BRAF Inhibitor Resistance Confers Increased Sensitivity to Mitotic Inhibitors.

Single agent and combination therapy with BRAFV600E/K and MEK inhibitors have remarkable efficacy against melanoma tumors with activating BRAF mutations, but in most cases BRAF inhibitor (BRAFi) resistance eventually develops. One resistance mechanism is reactivation of the ERK pathway. However, only about half of BRAFi resistance is due to ERK reactivation. The purpose of this study is to uncover pharmacological vulnerabilities of BRAFi-resistant melanoma cells, with the goal of identifying new therapeutic options for patients whose tumors have developed resistance to BRAFi/MEKi therapy. We screened a well-annotated compound library against a panel of isogenic pairs of parental and BRAFi-resistant melanoma cell lines to identify classes of compounds that selectively target BRAFi-resistant cells over their BRAFi-sensitive counterparts. Two distinct patterns of increased sensitivity to classes of pharmacological inhibitors emerged. In two cell line pairs, BRAFi resistance conferred increased sensitivity to compounds that share the property of cell cycle arrest at M-phase, including inhibitors of aurora kinase (AURK), polo-like kinase (PLK), tubulin, and kinesin. Live cell microscopy, used to track mitosis in real time, revealed that parental but not BRAFi-resistant melanoma cells were able to exit from compound-induced mitotic arrest through mitotic slippage, thus escaping death. Consistent with the key role of Cyclin B1 levels in regulating mitosis at the spindle checkpoint in arrested cells, we found lower Cyclin B1 levels in parental compared with BRAFi-resistant melanoma cells, suggesting that inability to down-regulate Cyclin B1 expression levels may explain the increased vulnerability of resistant cells to mitotic inhibitors. Another BRAFi-resistant cell line showed increased sensitivity to Chk1/2 inhibitors, which was associated with an accumulation of DNA damage, resulting in mitotic failure. This study demonstrates that BRAFi-resistance, in at least a subset of melanoma cells, confers vulnerability to pharmacological disruption of mitosis and suggests a targeted synthetic lethal approach for overcoming resistance to BRAF/MEK-directed therapies.

Therapeutic potential of targeting mixed lineage kinases in cancer and inflammation.

Dysregulation of intracellular signaling pathways is a key attribute of diseases associated with chronic inflammation, including cancer. Mitogen activated protein kinases have emerged as critical conduits of intracellular signal transmission, yet due to their ubiquitous roles in cellular processes, their direct inhibition may lead to undesired effects, thus limiting their usefulness as therapeutic targets. Mixed lineage kinases (MLKs) are mitogen-activated protein kinase kinase kinases (MAP3Ks) that interact with scaffolding proteins and function upstream of p38, JNK, ERK, and NF-kappaB to mediate diverse cellular signals. Studies involving gene silencing, genetically engineered mouse models, and small molecule inhibitors suggest that MLKs are critical in tumor progression as well as in inflammatory processes. Recent advances indicate that they may be useful targets in some types of cancer and in diseases driven by chronic inflammation including neurodegenerative diseases and metabolic diseases such as nonalcoholic steatohepatitis. This review describes existing MLK inhibitors, the roles of MLKs in various aspects of tumor progression and in the control of inflammatory processes, and the potential for therapeutic targeting of MLKs.

Correction: The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop.

[This corrects the article DOI: 10.18632/oncotarget.26047.].

The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop.

Activating protein-1 (AP-1) family members, especially Fra-1 and c-Jun, are highly expressed in invasive cancers and can mediate enhanced migration and proliferation. The aim of this study was to explore the significance of elevated levels of AP-1 family members under conditions that restrict growth. We observed that invasive MDA-MB-231 cells express high levels of Fra-1, c-Jun, and Jun-D during serum starvation and throughout the cell cycle compared to non-tumorigenic and non-invasive cell lines. We then analyzed Fra-1 levels in additional breast and other cancer cell lines. We found breast and lung cancer cells with higher levels of Fra-1 during serum starvation had relatively higher ability to proliferate and migrate under these conditions. Utilizing a dominant negative construct of AP-1, we demonstrated that proliferation and migration of MDA-MB-231 in the absence of serum requires AP-1 activity. Finally, we observed that MDA-MB-231 cells secrete factors(s) that induce Fra-1 expression and migration in non-tumorigenic and non-metastatic cells and that both the expression of and response to these factors require AP-1 activity. These results suggest the presence of an autocrine/paracrine loop that maintains high Fra-1 levels in aggressive cancer cells, enhancing their proliferative and metastatic ability and affecting neighbors to alter the tumor environment.

Targeting mixed lineage kinases in ER-positive breast cancer cells leads to G2/M cell cycle arrest and apoptosis.

Estrogen receptor (ER)-positive tumors represent the most common type of breast cancer, and ER-targeted therapies such as antiestrogens and aromatase inhibitors have therefore been widely used in breast cancer treatment. While many patients have benefited from these therapies, both innate and acquired resistance continue to be causes of treatment failure. Novel targeted therapeutics that could be used alone or in combination with endocrine agents to treat resistant tumors or to prevent their development are therefore needed. In this report, we examined the effects of inhibiting mixed-lineage kinase (MLK) activity on ER-positive breast cancer cells and non-tumorigenic mammary epithelial cells. Inhibition of MLK activity with the pan-MLK inhibitor CEP-1347 blocked cell cycle progression in G2 and early M phase, and induced apoptosis in three ER-positive breast cancer cell lines, including one with acquired antiestrogen resistance. In contrast, it had no effect on the cell cycle or apoptosis in two non-tumorigenic mammary epithelial cell lines. CEP-1347 treatment did not decrease the level of active ERK or p38 in any of the cell lines tested. However, it resulted in decreased JNK and NF-κB activity in the breast cancer cell lines. A JNK inhibitor mimicked the effects of CEP-1347 in breast cancer cells, and overexpression of c-Jun rescued CEP-1347-induced Bax expression. These results indicate that proliferation and survival of ER-positive breast cancer cells are highly dependent on MLK activity, and suggest that MLK inhibitors may have therapeutic efficacy for ER-positive breast tumors, including ones that are resistant to current endocrine therapies.

The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells.

MEK Partner 1 (MP1 or MAPKSP1) is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1's functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER) positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.

Enzyme replacement therapy for mucopolysaccharidosis VI: Growth and pubertal development in patients treated with recombinant human N-acetylgalactosamine 4-sulfatase.

BACKGROUND AND METHODS: Growth failure is characteristic of untreated mucopolysaccharidosis type VI (MPS VI: Maroteaux-Lamy syndrome). Growth was studied in fifty-six MPS VI patients (5 to 29 years old) prior to and for up to 240 weeks of weekly infusions of recombinant human arylsulfatase B (rhASB) at 1 mg/kg during Phase 1/2, Phase 2, Phase 3 or Phase 3 Extension clinical trials. Height, weight, and Tanner stage data were collected. Pooled data were analyzed to determine mean height increase by treatment week, growth impacts of pubertal status, baseline urinary GAG, and age at treatment initiation. Growth rate for approximately 2 years prior to and following treatment initiation was analyzed using longitudinal modeling. RESULTS: Mean height increased by 2.9 cm after 48 weeks and 4.3 cm after 96 weeks on enzyme replacement therapy (ERT). Growth on ERT was not correlated with baseline urinary GAG. Patients under 16 years of age showed greatest increases in height on treatment. Model results based on pooled data showed significant improvement in growth rate during 96 weeks of ERT when compared to the equivalent pretreatment time period. Delayed pubertal onset or progression was noted in 10 patients entering the clinical trials; all of whom showed progression of at least one Tanner stage during 2 years on ERT, and 6 of whom (60%) completed puberty. CONCLUSION: Analysis of mean height by treatment week and longitudinal modeling demonstrate significant increase in height and growth rate in MPS VI patients receiving long-term ERT. This impact was greatest in patients aged below 16 years. Height increase may result from bone growth and/or reduction in joint contractures. Bone growth and resolution of delayed puberty may be related to improvements in general health, bone cell health, nutrition, endocrine gland function and reduced inflammation.

Signal transducer and activator of transcription 5a mediates mammary ductal branching and proliferation in the nulliparous mouse.

Signal transducer and activator of transcription (Stat)5a is a critical regulator of mammary gland development. Previous studies have focused on Stat5a's role in the late pregnant and lactating gland, and although active Stat5a is detectable in mammary epithelial cells in virgin mice, little is known about its role during early mammary gland development. In this report, we compare mammary gland morphology in pubertal and adult nulliparous wild-type and Stat5a-/- mice. The Stat5a-null mammary glands exhibited defects in secondary and side branching, providing evidence that Stat5a regulates these processes. In addition, Stat5a-/- mammary glands displayed an attenuated proliferative response to pregnancy levels of estrogen plus progesterone (E+P), suggesting that it plays an important role in early pregnancy. Finally, we examined one potential mediator of Stat5a's effects, receptor activator of nuclear factor-kappaB ligand (RANKL). Stat5a-/- mammary glands were defective in inducing RANKL in response to E+P treatment. In addition, regulation of several reported RANKL targets, including inhibitor of DNA binding 2 (Id2), cyclin D1, and the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), was altered in Stat5a-/- mammary cells, suggesting that one or more of these proteins mediate the effects of Stat5a in E+P-treated mammary epithelial cells.

Progesterone receptor A-regulated gene expression in mammary organoid cultures.

Progesterone, through the progesterone receptor (PR), promotes development of the normal mammary gland and is implicated in the etiology of breast cancer. We identified PRA-regulated genes by microarray analysis of cultured epithelial organoids derived from pubertal and adult mouse mammary glands, developmental stages with differing progesterone responsiveness. Microarray analysis showed significant progestin (R5020)-regulation of 162 genes in pubertal organoids and 104 genes in adult organoids, with 68 genes regulated at both developmental stages. Greater induction of receptor activator of NFkappaB ligand and calcitonin expression was observed in adult organoids, suggesting possible roles in the differential progesterone responsiveness of the adult and pubertal mammary glands. Analysis of the R5020-responsive transcriptome revealed several enriched biological processes including cell adhesion, immune response, and survival. R5020 both induced Agtr1 and potentiated angiotensin II-stimulated proliferation, highlighting the functional significance of the latter process. Striking up-regulation of genes involved in innate immunity processes included the leukocyte chemoattractants serum amyloid A1, 2 and 3 (Saa1, 2, 3). In vivo analysis revealed that progesterone treatment increased SAA1 protein expression and leukocyte density in mammary gland regions undergoing epithelial expansion. These studies reveal novel targets of PRA in mammary epithelial cells and novel linkages of progesterone action during mammary gland development.

Development of chemosensitivity in neurons from the nucleus tractus solitarii (NTS) of neonatal rats.

We studied the development of chemosensitivity during the neonatal period in rat nucleus tractus solitarii (NTS) neurons. We determined the percentage of neurons activated by hypercapnia (15% CO(2)) and assessed the magnitude of the response by calculating the chemosensitivity index (CI). There were no differences in the percentage of neurons that were inhibited (9%) or activated (44.8%) by hypercapnia or in the magnitude of the activated response (CI 164+/-4.9%) in NTS neurons from neonatal rats of all ages. To assess the degree of intrinsic chemosensitivity in these neurons we used chemical synaptic block medium and the gap junction blocker carbenoxolone. Chemical synaptic block medium slightly decreased basal firing rate but did not affect the percentage of NTS neurons that responded to hypercapnia at any neonatal age. However, in neonates aged

Isolated adrenocorticotropic hormone deficiency presenting as an acute neurologic emergency in a peripubertal girl.

Isolated adrenocorticotropic hormone (ACTH) deficiency (IAD) is extraordinarily rare, and the clinical manifestations of its accompanying adrenal insufficiency are diverse. Early-onset forms of IAD have been linked to mutations in the Tpit transcription factor gene TPIT; however, the genetic basis of juvenile- or late-onset IAD is unknown. Herein, we describe a case of a peripubertal girl with IAD and a normal TPIT gene who presented with an acute neurologic emergency, demonstrating both the variable clinical presentation of IAD and the need for continued investigation into the molecular mechanisms underlying juvenile- and late-onset IAD.

Intrinsic chemosensitivity of individual nucleus tractus solitarius (NTS) and locus coeruleus (LC) neurons from neonatal rats.

Chemosensitive (CS) neurons are found in discrete brainstem regions, but whether the CS response of these neurons is due to intrinsic chemosensitivity of individual neurons or is mediated by changes in chemical and/or electrical synaptic input is largely unknown. We studied the effect of synaptic blockade (11.4 mM Mg2+/0.2mM Ca2+) solution (SNB) and a gap junction uncoupling agent carbenoxolone (CAR--100 microM) on the response of neurons from two CS brainstem regions, the NTS and the LC. In NTS neurons, SNB decreased spontaneous firing rate (FR). We calculated the magnitude of the FR response to hypercapnic acidosis (HA; 15% CO2) using the Chemosensitivity Index (CI). The percentage of NTS neurons activated and CI were the same in the absence and presence of SNB. Blocking gap junctions with CAR did not significantly alter spontaneous FR. CAR did not alter the CI in NTS neurons and resulted in a small decrease in the percentage of activated neurons, which was most evident in NTS neurons from rats younger than postnatal day 10. In LC neurons, SNB resulted in an increase in spontaneous FR. As with NTS neurons, SNB did not alter the percentage of activated neurons or the CI in LC neurons. CAR resulted in a small increase in spontaneous FR in LC neurons. In contrast, CAR had a marked effect on the response of LC neurons to HA: a reduced percentage of CS LC neurons and decreased CI. In summary, both NTS and LC neurons appear to contain intrinsically CS neurons. CS neurons from the two regions receive different tonic input in slices (excitatory for NTS and inhibitory for LC); however, blocking chemical synaptic input does not affect the CS response in either region. In NTS neurons, gap junction coupling plays a small role in the CS response, but gap junctions play a major role in the chemosensitivity of many LC neurons.

Estrogen and progesterone are critical regulators of Stat5a expression in the mouse mammary gland.

Signal transducer and activator of transcription (Stat)5a is a well-established regulator of mammary gland development. Several pathways for activating Stat5a have been identified, but little is known about the mechanisms that regulate its expression in this tissue. In this report, we used immunofluorescent staining to examine Stat5a expression in mammary epithelial cells during normal development and in response to treatment with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very low levels in the prepubertal gland and was highly induced in a subset of luminal epithelial cells during puberty. The percentage of positive cells increased in adult virgin, pregnant, and lactating animals, dropped dramatically during involution, and then increased again after weaning. Ovariectomy ablated Stat5a expression in virgin animals, and treatment with both E and P was necessary to restore it. Double-labeling experiments in animals treated with E plus P for 3 d demonstrated that Stat5a was localized exclusively to cells containing both E and P receptors. Together, these results identify a novel role for E and P in inducing Stat5a expression in the virgin mammary gland and suggest that these hormones act at the cellular level through their cognate receptors.

Functional ablation of pRb activates Cdk2 and causes antiestrogen resistance in human breast cancer cells.

Estrogens are required for the proliferation of hormone dependent breast cancer cells, making estrogen receptor (ER) positive tumors amenable to endocrine therapies such as antiestrogens. However, resistance to these agents remains a significant cause of treatment failure. We previously demonstrated that inactivation of the retinoblastoma protein (pRb) family tumor suppressors causes antiestrogen resistance in MCF-7 cells, a widely studied model of estrogen responsive human breast cancers. In this study, we investigate the mechanism by which pRb inactivation leads to antiestrogen resistance. Cdk4 and cdk2 are two key cell cycle regulators that can phosphorylate and inactivate pRb, therefore we tested whether these kinases are required in cells lacking pRb function. pRb family members were inactivated in MCF-7 cells by expressing polyomavirus large tumor antigen (PyLT), and cdk activity was inhibited using the cdk inhibitors p16(INK4A) and p21(Waf1/Cip1). Cdk4 activity was no longer required in cells lacking functional pRb, while cdk2 activity was required for proliferation in both the presence and absence of pRb function. Using inducible PyLT cell lines, we further demonstrated that pRb inactivation leads to increased cyclin A expression, cdk2 activation and proliferation in antiestrogen arrested cells. These results demonstrate that antiestrogens do not inhibit cdk2 activity or proliferation of MCF-7 cells in the absence of pRb family function, and suggest that antiestrogen resistant breast cancer cells resulting from pRb pathway inactivation would be susceptible to therapies that target cdk2.

Neonatal maturation of the hypercapnic ventilatory response and central neural CO2 chemosensitivity.

The ventilatory response to CO2 changes as a function of neonatal development. In rats, a ventilatory response to CO2 is present in the first 5 days of life, but this ventilatory response to CO2 wanes and reaches its lowest point around postnatal day 8. Subsequently, the ventilatory response to CO2 rises towards adult levels. Similar patterns in the ventilatory response to CO2 are seen in some other species, although some animals do not exhibit all of these phases. Different developmental patterns of the ventilatory response to CO2 may be related to the state of development of the animal at birth. The triphasic pattern of responsiveness (early decline, a nadir, and subsequent achievement of adult levels of responsiveness) may arise from the development of several processes, including central neural mechanisms, gas exchange, the neuromuscular junction, respiratory muscles and respiratory mechanics. We only discuss central neural mechanisms here, including altered CO2 sensitivity of neurons among the various sites of central CO2 chemosensitivity, changes in astrocytic function during development, the maturation of electrical and chemical synaptic mechanisms (both inhibitory and excitatory mechanisms) or changes in the integration of chemosensory information originating from peripheral and multiple central CO2 chemosensory sites. Among these central processes, the maturation of synaptic mechanisms seems most important and the relative maturation of synaptic processes may also determine how plastic the response to CO2 is at any particular age.

c-Myc suppresses p21WAF1/CIP1 expression during estrogen signaling and antiestrogen resistance in human breast cancer cells.

Estrogen rapidly induces expression of the proto-oncogene c-myc. c-Myc is required for estrogen-stimulated proliferation of breast cancer cells, and deregulated c-Myc expression has been implicated in antiestrogen resistance. In this report, we investigate the mechanism(s) by which c-Myc mediates estrogen-stimulated proliferation and contributes to cell cycle progression in the presence of antiestrogen. The MCF-7 cell line is a model of estrogen-dependent, antiestrogen-sensitive human breast cancer. Using stable MCF-7 derivatives with inducible c-Myc expression, we demonstrated that in antiestrogen-treated cells, the elevated mRNA and protein levels of p21(WAF1/CIP1), a cell cycle inhibitor, decreased upon either c-Myc induction or estrogen treatment. Expression of p21 blocked c-Myc-mediated cell cycle progression in the presence of antiestrogen, suggesting that the decrease in p21 is necessary for this process. Using RNA interference to suppress c-Myc expression, we further established that c-Myc is required for estrogen-mediated decreases in p21(WAF1/CIP1). Finally, we observed that neither c-Myc nor p21(WAF1/CIP1) is regulated by estrogen or antiestrogen in an antiestrogen-resistant MCF-7 derivative. The p21 levels in the antiestrogen-resistant cells increased when c-Myc expression was suppressed, suggesting that loss of p21 regulation was a consequence of constitutive c-Myc expression. Together, these studies implicate p21(WAF1/CIP1) as an important target of c-Myc in breast cancer cells and provide a link between estrogen, c-Myc, and the cell cycle machinery. They further suggest that aberrant c-Myc expression, which is frequently observed in human breast cancers, can contribute to antiestrogen resistance by altering p21(WAF1/CIP1) regulation.

Estrogen increases brain expression of the mRNA encoding transthyretin, an amyloid beta scavenger protein.

Estrogen replacement therapy in postmenopausal women is associated with a reduced risk of Alzheimer's Disease (AD). The multiple mechanisms by which estrogen protects against AD are still unknown. To conduct a broad screen for estrogen-regulated AD-related genes in the brain, we used cDNA array assays of brain mRNA samples from ovariectomized (ovx) adult female mice treated with either 17beta-estradiol or vehicle at 1 or 5 weeks post-ovx. The gene encoding transthyretin (TTR), which has been reported to scavenge amyloid beta peptides and reduce amyloid plaque formation, is increased by estradiol treatment at both 1 and 5 weeks post-ovx. Northern blot analyses and RNase protection assays performed on whole brain samples obtained from estradiol- or vehicle-treated mice confirmed the cDNA array assays showing a significant increase in TTR mRNA with estradiol treatment. Qualitative in situ hybridization or immunocytochemistry performed on brain sections demonstrated that TTR mRNA is expressed only in choroid plexus and leptomeninges, and that both estrogen receptor proteins, alpha and beta, are present in choroid plexus cells. These novel findings suggest that estrogen may reduce the risk of AD by acting on choroid plexus cells to increase TTR gene expression, leading to enhanced sequestration and reduced aggregation of amyloid beta peptides.

Expression of an estrogen receptor variant lacking exon 3 in derivatives of MCF-7 cells with acquired estrogen independence or tamoxifen resistance.

The estrogen receptor (ER) plays important roles in the development and progression of breast cancer, and is a major target for tumor therapy. In this study, we investigated ER function in two derivatives of MCF-7 cells that were selected for their ability to proliferate in the absence of estrogen or in the presence of the antiestrogen, tamoxifen. Reporter gene assays indicated decreased ER activity in both cells lines, although the activity remaining retained responsiveness to both estrogen and tamoxifen. The decreased ER activity correlated with expression of a 61 kDa variant ER protein, and sequencing of RT-PCR products indicated that this variant was the product of an exon 3 deletion (ERDeltaE3). To study its effects on cell proliferation, ERDeltaE3 cDNA was stably transfected into both the MCF-7 cell line and its estrogen-independent/tamoxifen-sensitive derivative MCF-7/LCC1 (LCC1), and the phenotypes of transfectants were examined. Expression of ERDeltaE3 was not sustainable in MCF-7 cells, but was maintained for at least 17 passages in LCC1 cells. These results are in agreement with previous reports that ERDeltaE3 inhibits wild-type ER activity and negatively regulates proliferation of MCF-7 cells. They further suggest that the alteration that leads to estrogen independence in LCC1 cells allows for sustained expression of ERDeltaE3, and that additional changes are required to confer tamoxifen resistance to these cells.