Divya Khaitan

The Melanoma‐Upregulated Long Noncoding RNA SPRY4-IT1 Modulates Apoptosis and Invasion

The identification of cancer-associated long noncoding RNAs (lncRNAs) and the investigation of their molecular and biological functions are important to understand the molecular biology of cancer and its progression. Although the functions of lncRNAs and the mechanisms regulating their expression are largely unknown, recent studies are beginning to unravel their importance in human health and disease. Here, we report that a number of lncRNAs are differentially expressed in melanoma cell lines in comparison to melanocytes and keratinocyte controls. One of these lncRNAs, SPRY4-IT1 (GenBank accession ID AK024556), is derived from an intron of the SPRY4 gene and is predicted to contain several long hairpins in its secondary structure. RNA-FISH analysis showed that SPRY4-IT1 is predominantly localized in the cytoplasm of melanoma cells, and SPRY4-IT1 RNAi knockdown results in defects in cell growth, differentiation, and higher rates of apoptosis in melanoma cell lines. Differential expression of both SPRY4 and SPRY4-IT1 was also detected in vivo, in 30 distinct patient samples, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic melanoma. The elevated expression of SPRY4-IT1 in melanoma cells compared to melanocytes, its accumulation in cell cytoplasm, and effects on cell dynamics, including increased rate of wound closure on SPRY4-IT1 overexpression, suggest that the higher expression of SPRY4-IT1 may have an important role in the molecular etiology of human melanoma.

The Regulation of miRNA-211 Expression and Its Role in Melanoma Cell Invasiveness

The immediate molecular mechanisms behind invasive melanoma are poorly understood. Recent studies implicate microRNAs (miRNAs) as important agents in melanoma and other cancers. To investigate the role of miRNAs in melanoma, we subjected human melanoma cell lines to miRNA expression profiling, and report a range of variations in several miRNAs. Specifically, compared with expression levels in melanocytes, levels of miR-211 were consistently reduced in all eight non-pigmented melanoma cell lines we examined; they were also reduced in 21 out of 30 distinct melanoma samples from patients, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic. The levels of several predicted target mRNAs of miR-211 were reduced in melanoma cell lines that ectopically expressed miR-211. In vivo target cleavage assays confirmed one such target mRNA encoded by KCNMA1. Mutating the miR-211 binding site seed sequences at the KCNMA1 3′-UTR abolished target cleavage. KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels. Two different melanoma cell lines ectopically expressing miR-211 exhibited significant growth inhibition and reduced invasiveness compared with the respective parental melanoma cell lines. An shRNA against KCNMA1 mRNA also demonstrated similar effects on melanoma cells. miR-211 is encoded within the sixth intron of TRPM1, a candidate suppressor of melanoma metastasis. The transcription factor MITF, important for melanocyte development and function, is needed for high TRPM1 expression. MITF is also needed for miR-211 expression, suggesting that the tumor-suppressor activities of MITF and/or TRPM1 may at least partially be due to miR-211s negative post transcriptional effects on the KCNMA1 transcript. Given previous reports of high KCNMA1 levels in metastasizing melanoma, prostate cancer and glioma, our findings that miR-211 is a direct posttranscriptional regulator of KCNMA1 expression as well as the dependence of this miRNAs expression on MITF activity, establishes miR-211 as an important regulatory agent in human melanoma.

Role of KCNMA1 gene in breast cancer invasion and metastasis to brain

BackgroundThe prognosis for patients with breast tumor metastases to brain is extremely poor. Identification of prognostic molecular markers of the metastatic process is critical for designing therapeutic modalities for reducing the occurrence of metastasis. Although ubiquitously present in most human organs, large-conductance calcium- and voltage-activated potassium channel (BKCa) channels are significantly upregulated in breast cancer cells. In this study we investigated the role of KCNMA1 gene that encodes for the pore-forming α-subunit of BKCa channels in breast cancer metastasis and invasion.MethodsWe performed Global exon array to study the expression of KCNMA1 in metastatic breast cancer to brain, compared its expression in primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immunohistochemistry was performed to study the expression and localization of BKCa channel protein in primary and metastatic breast cancer tissues and breast cancer cell lines. We performed matrigel invasion, transendothelial migration and membrane potential assays in established lines of normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether BKCa channel inhibition attenuates breast tumor invasion and metastasis using KCNMA1 knockdown with siRNA and biochemical inhibition with Iberiotoxin (IBTX).ResultsThe Global exon array and RT-PCR showed higher KCNMA1 expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased BKCa channel activity, leading to greater invasiveness and transendothelial migration, both of which could be attenuated by blocking KCNMA1.ConclusionDetermining the relative abundance of BKCa channel expression in breast cancer metastatic to brain and the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy.

Epigenetic regulation of microRNA genes and the role of miR-34b in cell invasion and motility in human melanoma.

Invasive melanoma is the most lethal form of skin cancer. The treatment of melanoma-derived cell lines with 5-aza-2-deoxycytidine (5-Aza-dC) markedly increases the expression of several miRNAs, suggesting that the miRNA-encoding genes might be epigenetically regulated, either directly or indirectly, by DNA methylation. We have identified a group of epigenetically regulated miRNA genes in melanoma cells, and have confirmed that the upstream CpG island sequences of several such miRNA genes are hypermethylated in cell lines derived from different stages of melanoma, but not in melanocytes and keratinocytes. We used direct DNA bisulfite and immunoprecipitated DNA (Methyl-DIP) to identify changes in CpG island methylation in distinct melanoma patient samples classified as primary in situ, regional metastatic, and distant metastatic. Two melanoma cell lines (WM1552C and A375 derived from stage 3 and stage 4 human melanoma, respectively) were engineered to ectopically express one of the epigenetically modified miRNA: miR-34b. Expression of miR-34b reduced cell invasion and motility rates of both WM1552C and A375, suggesting that the enhanced cell invasiveness and motility observed in metastatic melanoma cells may be related to their reduced expression of miR-34b. Total RNA isolated from control or miR-34b-expressing WM1552C cells was subjected to deep sequencing to identify gene networks around miR-34b. We identified network modules that are potentially regulated by miR-34b, and which suggest a mechanism for the role of miR-34b in regulating normal cell motility and cytokinesis.

Modulation of KCa channels increases anticancer drug delivery to brain tumors and prolongs survival in xenograft model.

Most anticancer drugs fail to impact patient survival since they fail to cross the blood-brain tumor barrier (BTB) at therapeutic levels. For example, Temozolomide (TMZ) exhibits some anti-tumor activity against brain tumors, so does Trastuzumab (Herceptin, Her-2 inhibitor), which might be effective against Her2 neu overexpressing gliomas. Nevertheless, intact BTB and active efflux system may prevent their entry to brain tumors. Previously we have shown that potassium channel agonists increased carboplatin and Her-2 neu antibody delivery in animal glioma models. Here, we studied whether potassium channel agonist increase TMZ and Herceptin delivery across the BTB to elicit anti-tumor activity and increase survival in nude mice with human glial tumor. The KCa channel activity and expression was also evaluated in human glioma tissues. We administered NS-1619, calcium-dependent potassium (KCa) channel agonist, with [14C]-TMZ, and quantified TMZ delivery. The results clearly demonstrate that when given systemically both TMZ and Herceptin do not cross the BTB in significant amounts, however, NS-1619 co-infusion with [14C]-TMZ and Herceptin resulted in enhanced drug delivery to brain-tumor cells. The combination treatment of TMZ and Herceptin also showed improved anti-tumor effect which was more prominent than that of either treatment alone in increasing the survival in mice with brain tumor, when co-infused with KCa channel agonists. In conclusion, KCa channel agonists may benefit brain tumor patients by increasing anti-neoplastic agent’s delivery to brain tumors. A clinical outcome of this research is the discovery of a novel drug delivery system that circumvents the BBB/BTB to benefit brain tumor patients.

Activation of KATP channels increases anticancer drug delivery to brain tumors and survival

Several anticancer drugs are ineffective against brain tumor and do not impact patient survival because they fail to cross the blood-brain tumor barrier (BTB) effective levels. One such agent temozolomide is commonly used in brain tumor patients, which works better when combined with radiation or other anticancer agents. Likewise, trastuzumab (Herceptin, Her-2 inhibitor), which might be effective against Her2/neu over expressing gliomas may work well when combined with temozolomide. Nonetheless, both drugs do not cross the BTB to significantly impact patient survival. Beforehand we showed that potassium channel agonists when intracarotidly administered increased carboplatin and Her-2 antibody delivery in animal glioma models by triggering formation of brain vascular endothelial transcytotic vesicles. In this study, we investigated whether, intravenously administered, ATP-sensitive potassium channel (K(ATP)) activator (minoxidil sulfate; MS) increases temozolomide and Herceptin delivery to brain tumors to induce anti-tumor activity and increase survival in nude mice with Glioblastoma multiforme (GBM) cells. The results clearly demonstrate that when given intravenously temozolomide crosses BTB at a relatively low amount while Herceptin failed to cross the BTB. However, MS co-infusion with [(14)C]-temozolomide or fluorescently labeled-Herceptin resulted in improved and selective drug delivery to brain tumor. We also showed that combination treatment with temozolomide and Herceptin has enhanced anti-tumor effect which was more prominent than that of either treatment alone in increasing the survival in mice with GBM when co-infused with MS. Therefore, brain tumor patients may be benefited when anti-neoplastic agent delivery is increased selectively to the brain tumors using KATP channel agonists.

Recent advances in understanding of blood–brain tumor barrier (BTB) permeability mechanisms that enable better detection and treatment of brain tumors

Abstract We reviewed studies on biology of blood–brain tumor barrier (BTB) permeability regulation in brain tumors. Recent developments in molecular imaging probes using nanotechnology for functional imaging should improve the efficacy of targeted treatment of brain tumors. We reviewed studies of leading researchers who have identified and developed nanomedicines and nanoimaging agents. They have combined specialized molecules and nanospheres to carry pay load of anticancer or imaging agents across the blood–brain barrier or BTB brain tumors. Unique biomarkers on BTB for increased imaging agents’ and anticancer drug delivery to brain tumors were discussed. For instance, we targeted calcium-activated potassium channels (BKCa) and adenosine triphosphate (ATP)-sensitive potassium channels (KATP) for enhanced delivery of antineoplastic drugs and imaging agents. We also discussed nanomedicines and multimeric molecular imaging strategies, including development of peripheral benzodiazepine receptors ligand-PK11195 for better metastatic brain tumor detection. Such agents with flurophores or therapeutics encapsulated in nanospheres are useful in detection of brain tumors, treatment, and monitoring.

Targeting Brain Tumors with Nanomedicines: Overcoming Blood Brain Barrier Challenges

BACKGROUNDnThis review elucidates ongoing research, which show improved delivery of anticancer drugs alone and/ or enclosed in carriers collectively called nanomedicines to cross the BBB/ BTB to kill tumor cells and impact patient survival. We highlighted various advances in understanding the mechanism of BTB function that has an impact on anticancer therapeutics delivery. We discussed latest breakthroughs in developing pharmaceutical strategies, including nanomedicines and delivering them across BTB for brain tumor management and treatment.nnnMETHODSnWe performed an extensive literature search and highlighted important studies on the regulation of BTB permeability with respect to nanotech-based nanomedicines for targeted treatment of brain tumors. We have reviewed research articles that describe the development of specialized molecules and nanospheres, which carry payload of anticancer agents to brain tumor cells across the BBB/ BTB and avoid drug efflux systems. We highlighted research on the identification and development of targeted anti-cancer drug delivery to brain tumors. In addition, we discussed multimeric molecular therapeutics and nanomedicines that were encapsulated in nanospheres for treatment and monitoring of brain tumors.nnnRESULTSnIn this context, we quoted our research on large conductance calcium-activated potassium channels (BKCa) and ATP-dependent potassium channels (KATP) as portals of enhanced antineoplastic drugs delivery. We showed that several innovative drug delivery agents such as liposomes, polymeric nanoparticles, dendrimers and many such tools can be utilized to improve anticancer drugs and nanomedicines across the BTB to reach brain tumor cells.nnnCONCLUSIONnThis review might interest both academic and drug company scientists involved in drug delivery to brain tumors. We further seek to present evidence that BTB modulators can be clinically developed as combination drug or/ and as stand-alone anticancer drugs. Eventually, it is expected that unrelenting effort from the scientific community in developing novel drug delivery methods should increase the survival rate of brain tumor patients, which is dismally low presently.

The melanoma-upregulated long noncoding RNA SPRY4-IN1 modulates apoptosis and invasion

Resumen del poster presentado al 36th FEBS Congress celebrado en Torino (Italia) del 25 al 30 de Junio de 2011.-- et al.

An Alternative Splice Variant of KCNMA1 Drives Breast Cancer Metastasis and Invasion.

Background: We have recently shown that KCNMA1, which encodes the pore forming α-subunit of large-conductance calcium-activated voltage-sensitive potassium (BK Ca ) channel, plays a highly critical role in breast tumor cells metastatic to brain and somewhat less critical role in primary breast cancer cell metastasis. The resulting functional heterogeneity of BK Ca channels is attributed generally to alternative splicing of KCNMA1 that generate BK Ca channel isoform with altered channel properties. We have identified a KCNMA1 splice variant (KCNMA1vE22) expressed in breast cancer cell line (MDA-MB-361) metastatic to brain with a deletion of 108 bp in exon 22 between the S9 and S10 protein subunit (C-terminus). In this study we investigated the biological function of KCNMA1vE22 in vitro and in vivo. Methods: We validated the results from the breast tumor cell lines data with human normal breast tissue, primary, non-metastatic and metastatic breast tumors by RT-PCR. The KCNMA1vE22 was identified from A-172 tumor cells and cloned into mammalian expression vector (pcDNA6). MCF-7 cells (non-metastatic, null type for KCNMA1vE22) and HEK cells (null type for KCNMA1 and KCNMA1vE22) were transfected with pcDNA6/KCNMA1vE22 and stable clones selected. The expression of KCNMA1 and KCNMA1vE22 was confirmed using RT-PCR, western blot and qPCR. The biological role of the splice variant was studied using parental and transfected MCF-7 cells by cell proliferation, matrigel invasion, transendothelial migration and membrane potential assays in vitro and tumorogenecity using s.c xenograft mouse model.Results: Based on the preliminary screening of breast tumor samples, KCNMA1vE22 was undetectable in primary as well as systemic metastatic breast tumor samples. Stably transfected HEK and MCF-7 cells showed 2.5 to 3-fold increase in KCNMA1 mRNA expression, which corroborated with the increased protein levels and BK Ca channel activity. In addition MCF-7 cells expressing KCNMA1vE22 showed a eight-fold increase in invasion while a three-fold increase in TEM was observed compared to parental MCF-7 cells. A marked increase in the proliferation rate of KCNAM1vE22- transfected cells compared to MCF-7 alone was observed, suggesting that KCNAM1vE22 has a profound effect on breast cancer cell proliferation. The in vitro results were validated by subcutaneous mouse model experiment. We found statistically significant increase in mean tumor volume in mice with KCNMA1vE22 transfected compared with parental MCF-7 cells.Conclusions: Our results clearly show that KCNMA1vE22 promotes breast cancer cell invasion, and possibly metastasis to brain. Perhaps the discovery and validation of brain specific metastasis-associated KCNMA1 alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. The variant KCNMA1vE22 that we have discovered potentially may fill the gap to serve as a new generation of biomarker of breast cancer metastasis to brain. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6166.