Two Sides of the Same Coin: The Role of Developmental pathways and pluripotency factors in normal mammary stem cells and breast cancer metastasis.

Breast cancer initiation and progression are often observed as the result of dysregulation of normal developmental processes and pathways. Studies focused on normal mammary stem/progenitor cell activity have led to an understanding of how breast cancer cells acquire stemness-associated properties including tumor initiation, survival and multi-lineage differentiation into heterogeneous tumors that become difficult to target therapeutically. Importantly, more recent investigations have provided valuable insight into how key developmental regulators can impact multiple phases of metastasis, where they are repurposed to not only promote metastatic phenotypes such as migration, invasion and EMT at the primary site, but also to regulate the survival, initiation and maintenance of metastatic lesions at secondary organs. Herein, we discuss findings that have led to a better understanding of how embryonic and pluripotency factors contribute not only to normal mammary development, but also to metastatic progression. We further examine the therapeutic potential of targeting these developmental pathways, and discuss how a better understanding of compensatory mechanisms, crosstalk between pathways, and novel experimental models could provide critical insight into how we might exploit embryonic and pluripotency regulators to inhibit tumor progression and metastasis.

Mitoxantrone in relapsing-remitting and rapidly progressive multiple sclerosis: Ten-year clinical outcomes post-treatment with mitoxantrone.

BACKGROUND: Mitoxantrone (MTX) has been used as an effective disease modifying treatment (DMT) in multiple sclerosis (MS). Evidence from studies demonstrates benefits of reduced relapse rates, MRI disease activity and disability progression in patients treated with MTX. While effective, MTX use has been limited due to potential adverse effects (AE) ranging from mild to potentially life-threatening AEs such as cardiotoxicity, bone marrow suppression and hematological malignancies. In this study we aimed to review the long-term clinical efficacy, tolerability, and AE profile of treatment with MTX in patients both with relapsing-remitting and rapidly progressive MS over a 10-year follow-up period. METHODS: We collected prospective data of 70 patients with relapsing-remitting and rapidly progressive MS treated with MTX and followed-up over a 10-year period. Expanded disability status scale (EDSS) scores and annualized relapse rates (ARR) were assessed 1 year prior to MTX treatment, and at different time points (1, 2, 3, 5 and 10 years) during follow-up. We recorded the time to first relapse and 0.5-point EDSS increase to assess efficacy. We also obtained frequency data on AEs and patients withdrawn from treatment. RESULTS: 70 patients were started on treatment with MTX with 53 patients (34 relapsing-remitting MS, 19 progressive disease) completing the course. Mean EDSS progressed from 5.5 to 6.5 in the relapsing-remitting group and 6.7 to 9.0 in the progressive group over the study period. ARR in the RRMS group reduced at all time points from 2.2 prior to MTX to 0.3 by year 10. We reported 3 significant AEs, one chicken pox and subsequent acute promyelocytic leukemia, one left ventricular systolic dysfunction, one pancytopenia. The commonest AE reported was nausea/vomiting in 28 (40%) patients. Seventeen patients (5 relapsing-remitting, 12 progressive disease) stopped treatment. In fifteen (87%) of these this was due to lack of efficacy. In the remaining 2 patients, MTX was stopped due to one patient developing chicken pox and the other developing first-degree heart block. CONCLUSION: Our study demonstrated that MTX is an effective disease modifying treatment for relapsing-remitting MS with a well-established risk profile. While MTX is now used less frequently, many MS and neurology services continue to follow-up patients who have been treated with MTX previously. Therefore, understanding the long-term effects risks and benefits remains relevant in this patient group. MTX is also a low-cost treatment in comparison to other high efficacy MS disease-modifying treatments and this may be beneficial in low resource settings.

Patient perspective on decisions to switch disease-modifying treatments in relapsing-remitting multiple sclerosis.

BACKGROUND: There are now large cohorts of people with relapsing-remitting multiple sclerosis (pwRRMS) who have taken several Disease-Modifying Treatments (DMTs). Studies about switching DMTs mostly focus on clinical outcomes rather than patients' decision-making. Neurologists are now required to support decisions at various times during the relapsing disease course and they do so with concerns about DMTs risks. This qualitative study investigates how pwRRMS weigh up the pros and cons of DMTs, focusing on perceptions of effectiveness and risks when new treatments are considered. OBJECTIVE: To increase understanding of people's experiences of decision-making when switching DMTs. METHODS: 30 semi-structured interviews were conducted with pwRRMS in England. 16 participants had switched DMT and their experiences were compared with those who had only taken one DMT. Interviews were analysed thematically to answer: what main factors influence people's decision-making to switch DMTs and why? RESULTS: Of the 16 participants with experience of switching DMT, eight had taken two or more DMTs; eight had taken three or more. Two was the DMT median. This study demonstrated that despite the term "switching" implying that similar treatments are inter-changeable, for pwRRMS taking new treatments involves different emotions, routines, risks, prognosis and communication experiences. Two meta themes identified were: 1) A distinctive, rapid and emotional decision-making process where old emotions related to MS prognosis are revisited. 2) Switching has a different impact on communication for escalation or de-escalation processes. CONCLUSION: Switching DMT involves different routines, risks, prognosis and communication experiences. These decisions are emotionally difficult because of the fear about transitioning to secondary progressive MS, and DMT effectiveness uncertainty. Patient centred decision aids should include information about first and consecutive treatment decisions.

Expression of Six1 homeobox gene during development of the mouse submandibular salivary gland.

BACKGROUND: Members of the Six family of homeoproteins are expressed in numerous tissues during vertebrate embryogenesis, and are critical regulators of both cell proliferation and survival. Here we report the temporal and spatial expression of Six1 during maturation of the mouse submandibular salivary gland (SSG) from embryonic day 18.5 (E18.5) to postnatal day 28. Additionally, we examine the role of Six1 during SSG development using Six1-deficient mice. METHODS: Six1 expression was assessed by reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence. Proliferation was measured by bromodeoxyuridine (BrdU) incorporation index, and apoptosis was evaluated by TUNEL assay. RESULTS: Six1 mRNA and protein levels are high in the epithelial SSG cells at E18.5 and decrease progressively in the postnatal maturing SSG. Although SSGs from Six1(-/-) embryos are significantly smaller than wild type SSGs, the histological structures of the SSG acini and ducts are similar. Six1(-/-) salivary epithelial cells exhibit an intrinsic defect in cell proliferation accompanied by a significant reduction in the Six1 target gene cyclin A1, previously shown to be a critical mediator of Six1-induced proliferation. CONCLUSION: Our results suggest that the reduction in size of Six1(-/-) SSGs is result of a decrease in cell proliferation during development/maturation.

Use of coping strategies in multiple sclerosis: Association with demographic and disease-related characteristics✰.

BACKGROUND: Coping positively and negatively influences psychosocial and other outcomes in multiple sclerosis (MS), but there is conflicting evidence about the use of different coping strategies and their associations with demographic and disease characteristics. Our aims were to examine which coping strategies are used by a large sample of people with MS, then to identify any associations between demographic and disease related factors with use of individual coping strategies. METHODS: Participants in the Trajectories of Outcomes in Neurological Conditions (TONiC) study completed the Coping Orientations to Problems Experienced (COPE60) questionnaire. Relationships between demographic and clinical characteristics and coping strategies were examined by multiple ordinal logistic regression to assess the effect of each potential predictor after adjustment for other possible covariates. RESULTS: From 722 patients, the most commonly used strategy was Acceptance, followed by Active Coping, Planning and Positive Reinterpretation and Growth. All but two strategies showed significant associations with demographic and clinical characteristics. The most marked effects were found for Restraint, with people in employment 2.1 times as likely to utilise this strategy compared to those unemployed, and Seeking of Emotional Social Support and Focus on and Venting of Emotions, which were utilised twice as much by women compared to men. Behavioural and Mental Disengagement were highly associated with greater disability and not being in employment. CONCLUSION: Clinicians should be aware of several disease and demographic characteristics that are associated with use of potentially maladaptive coping strategies.

Cell cycle regulation of the human Six1 homeoprotein is mediated by APC(Cdh1).

The Six1 homeoprotein is an important mediator of normal development, where it is critical for the proliferation of precursor cell populations that ultimately constitute the muscle, kidney and inner ear, among other organs. Interestingly, its overexpression has been observed in numerous cancers, where it contributes to the proliferative and metastatic ability of the cancer cells. Here we show that Six1 not only regulates the cell cycle, but is itself regulated throughout the cell cycle via ubiquitin-mediated proteolysis. The protein is present from the G(1)/S boundary until mitosis, when it is degraded via the anaphase-promoting complex (APC) with its activating subunit Cdh1. However, unlike most identified APC(Cdh1) targets, Six1 does not contain functional destruction or KEN box motifs that are necessary for its degradation. Instead, the Six1 protein contains multiple, as yet undefined, sequences within its N- and C-termini responsible for its degradation, including an N-terminal region that binds to Cdh1. Cell cycle regulation of Six1 occurs both transcriptionally and post-translationally via phosphorylation; therefore, this study demonstrates a third and novel mechanism of cell cycle-specific regulation of Six1, underscoring the importance of confining its activity to a defined cell cycle window from the G(1)/S boundary to early mitosis.

Multi-system complications of hypothermia: a case of recurrent episodic hypothermia with a review of the pathophysiology of hypothermia.

A 61-year-old woman with secondary progressive multiple sclerosis presented on six occasions over a 2-year period with severe hypothermia (31-33.5 degrees C). This resulted in numerous multi-system complications comprising acute pancreatitis, hepatitis, gastrointestinal haemorrhage, psychiatric disturbance, bradycardia, paradoxical sweating, thrombocytopenia, anaemia and raised inflammatory markers. Septic screens were consistently normal. On each occasion she was successfully treated with passive external rewarming and made a complete recovery. This is the first reported case of such extensive sequelae in a single patient with recurrent hypothermic episodes. This unusual patient provides an invaluable insight into the natural history and pathophysiology of hypothermia. The case report is followed by a review of dysfunctional thermoregulation and pathophysiology of hypothermia-induced multi-system complications. A key learning point is to recognise that the clinical manifestations of hypothermia may be widespread and serious but are nonetheless reversible. In addition, one should consider the differential diagnosis of covert hypothermia in those patients with episodic confusion, as hypothermia is under-recognised, particularly in older people, who are prone to accidental hypothermia, and in those with common neurological conditions, such as stroke, head injury and multiple sclerosis, that may have suboptimal thermoregulation.

The miR-106b-25 cluster mediates breast tumor initiation through activation of NOTCH1 via direct repression of NEDD4L.

Tumor-initiating cells (TIC) represent a subset of tumor cells with increased self-renewal capability. TICs display resistance to frontline cancer treatment and retain the ability to repopulate a tumor after therapy, leading to cancer relapse. NOTCH signaling has been identified as an important driver of the TIC population, yet mechanisms governing regulation of this pathway in cancer remain to be fully elucidated. Here we identify a novel mechanism of NOTCH regulation and TIC induction in breast cancer via the miR-106b-25 miRNA cluster. We show that the miR-106b-25 cluster upregulates NOTCH1 in multiple breast cancer cell lines, representing both estrogen receptor (ER+) and triple negative breast cancer (TNBC) through direct repression of the E3 ubiquitin ligase, NEDD4L. We further show that upregulation of NOTCH1 is necessary for TIC induction downstream of miR-106b-25 in both ER + and TNBC breast cancer cells, and that re-expression of NEDD4L is sufficient to reverse miR106b-25-mediated NOTCH1 upregulation and TIC induction. Importantly, we demonstrate a significant positive correlation between miR-106b-25 and NOTCH1 protein, yet a significant inverse correlation between miR-106b-25 and NEDD4L mRNA in human breast cancer, suggesting a critical role for the miR106b-25/NEDD4L/NOTCH1 axis in the disease. Further, we show for the first time that NEDD4L expression alone is significantly associated with a better relapse-free prognosis for breast cancer patients. These data expand our knowledge of the mechanisms underlying NOTCH activation and TIC induction in breast cancer, and may provide new avenues for the development of therapies targeting this resistant subset of tumor cells.

Assessment of susceptibility of the poultry red mite Dermanyssus gallinae (Acari: Dermanyssidae) to some acaricides using an adapted filter paper based bioassay.

A previously described bioassay was modified to assess the response of the poultry red mite, an important ectoparasite of fowl, to a selected group of acaricides. The adapted bioassay is simple to use, escape-proof and provides data that can be subjected to probit analysis. The reproducibility of the method was assessed by three tests with alpha-cypermethrin against a reference strain, which produced dose-response lines that did not differ significantly (chi(2)=1.39, 2 d.f., p=0.50), and had a derived common slope of 1.89. In addition, a limited evaluation study enabled assessment of response to commercial formulations of bifenthrin, bifenthrin+malathion and cypermethrin by field populations of the poultry red mite.

Drg-1 as a differentiation-related, putative metastatic suppressor gene in human colon cancer.

A gene related to cell differentiation was identified by differential display as a candidate suppressor of metastases in colon cancer. This gene, with a full-length cDNA of 3 kb, is expressed in normal colon and primary colon cancer tissues and cell lines but not in their metastatic counterparts. A GenBank search found that it is identical to a recently cloned gene, differentiation-related gene-1 (Drg-1), isolated from differentiated HT-29 colon cancer cells. Stable transfection of the SW620 metastatic colon cancer cell line with Drg-1 cDNA induced morphological changes consistent with differentiation and up-regulated the expression of several colonic epithelial cell differentiation markers (alkaline phosphatase, carcinoembryonic antigen, and E-cadherin). Moreover, the expression of Drg-1 is controlled by several known cell differentiation reagents, such as ligands of peroxisome proliferator-activated receptor gamma (troglitazone and BRL46593) and of retinoid X receptor (LG268), and histone deacetylase inhibitors (trichostatin A, suberoylanilide hydroxamic acid, and tributyrin). A synergistic induction of Drg-1 expression was seen with the combination of tributyrin and a low dose of 5'-aza-2'-dexoycytidine (100 nM), an inhibitor of DNA methylation. Functional studies revealed that overexpression of Drg-1 in metastatic colon cancer cells reduced in vitro invasion through Matrigel and suppressed in vivo liver metastases in nude mice. We propose that Drg-1 suppresses colon cancer metastasis by inducing colon cancer cell differentiation and partially reversing the metastatic phenotype.

Interaction of metastasis associated Mts1 protein with nonmuscle myosin.

The mts1 gene codes for a 101 amino acid protein which belongs to the subfamily of S100 Ca(2+)-binding proteins and is overexpressed in metastatic cancers as compared to their nonmetastatic counterparts. While the Mts1 protein is putatively involved in cytoskeletal-membrane interactions, its exact physiological role is not known. In order to gain insight into the biological function of Mts1, its expression was monitored throughout mouse embryogenesis as well as in normal adult mouse tissues. In situ hybridizations of mouse embryos showed expression of mts1 mRNA to be highest in trophoblast cells of a day 8 old embryo. In normal adult mouse tissues, immunocytochemistry revealed Mts1 expression in a T-cell specific pattern in both the thymus and spleen. These results indicate that the expression of mts1 is prevalent in motile cell types, and to further elucidate the role of Mts1, gel overlay and GST-fusion protein experiments were performed to identify proteins that interact with the Mts1 protein. Nonmuscle myosin II was found to interact with Mts1, suggesting a role for Mts1 in the process of motility and lending credence to the hypothesis that Mts1 may enhance metastatic potential by increasing the motility of cancerous cells.

Expression of Mts1, a metastasis-associated gene, increases motility but not invasion of a nonmetastatic mouse mammary adenocarcinoma cell line.

The mts1 gene codes for a 101 amino acid protein belonging to the S100 subfamily of Ca(2+)-binding proteins. Mts1 is overexpressed in metastatic cancers as compared to their nonmetastatic counterparts, and although mts1 is known to be involved in the metastatic phenotype (Davies et al., 1993; Grigorian et al., 1993), the role mts1 plays in this process is not clearly understood. In order to determine what role mts1 plays in the process of metastasis, we have performed transfection studies on nonmetastatic and metastatic mouse mammary adenocarcinoma cell lines, CSML0 and CSML100, respectively (Senin et al., 1983, 1984). The metastatic variant, CSML100, expresses high levels of mts1, whereas the nonmetastatic variant, CSML0, expresses almost no mts1. CSML0 cells transfected with mts1 were assessed in in vitro motility and invasion assays, as well as in vivo metastasis assays to determine the role of mts1 in these processes. Cell lines expressing mts1 display an altered morphology as well as increased motility in modified Boyden chemotaxis chambers. However, no significant increase in in vitro invasion or in in vivo metastasis was observed. Therefore, the presence of mts1 may be important for metastasis by increasing motility, but may not be sufficient for invasion in vitro or metastasis in vivo. Very low levels of type IV collagenase activities were observed in CSML0 cells and the transfectants, as opposed to the highly metastatic CSML100 cells, where high levels of type IV collagenase activities were observed. It is possible that the presence of these proteases in addition to mts1 may be responsible for the high metastatic potential of the CSML100 in vivo.

Characterization of a positive regulatory element in the mts1 gene.

The first intron of the mts1 gene, a gene that is selectively expressed in metastatic cells and in normal cells that are motile, was found to be highly homologous to the CD3 delta enhancer element. Because of the homology between the CD3 delta enhancer and the first intron of mts1, we analysed the first intron of the mts1 gene to determine whether it functions as a transcriptional regulatory element. Highly metastatic CSML-100 cells transfected with chloramphenicol acetyl transferase-containing plasmids demonstrated the ability of the mts1 first intron to function as a positive regulatory element. In vitro footprinting analysis using extracts from CSML-0 cells (which express mts1 at low levels) or CSML-100 cells (which express mts1 at high levels) identified a protected 16-nucleotide element in the first intron of mts1, regardless of the extract used. However, in vivo footprinting analysis of the same region identified the protected 16-nucleotide fragment only in the mts1 intron from CSML-100 cells, not from CSML-0 cells. Differences in the methylation pattern of the mts1 gene in CSML-100 cells and CSML-0 cells are known to exist, and may in part be responsible for the mts1 footprinting differences observed in vivo from the different cell lines.

A simple pump-assisted method for collecting live, undamaged Psoroptes ovis from sheep using circulating saline.

The availability of large numbers of undamaged sheep scab mites, Psoroptes ovis, would be beneficial for discovery screening and development trials. There are several reported procedures for removing scab mites from sheep but they have limitations. To overcome this, a simple but versatile method employing the use of pumped saline was developed to remove all stages of the P. ovis mite from sheep. The method takes no more than 2 min to remove mites from the selected site with relative ease and is not affected by the condition of the fleece or lesion. The number of mites removed with the new method was 5-10 times more than detected by visual examination. These mites were undamaged and survived off-host for up to 16 days. The robust, portable equipment is easy to use under field conditions, making this method suitable for use as a diagnostic tool for early detection and monitoring of scab mites thus providing opportunities for development of novel alternative control strategies.

Health status and quality of life of people with multiple sclerosis.

PURPOSE: The aim of this study was to ascertain the health status and quality of life of a community based cohort of people with multiple sclerosis. METHOD: A postal questionnaire with self-completed measures of impairment, disability, physical dependency and quality of life was sent to a random sample of 203 people with multiple sclerosis from a population register. The sample was stratified according to five disease courses. The population register is of the prevalent population of 760 people with multiple sclerosis resident in the Leeds Health Authority. The register used multiple sources of ascertainment and is prospectively maintained with new incident cases. RESULTS: The estimated mean age of people with multiple sclerosis is 46 years (SE: 0.85), and mean duration of disease is 14.4 years (SE: 0.69). Almost four in five (78 %) are female, and one in six (17%) live alone. Impairments of balance, vision and memory are common and in all cases there is little difference in the frequency between disease course groups. In contrast, impairments of bladder and bowel are more common in those with a progressive disease course. Disability is more common in those with a progressive disease course but all scores on the SF36 Physical Function scale are low and demonstrate the disabling consequences of the disease, irrespective of disease course. These consequences must contribute to the fact that over two-thirds (68 %) were not employed at the time of the survey. Quality of life does not differ across disease course groups, but rather varies by age and duration. CONCLUSIONS: People with multiple sclerosis experience a range of impairments and disabilities. Those with progressive disease courses experience greater levels of impairment and disability than other groups. There is not a straightforward exchange between health status and quality of life. A measure of subjective quality of life may reflect adjustment to disease, such that, for example, the longer the duration, the older the individual, the more likely the person will report a relatively good quality of life.

Bacterial meningitis in developing countries.

PIP: Hemophilus influenza, Streptococcus pneumoniae, and Neisseria meningitidis account for over 75% of all cases of bacterial meningitis. S. pneumoniae is the commonest causative organism in many developing countries, particularly in Africa. In developing countries overall case fatality rates of 33-44% have been reported, rising to over 60% in adult groups. S. pneumoniae accounts for the highest mortality worldwide. Sequela rates of 22-26% of survivors have been found in African studies, mostly of a neurological nature. There have been few reports of AIDS-related bacterial meningitis in the USA, and a recent study from Uganda found no association between HIV infection and meningococcal meningitis. Stronger associations have been found between opportunistic infections, both viral (cytomegalovirus, herpes virus) and non-viral (TB, Toxoplasma gondii, Cryptococcus neoformans). A lumbar puncture and analysis of the cerebrospinal fluid should be performed on suspected cases unless there is suspicion of impending coning (decreasing consciousness or focal neurological signs). The intramuscular administration of chloramphenicol alone is comparable with intravenous use, and can be given as a shorter course of therapy (2 or 3 days) followed by an oral course. The use of adjunct therapy with corticosteroids in children is now commonplace in the USA and Europe. It appears reasonable to use dexamethasone, given early and in high dosage (0.15 mg/kg 6 hourly for 4 days), in those patients who are severely ill. Rifampicin is effective for chemoprophylaxis (10 mg/kg twice daily for 2 days for meningococcal contacts, 20 mg/kg once daily for 4 days for hemophilus contacts, maximum 600 mg per dose). The recent development and introduction of conjugate vaccines for H. influenza (HIB) has led to rapid reductions in the incidence of hemophilus meningitis in many European countries. An important step in improving prognosis is to increase awareness in both health workers and the public, to encourage early hospital referral, and early antibiotic therapy.^ieng

On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic antibodies against TRAIL death receptors (DR) kill tumor cells while causing virtually no damage to normal cells. Several novel drugs targeting TRAIL receptors are currently in clinical trials. However, TRAIL resistance is a common obstacle in TRAIL-based therapy and limits the efficiency of these drugs. In this review article we discuss different mechanisms of TRAIL resistance, and how they can be predicted and therapeutically circumvented. In addition, we provide a brief overview of all TRAIL-based clinical trials conducted so far. It is apparent that although the effects of TRAIL therapy are disappointingly modest overall, a small subset of patients responds very well to TRAIL. We argue that the true potential of targeting TRAIL DRs in cancer can only be reached when we find efficient ways to select for those patients that are most likely to benefit from the treatment. To achieve this, it is crucial to identify biomarkers that can help us predict TRAIL sensitivity.

Eya2 is required to mediate the pro-metastatic functions of Six1 via the induction of TGF-ß signaling, epithelial-mesenchymal transition, and cancer stem cell properties.

Six1 is a critical regulator of embryonic development that requires interaction with the Eya family of proteins (Eya1-4) to activate the transcription of genes involved in neurogenesis, myogenesis and nephrogenesis. Although expression of Six1 and Eya family members is predominantly observed in development, their overexpression is observed in numerous cancers. Importantly, both Six1 and Eya have independently been shown to mediate breast cancer metastasis, but whether they functionally interact during tumor progression has not been explored. Herein, we demonstrate that knockdown of Eya2 in MCF7 mammary carcinoma cells reverses the ability of Six1 to induce transforming growth factor-ß signaling, as well as to induce characteristics associated with epithelial-mesenchymal transition and cancer stem cells, suggesting that Six1 is dependent on Eya2 to mediate numerous pro-metastatic characteristics. The importance of the Six1-Eya interaction in human breast cancer is underscored by the finding that high levels of Six1 correlate with shortened time to relapse and metastasis as well as decreased survival only when co-expressed with high levels of Eya2. Overall, these data implicate Eya2 as a necessary co-factor for many of the metastasis promoting functions of Six1, suggesting that targeting the Six1-Eya interaction may inhibit breast cancer progression. As Six1 and Eya2 are not highly expressed in most adult tissues, the Six1-Eya interaction may be a valuable future therapeutic target whose inhibition would be expected to impair breast cancer progression while conferring limited side effects.

The miR-106b-25 cluster targets Smad7, activates TGF-ß signaling, and induces EMT and tumor initiating cell characteristics downstream of Six1 in human breast cancer.

The role of TGF-ß signaling in tumorigenesis is paradoxical: it can be tumor suppressive or tumor promotional, depending on context. The metastatic regulator, Six1, was recently shown to mediate this switch, providing a novel means to explain this elusive 'TGF-ß paradox'. Herein, we identify a mechanism by which Six1 activates the tumor promotional arm of TGF-ß signaling, via its ability to upregulate the miR-106b-25 microRNA cluster, and further identify a novel function for this cluster of microRNAs. Although expression of the miR-106b-25 cluster is known to overcome TGF-ß-mediated growth suppression via targeting p21 and BIM, we demonstrate for the first time that this same cluster can additionally target the inhibitory Smad7 protein, resulting in increased levels of the TGF-ß type I receptor and downstream activation of TGF-ß signaling. We further show that the miR-106b-25 cluster is sufficient to induce an epithelial-to-mesenchymal transition and a tumor initiating cell phenotype, and that it is required downstream of Six1 to induce these phenotypes. Finally, we demonstrate a significant correlation between miR-106b, Six1, and activated TGF-ß signaling in human breast cancers, and further show that high levels of miR-106b and miR-93 in breast tumors significantly predicts shortened time to relapse. These findings expand the spectrum of oncogenic functions of miR-106b-25, and may provide a novel molecular explanation, through the Six1 regulated miR-106b-25 cluster, by which TGF-ß signaling shifts from tumor suppressive to tumor promoting.