While the HER2-targeting agents trastuzumab and lapatinib have improved the survival of patients with HER2-positive breast cancer, resistance to these targeted therapies is a major challenge. To investigate mechanisms of acquired lapatinib resistance, we generated acquired lapatinib resistance cell models by extended exposure of two HER2-positive breast cancer cell lines to lapatinib. Genomic and proteomic analyses revealed that lapatinib-resistant breast cancer cells gained additional PI3K activation through activating mutation in PI3K p110α and/or increasing protein expression of existing mutant p110α. p110α protein up-regulation in lapatinib-resistant cells occurred through gene amplification or post-transcriptional upregulation. Knockdown of p110α, but not p110β, the other PI3K catalytic subunit present in epithelial cells, inhibited proliferation of lapatinib-resistant cells, especially when combined with lapatinib. Lapatinib-resistant xenograft growth was inhibited persistently by combination treatment with the p110α-selective PI3K inhibitor BYL719 and lapatinib; the drug combination was also well-tolerated in mice. Mechanistically, the combination of lapatinib plus BYL719 more effectively inhibited Akt phosphorylation and, surprisingly, Erk phosphorylation, than either drug alone in the resistance model. These findings indicate that lapatinib resistance can occur through p110α protein upregulation-mediated, and/or mutation-induced, PI3K activation. Moreover, a combinatorial targeted therapy, lapatinib plus BYL719, effectively overcame lapatinib resistance in vivo and could be further tested in clinical trials. Finally, our findings indicate that p110β may be dispensable for lapatinib resistance in some cases. This allows the usage of p110α-specific PI3K inhibitors and thus may spare patients the toxicities of pan-PI3K inhibition to allow maximal dosage and efficacy.
14-3-3ζ is overexpressed in over 40% of breast cancers but its pathophysiological relevance to tumorigenesis has not been established. Here we show that 14-3-3ζ overexpression is sufficient to induce tumorigenesis in a transgenic mouse model of breast cancer. MMTV-LTR promoter driven HA-14-3-3ζ transgenic mice (MMTV-HA-14-3-3ζ) developed mammary tumors whereas control mice did not. Whey acidic protein promoter driven HA-14-3-3ζ transgenic mice (WAP-HA-14-3-3ζ) developed hyperplastic lesions and showed increased susceptibility to carcinogen-induced tumorigenesis. When crossed with MMTV-neu transgenic mice, 14-3-3ζ.neu transgenic mice exhibited accelerated mammary tumorigenesis and metastasis compared to MMTV-neu mice. Mechanistically, 14-3-3ζ overexpression enhanced MAPK/c-Jun signaling leading to increased miR-221 transcription, which inhibited p27 CDKI translation, and consequently, promoted cell proliferation. Importantly, this 14-3-3ζ/miR-221/p27/proliferation axis is also functioning in patients' breast tumors and associates with high grade cancers. Taken together, our findings show that 14-3-3ζ overexpression has a causal role in mammary tumorigenesis and progression, acting through miR-221 in cooperation with known oncogenic events to drive neoplastic cell proliferation.
Breast cancer; 14-3-3ζ; microRNA; transgenic mice
Despite better control of early stage disease and improved overall survival of patients with breast cancer, the incidence of life-threatening brain metastases continues to increase in some of these patients. Unfortunately, other than palliative treatments there is no effective therapy for this condition. In this study, we reveal a critical role for Src activation in promoting brain metastasis in a preclinical model of breast cancer, and we show how a Src-targeting combinatorial regimen can treat HER2+ brain metastases in this model. We found that Src was hyperactivated in brain-seeking breast cancer cells derived from human cell lines or from patients’ brain metastases. Mechanistically, Src activation promoted tumor cell extravasation into the brain parenchyma via permeabilization of the blood-brain barrier. When combined with the EGFR/HER2 dual-targeting drug lapatinib, a Src-targeting combinatorial regimen prevented outgrowth of disseminated breast cancer cells_through the induction of cell cycle arrest. More importantly, this combinatorial regimen inhibited the outgrowth of established experimental brain metastases, prolonging the survival of metastases-bearing mice. Our results provide a rationale for clinical evaluation of Src-targeting regimens to treat breast cancer patients suffering from brain metastasis.
Src; Breast cancer; Brain metastasis; Blood brain barrier; Lapatinib; Saracatinib
Sirtuin 6 (SIRT6) is associated with longevity and is also a tumor suppressor. Identification of molecular regulators of SIRT6 might enable its activation therapeutically in cancer patients. In various breast cancer cell lines, we found that SIRT6 was phosphorylated at Ser338 by the kinase AKT1, which induced the interaction and ubiquitination of SIRT6 by MDM2, targeting SIRT6 for protease-dependent degradation. The survival of breast cancer patients positively correlated with the abundance of SIRT6 and inversely correlated with the phosphorylation of SIRT6 at Ser338. In a panel of breast tumor biopsies, SIRT6 abundance inversely correlated with the abundance of phosphorylated AKT. Inhibiting AKT or preventing SIRT6 phosphorylation by mutating Ser338 prevented the degradation of SIRT6 mediated by MDM2, suppressed the proliferation of breast cancer cells in culture, and inhibited the growth of breast tumor xenografts in mice. Overexpressing MDM2 decreased the abundance of SIRT6 in cells, whereas overexpressing an E3 ligase–deficient MDM2 or knocking down endogenous MDM2 increased SIRT6 abundance. Trastuzumab (known as Herceptin) is a drug that targets a specific receptor common in some breast cancers, and knocking down SIRT6 increased the survival of a breast cancer cell exposed to trastuzumab. Overexpression of a nonphosphorylatable SIRT6 mutant increased trastuzumab sensitivity in a resistant breast cancer cell line. Thus, stabilizing SIRT6 may be a clinical strategy for overcoming trastuzumab resistance in breast cancer patients.
Breast cancer is the second-leading cause of oncology-related death in US women. Of all invasive breast cancers, patients with tumors lacking expression of the estrogen and progesterone hormone receptors and overexpression of human epidermal growth factor receptor 2 have the poorest clinical prognosis. These referred to as triple-negative breast cancer (TNBC) represent an aggressive form of disease that is marked by early-onset metastasis, high tumor recurrence rate, and low overall survival during the first three years post-diagnosis. In this report, we discuss a novel model of early-onset TNBC metastasis to bone and lungs, derived from MDA-MB-231 cells. Breast cancer cells injected intravenously produced rapid, osteolytic metastases in long bones and spines of athymic nude mice, with concurrent metastasis to lungs, liver, and soft tissues. From the bone metastases, we developed a highly metastatic luciferase-tagged cell line variant named MDA-231-LUC Met. In this report, we demonstrate that the Akt/mTOR/S6K1 axis is hyperactivated in these cells, leading to a dramatic increase in phosphorylation of S6 ribosomal protein at Ser235/236. Lastly, we provide evidence that inhibition of the furthest downstream kinase in the mTOR pathway, S6K1, with a highly specific inhibitor PF-4708671 inhibits cell migration, and thus may provide a potent anti-metastatic adjuvant therapy approach.
TNBC; metastasis; bone; S6K1; S6
Activation of K-ras and inactivation of p16 are the most frequently identified genetic alterations in human pancreatic epithelial adenocarcinoma (PDAC). Mouse models engineered with mutant K-ras and deleted p16 recapitulate key pathological features of PDAC. However, a human cell culture transformation model that recapitulates the human pancreatic molecular carcinogenesis is lacking. In this study, we investigated the role of p16 in hTERT-immortalized human pancreatic epithelial nestin-expressing (HPNE) cells expressing mutant K-ras (K-rasG12V). We found that expression of p16 was induced by oncogenic K-ras in these HPNE cells and that silencing of this induced p16 expression resulted in tumorigenic transformation and development of metastatic PDAC in an orthotopic xenograft mouse model. Our results revealed that PI3K/Akt, ERK1/2 pathways and TGFα signaling were activated by K-ras and involved in the malignant transformation of human pancreatic cells. Also, p38/MAPK pathway was involved in p16 up-regulation. Thus, our findings establish an experimental cell-based model for dissecting signaling pathways in the development of human PDAC. This model provides an important tool for studying the molecular basis of PDAC development and gaining insight into signaling mechanisms and potential new therapeutic targets for altered oncogenic signaling pathways in PDAC.
Trastuzumab is a successful rationally designed ERBB2-targeted therapy. However, about half of individuals with ERBB2-overexpressing breast cancer do not respond to trastuzumab-based therapies, owing to various resistance mechanisms. Clinically applicable regimens for overcoming trastuzumab resistance of different mechanisms are not yet available. We show that the nonreceptor tyrosine kinase c-SRC (SRC) is a key modulator of trastuzumab response and a common node downstream of multiple trastuzumab resistance pathways. We find that SRC is activated in both acquired and de novo trastuzumab-resistant cells and uncover a novel mechanism of SRC regulation involving dephosphorylation by PTEN. Increased SRC activation conferred considerable trastuzumab resistance in breast cancer cells and correlated with trastuzumab resistance in patients. Targeting SRC in combination with trastuzumab sensitized multiple lines of trastuzumab-resistant cells to trastuzumab and eliminated trastuzumab-resistant tumors in vivo, suggesting the potential clinical application of this strategy to overcome trastuzumab resistance.
Brain metastasis is a common cause of mortality in cancer patients, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF-IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that IGF-IR is constitutively autophosphorylated in brain-seeking breast cancer sublines. Knockdown of IGF-IR results in a decrease of phospho-AKT and phospho-p70s6k, as well as decreased migration and invasion of MDA-MB-231Br brain-seeking cells. In addition, transient ablation of IGFBP3, which is overexpressed in brain-seeking cells, blocks IGF-IR activation. Using an in vivo experimental brain metastasis model, we show that IGF-IR knockdown brain-seeking cells have reduced potential to establish brain metastases. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.
Trastuzumab is an iconic rationally designed targeted therapy for HER2-positive breast cancers. However, the low response rate and development of resistance call for novel approaches for the treatment of patients. Here, we report that concurrent targeting of tumor cells and activation of T cells in the tumor microenvironment results in a synergistic inhibitory effect on tumor growth and overcomes resistance in two distinct PTEN loss–mediated trastuzumab-resistant mammary tumor mouse models. In vivo combination treatment with HER2/Neu antibody and Akt inhibitor triciribine effectively inhibited tumor growth in both models via inhibiting PI3K/AKT and mitogen-activated protein kinase signaling accompanied by increased T-cell infiltration in the tumor microenvironment. We showed that both CD8+ and CD4+ T cells were essential to the optimal antitumor effect of this combination treatment in an IFN- γ–dependent manner. Importantly, the antitumor activities of HER2/Neu antibody and triciribine combination treatment were further improved when coinhibitory receptor cytotoxic T-lymphocyte–associated antigen 4 was blocked to enhance the T-cell response. Our data indicate that multitargeted combinatorial therapies targeting tumor cells and concomitantly enhancing T-cell response in the tumor microenvironment could cooperate to exert maximal therapeutic activity, suggesting a promising clinical strategy for treating trastuzumab-resistant breast cancers and other advanced malignancies.
Src is a non-receptor tyrosine kinase that is deregulated in many types of cancer. Decades of research have revealed the crucial role of Src in many aspects of tumor development, including proliferation, survival, adhesion, migration, invasion and, most importantly, metastasis, in multiple tumor types. Despite extensive pre-clinical evidence which warrants targeting Src as a promising therapeutic approach for cancer, Src inhibitor(s) show only minimal therapeutic activity in various types of solid tumors as a single agent in recent early-phase clinical trials. In this review, we highlight the most recent advances from preclinical studies and clinical trials that shed light on potential clinical use of Src inhibitor-containing combinatorial regimens in overcoming resistance to current anti-cancer therapies and in preventing metastatic recurrence.
Akt kinase plays a central role in cell growth, metabolism and tumorigenesis. Although TRAF6 E3 ligase orchestrates IGF-1-mediated Akt ubiquitination and activation, it is unclear whether TRAF6 is involved in Akt activation by other growth factor receptors as well. Here we show that Akt ubiquitination is also induced by activation of ErbB receptors; unexpectedly, Skp2 SCF complex, but not TRAF6, is a critical E3 ligase for ErbB receptor-mediated Akt ubiquitination and membrane recruitment. Interestingly, Skp2 deficiency impairs Akt activation, Glut1 expression, glucose uptake and glycolysis, and breast cancer progression in various tumor models. Moreover, Skp2 overexpression correlates with Akt activation, breast cancer metastasis, and serves as a marker for poor prognosis in Her2-positive patients. Finally, we showed that Skp2 silencing sensitizes Her2-overexpressing tumors to Herceptin treatment. Our study suggests that distinct E3 ligases are utilized by diverse growth factors for Akt ubiquitination and activation.
Breast cancer initiating cells (BCICs), which can fully recapitulate the tumor origin and are often resistant to chemo- and radiotherapy, are currently considered as a major obstacle for breast cancer treatment. Here, we show that BIKDD, a constitutively active mutant form of proapoptotic gene, BIK, effectively induces apoptosis of breast cancer cells and synergizes with lapatinib. Most importantly, BikDD significantly reduced BCICs through co-antagonism of its binding partners Bcl-2, Bcl-xL and Mcl-1, suggesting a potential therapeutic strategy targeting BCICs. Furthermore, we developed a cancer-specific targeting approach for breast cancer that selectively expresses BikDD in breast cancer cells including BCICs, and demonstrated its potent antitumor activity and synergism with lapatinib in vitro and in vivo.
The ubiquitously expressed 14-3-3 proteins regulate many pathways involved in transformation. Previously, we found that 14-3-3ζ overexpression increased Akt phosphorylation in human mammary epithelial cells. Here, we investigated the clinical relevance and molecular mechanism of 14-3-3ζ overexpression-mediated Akt phosphorylation and the potential impact on breast cancer progression. We found that 14-3-3ζ overexpression was significantly (P = 0.005) associated with increased Akt phosphorylation in human breast tumors. Additionally, 14-3-3ζ overexpression combined with strong Akt phosphorylation was significantly (P=0.01) associated with increased cancer recurrence in patients. In contrast, knockdown of 14-3-3ζ expression by siRNA in cancer cell lines and tumor xenografts reduced Akt phosphorylation. Furthermore, 14-3-3ζ enhanced Akt phosphorylation through activation of PI3K. Mechanistically, 14-3-3ζ bound to the p85 regulatory subunit of PI3K and increased PI3K translocation to the cell membrane. A single 14-3-3 binding motif encompassing serine 83 on p85 is largely responsible for 14-3-3ζ-mediated p85 binding and PI3K/Akt activation. Mutation of serine 83 to alanine on p85 inhibited 14-3-3ζ binding to the p85 subunit of PI3K, reduced PI3K membrane localization and activation, impeded anchorage independent growth and enhanced stress induced apoptosis. These findings revealed a novel mechanism by which 14-3-3ζ overexpression activates PI3K, a key node in the mitogenic signaling network known to promote malignancies in many cell types.
14-3-3ζ; breast cancer; PI3K; Akt
Trastuzumab resistance has been linked to activation of the phosphoinositol 3-kinase (PI3K) pathway. Phosphatase and tensin homolog (PTEN) is a dual phosphatase that counteracts the PI3K function; PTEN loss leads to activation of the Akt cascade and the downstream mammalian target of rapamycin (mTOR). Preclinical studies demonstrated that mTOR inhibition sensitized the response to trastuzumab in mice with HER2 overexpressing and PTEN-deficient breast xenografts. Our trial evaluated the safety and efficacy of the combination of everolimus and trastuzumab in women with HER2-overexpressing metastatic breast cancer (MBC) that progressed on trastuzumab-based therapy.
Patients and Methods
This represents a pooled analysis (n = 47), stemming from two trials that occurred concurrently in The University of Texas MD Anderson Cancer Center, Beth Israel Deaconess Medical Center, and Dana-Farber Cancer Institute. Patients with HER2-overexpressing MBC who had progressed on trastuzumab-based therapy received trastuzumab every 3 weeks in combination with daily everolimus.
Among 47 patients, the combination of everolimus and trastuzumab provided partial responses in seven patients (15%) and persistent stable disease (lasting 6 months or longer) in nine patients (19%), resulting in a clinical benefit rate of 34%. The median progression-free survival (PFS) was 4.1 month. Fatigue, infection, and mucositis were the predominant nonhematologic toxicities. Trastuzumab did not have significant influence on the pharmacokinetic profile of everolimus. Patients with PTEN loss demonstrated decreased overall survival (P = .048). However, PFS was not affected by PTEN loss.
Inhibition of mTOR results in clinical benefit and disease response in patients with trastuzumab-resistant HER2-overexpressing MBC.
Obesity is a well-established risk factor for cancer, accounting for up to 20% of cancer deaths in women. Studies of women with breast cancer have shown obesity to be associated with an increased risk of dying from breast cancer and increased risk of distant metastasis. While previous studies have focused on differences in circulating hormone levels as a cause for increased breast cancer incidence in post-menopausal women, few studies have focused on potential differences in the protein expression patterns of mammary epithelial cells obtained from obese versus non-obese women.
Protein expression was assessed by reverse phase protein microarray in mammary epithelial cells from 31 random periareolar fine needle aspirations performed on 26 high-risk women.
In this pilot and exploratory study, vimentin (unadjusted p=0.028) expression was significantly different between obese and non-obese women.
Vimentin is integral to adipocyte structure and function as well as to the epithelial-to-mesenchymal transition needed for cancer cell metastasis. Further research is needed to confirm this finding and determine the possible effects of the adipocyte microenvironment on the initiation and progression of breast cancer in high-risk women.
Differential protein expression patterns obtained from a future expanded study may serve to elaborate the underlying pathology of breast cancer initiation and progression in obese women and identify potential biomarkers of response to preventative interventions, such as dietary changes and exercise.
protein microarray; random periareolar fine needle aspiration; obesity; breast cancer risk assessment
Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1–CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1–CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1–mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management.
The expression of proinflammatory protein tissue transglutaminase 2 (TG2) is frequently upregulated in multiple cancer cell types. However, the exact role of TG2 in cancer cells is not well-understood. We recently initiated studies to determine the significance of TG2 in cancer cells and observed that sustained expression of TG2 resulted in epithelial-to-mesenchymal transition (EMT) and promoted cancer stem cell (CSC) traits in mammary epithelial cells. These results suggested that TG2 could serve as a promising therapeutic target for overcoming chemoresistance and inhibiting metastatic spread of cancer cells.
Using various mutant constructs, we analyzed the activity of TG2 that is essential for promoting the EMT-CSC phenotype.
Our results suggest that catalytically inactive TG2 (TG2-C277S) is as effective as wild-type TG2 (TG2-WT) in inducing the EMT-CSC in mammary epithelial cells. In contrast, overexpression of a GTP-binding-deficient mutant (TG2-R580A) was completely incompetent in this regard. Moreover, TG2-dependent activation of the proinflammatory transcription factor NF-κB is deemed essential for promoting the EMT-CSC phenotype in mammary epithelial cells.
Our results suggest that the transamidation activity of TG2 is not essential for promoting its oncogenic functions and provide a strong rationale for developing small-molecule inhibitors to block GTP-binding pockets of TG2. Such inhibitors may have great potential for inhibiting the TG2-regulated pathways, reversing drug resistance and inhibiting the metastasis of cancer cells.
While p21 is well known to inhibit cyclin-CDK activity in the nucleus and it has also been demonstrated to have oncogenic properties in different types of human cancers. In vitro studies showed that the oncogenic function of p21is closely related to its cytoplasmic localization. However, it is unclear whether cytoplasmic p21 contributes to tumorigenesis in vivo. To address this question, we generated transgenic mice expressing the Akt-phosphorylated form of p21 (p21T145D) in the mammary epithelium. The results showed that Akt-activated p21 was expressed in the cytoplasm of mammary epithelium. Overexpression of Akt-activated p21 accelerated tumor onset and promoted lung metastasis in MMTV/neu mice, providing evidence that p21, especially cytoplasmic phosphorylated p21, has an oncogenic role in promoting mammary tumorigenesis and metastasis.
p21; PKB/Akt; mammary tumorigenesis; lung metastasis
Importance of the field
The ubiquitously expressed 14-3-3ζ protein is involved in numerous important cellular pathways involved in cancer. Recent research suggests 14-3-3ζ may play a central role regulating multiple pathways responsible for cancer initiation and progression. This review will provide an overview of 14-3-3 proteins and address the role of 14-3-3ζ overexpression in cancer.
Areas covered in this review
The review covers the basic role of 14-3-3 in regulation of multiple pathways with a focus on 14-3-3ζ as a clinically relevant biomarker for cancer recurrence.
What the reader will gain
14-3-3ζ overexpression has been found in multiple cancers; however, the clinical implications were unclear. Recently, 14-3-3ζ has been identified as a biomarker for poor prognosis and chemoresistance in multiple tumor types, indicating a potential clinical application for using 14-3-3ζ in selecting treatment options and predicting cancer patients’ outcome.
Take home message
14-3-3ζ is a potential prognostic marker of cancer recurrence and predictive marker for therapeutic resistance. The overexpression of 14-3-3ζ in multiple cancers suggests that it may be a common target to intervene tumor progression; therefore, more efforts are needed for the development of 14-3-3 inhibitors.
14-3-3 zeta; apoptosis; cancer; chemoresistance; prognostic marker
Amplification or overexpression of MDM2 promotes a variety of human tumors by degrading tumor suppressor proteins such as p53. Phosphorylation of MDM2 on serines 166 and 186 by the survival kinase Akt inhibits p53-mediated apoptosis. However, it is unclear whether this pathway contributes to normal or malignant pathophysiology in vivo. To address these questions, we generated transgenic mice expressing the Akt-phosphorylated form of MDM2 (MDM2DDS166D/S186D) in the mammary epithelium. Activation of MDM2 delayed mammary gland involution and accelerated tumor progression in MMTV/neu transgenic mice by inhibiting apoptosis in a manner associated with decreased p53 expression. Our findings offer in vivo evidence that activation of MDM2 by Akt contributes to mammary development and tumorigenesis.
Epithelial mechenchymal transition (EMT) has recently been linked to stem cell phenotype1, 2. However, the molecular mechanism involving regulation of EMT and stemness remains elusive. Here, using genomic approaches, we discovered that tumor suppressor p53 plays a role in regulating both EMT and EMT-associated stem cell properties through transcriptional activation of miR-200c. p53 transactivates miR-200c through direct binding to the miR-200c promoter. Loss of p53 in mammary epithelial cells leads to decreased expression of miR-200c and activates EMT program, accompanied by increased mammary stem cell population. Re-expressing miR-200c suppresses genes that mediate EMT and stemness properties3, 4 and thereby reverts mesenchymal and stem cell-like phenotype caused by loss of p53 to differentiated epithelial cell phenotype. Furthermore, loss of p53 negatively correlates with miR-200c level but positively with increased expression of EMT and stemness markers as well as high tumor grade in a cohort of breast tumors. Together, this study elucidates a role of p53 in regulating EMT-MET (mechenchymal epithelial transition) and stemness or differentiation plasticity and reveals a potential therapeutic implication to suppress EMT associated-cancer stem cells through activation of p53-miR-200c pathway.
Cancer stem cells (CSCs) or tumor initiating cells (TICs) make up only a small fraction of total tumor cell population, but recent evidence suggests that they are responsible for tumor initiation and the maintenance of tumor growth. Whether CSCs/TICs originate from normal stem cells or result from the dedifferentiation of terminally differentiated cells remains unknown. Here we provide evidence that sustained expression of the proinflammatory protein tissue transglutaminase (TG2) confers stem cell like properties in non-transformed and transformed mammary epithelial cells. Sustained expression of TG2 was associated with increase in CD44high/CD24low/- subpopulation, increased ability of cells to form mammospheres, and acquisition of self-renewal ability. Mammospheres derived from TG2-transfected mammary epithelial cells (MCF10A) differentiated into complex secondary structures when grown in Matrigel cultures. Cells in these secondary structures differentiated into Muc1-positive (luminal marker) and integrin α6-positive (basal marker) cells in response to prolactin treatment. Highly aggressive MDA-231 and drug-resistant MCF-7/RT breast cancer cells, which express high basal levels of TG2, shared many traits with TG2-transfected MCF10A stem cells but unlike MCF10A-derived stem cells they failed to form the secondary structures and to differentiate into Muc1-positive luminal cells when grown in Matrigel culture. Downregulation of TG2 attenuated stem cell properties in both non-transformed and transformed mammary epithelial cells. Taken together, these results suggested a new function for TG2 and revealed a novel mechanism responsible for promoting the stem cell characteristics in adult mammary epithelial cells.
Significant progress has been achieved toward elucidating the molecular mechanisms that underlie breast cancer progression; yet, much less is known about the associated cellular biophysical traits. To this end, we use time-lapsed confocal microscopy to investigate the interplay among cell motility, three-dimensional (3D) matrix stiffness, matrix architecture, and transforming potential in a mammary epithelial cell (MEC) cancer progression series. We use a well characterized breast cancer progression model where human-derived MCF10A MECs overexpress either ErbB2, 14-3-3ζ, or both ErbB2 and 14-3-3ζ, with empty vector as a control. Cell motility assays showed that MECs overexpressing ErbB2 alone exhibited notably high migration speeds when cultured atop two-dimensional (2D) matrices, while overexpression of 14-3-3ζ alone most suppressed migration atop 2D matrices (as compared to non-transformed MECs). Our results also suggest that co-overexpression of the 14-3-3ζ and ErbB2 proteins facilitates cell migratory capacity in 3D matrices, as reflected in cell migration speed. Additionally, 3D matrices of sufficient stiffness can significantly hinder the migratory ability of partially transformed cells, but increased 3D matrix stiffness has a lesser effect on the aggressive migratory behavior exhibited by fully transformed cells that co-overexpress both ErbB2 and 14-3-3ζ. Finally, this study shows that for MECs possessing partial or full transforming potential, those overexpressing ErbB2 alone show the greatest sensitivity of cell migration speed to matrix architecture, while those overexpressing 14-3-3ζ alone exhibit the least sensitivity to matrix architecture. Given the current knowledge of breast cancer mechanobiology, these findings overall suggest that cell motility is governed by a complex interplay between matrix mechanics and transforming potential.
Metastasis accounts for 90% of cancer-related mortality. Brain metastases generally present during the late stages in the natural history of cancer progression. Recent advances in cancer treatment and management have resulted in better control of systemic disease metastatic to organs other than the brain and improved patient survival. However, patients who experience recurrent disease manifest an increasing number of brain metastases, which are usually refractory to therapies. To meet the new challenges of controlling brain metastasis, the research community has been tackling the problem with novel experimental models and research tools, which have led to an improved understanding of brain metastasis. The time-tested "seed-and-soil" hypothesis of metastasis indicates that successful outgrowth of deadly metastatic tumors depends on permissible interactions between the metastatic cancer cells and the site-specific microenvironment in the host organs. Consistently, recent studies indicate that the brain, the major component of the central nervous system, has unique physiological features that can determine the outcome of metastatic tumor growth. The current review summarizes recent discoveries on these tumor-brain interactions, and the potential clinical implications these novel findings could have for the better treatment of patients with brain metastasis.
Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.