

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.     

BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor,CTGF) expression

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, regulate a wide range of cellular responses including cell proliferation, differentiation, adhesion, migration, and apoptosis. BMP9, the latest BMP to be discovered, is reportedly expressed in a variety of human carcinoma cell lines, but the role of BMP9 in breast cancer has not been fully clarified. In a previous study, BMP9 was found to inhibit the growth, migration, and invasiveness of MDA-MB-231 breast cancer cells. In the current study, the effect of BMP9 on the bone metastasis of breast cancer cells was investigated. After absent or low expression of BMP9 was detected in the MDA-MB-231 breast cancer cells and breast non-tumor adjacent tissues using Western blot and immunohistochemistry, In our previous study, BMP9 could inhibit the proliferation and invasiveness of breast cancer cells MDA-MB-231 in vitro and in vivo. This paper shows that BMP9 inhibit the bone metastasis of breast cancer cells by activating the BMP/Smad signaling pathway and downregulating connective tissue growth factor (CTGF); however, when CTGF expression was maintained, the inhibitory effect of BMP9 on the MDA-MB-231 cells was abolished. Together, these observations indicate that BMP9 is an important mediator of breast cancer bone metastasis and a potential therapeutic target for treating this deadly disease.     

The mitochondrial protein C1qbp promotes cell proliferation,migration and resistance to cell death

Complement 1q-binding protein (C1qbp) is a mitochondrial protein reported to be upregulated in cancer. However, whether C1qbp plays a tumor suppressive or tumorigenic role in the progression of cancer is controversial. Moreover, the exact effects of C1qbp on cell proliferation, migration and death/survival have not been definitely proven. To this end, we comprehensively examined the effects of C1qbp on mitochondrial-dependent cell death, proliferation and migration in both normal and breast cancer cells using genetic gain- and loss-of-function approaches. In normal fibroblasts, overexpression of C1qbp protected the cells against staurosporine-induce apoptosis, increased proliferation, decreased cellular ATP and increased cell migration in a wound-healing assay. In contrast, the opposite effects were observed in fibroblasts depleted of C1qbp by RNA interference. C1qbp expression was found to be markedly elevated in 4 different human breast cancer cell lines as well as in ductal and adenocarcinoma tumors from breast cancer patients. Stable knockdown of C1qbp by shRNA in the aggressive MDA-MB-231 breast cancer cell line greatly reduced cell proliferation, increased ATP levels and decreased cell migration compared with control shRNA-transfected cells. Moreover, C1qbp knockdown elicited a significant increase in doxorubicin-induced apoptosis in the MDA-MB-231 cells. Finally, C1qbp upregulation was not restricted to breast cancer cells and tumors, as levels of C1qbp were also found to be significantly elevated in both human lung and colon cancer cell lines and carcinomas. Together, these results establish a pro-tumor, rather than antitumor, role for C1qbp and indicate that C1qbp could serve as a molecular target for cancer therapeutics.Key words: mitochondria, cell proliferation, cell migration, cell death, tumor cells     

Caveolin-1-enhanced motility and focal adhesion turnover require tyrosine-14 but not accumulation to the rear in metastatic cancer cells

Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells.     

Clinicopathological and biological significance of human voltage-gated proton channel Hv1 protein overexpression in breast cancer

In our previous work, we showed for the first time that the voltage-gated proton channel Hv1 is specifically expressed in highly metastatic human breast tumor tissues and cell lines. However, the contribution of Hv1 to breast carcinogenesis is not well known. In this study, we showed that Hv1 expression was significantly correlated with the tumor size (p = 0.001), tumor classification (p = 0.000), lymph node status (p = 0.000), clinical stage (p = 0.000), and Her-2 status (p = 0.045). High Hv1 expression was associated significantly with shorter overall (p = 0.000) and recurrence-free survival (p = 0.000). In vitro, knockdown of Hv1 expression in the highly metastatic MDA-MB-231 cells decreased the cell proliferation and invasiveness, inhibited the cell proton secretion and intracellular pH recovery, and blocked the cell capacity of acidifying extracellular milieu. Furthermore, the gelatinase activity in MDA-MB-231 cells that suppressed Hv1 was reduced. In vivo, the breast tumor size of the implantation of the MDA-MB-231 xenografts in nude mice that were knocked down by Hv1 was dramatically smaller than that in the control groups. The results demonstrated that the inhibition of Hv1 function via knockdown of Hv1 expression can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Based on these results, we came to the conclusion that Hv1 is a potential biomarker for prognosis of breast cancer and a potential target for anticancer drugs in breast cancer therapy.