Reversible promoter demethylation of PDGFD confers gemcitabine resistance through STAT3 activation and RRM1 upregulation.

Drug resistance is a major problem in cancer treatment with traditional or targeted therapeutics. Gemcitabine is approved for several human cancers and the first line treatment for locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, gemcitabine resistance frequently occurs and is a major problem in successful treatments of these cancers and the mechanism of gemcitabine resistance remains largely unknown. In this study, we identified 65 genes that had reversible methylation changes in their promoters in gemcitabine resistant PDAC cells using whole genome Reduced Representation Bisulfite Sequencing analyses. One of these genes, PDGFD, was further studied in detail for its reversible epigenetic regulation in expression and shown to contribute to gemcitabine resistance in vitro and in vivo via stimulating STAT3 signaling in both autocrine and paracrine manners to upregulate RRM1 expression. Analyses of TCGA datasets showed that PDGFD positively associates with poor outcome of PDAC patients. Together, we conclude that the reversible epigenetic upregulation plays an important role in gemcitabine resistance development and targeting PDGFD signaling alleviates gemcitabine resistance for PDAC treatment.

The CXCR4-Dependent LASP1-Ago2 Interaction in Triple-Negative Breast Cancer.

The CXCR4-LASP1 axis is an emerging target in the field of breast cancer metastasis. C-X-C chemokine receptor type 4 (CXCR4) mediates directed cell migration when activated by its cognate ligand CXCL12. LIM and SH3 Protein 1 (LASP1) is a critical node in the CXCR4 signaling pathway, as its deficiency blocks CXCR4-dependent Matrigel invasion. The mechanism by which LASP1 facilitates this invasive ability of tumor cells when CXCR4 is activated is unknown. Our previous proteomics work had revealed several components of the RNA interference (RNAi) machinery as being potential LASP1 interacting proteins. Here we report that argonaute 2 (Ago2), a protein with central involvement in RNAi, associates with LASP1 in triple-negative breast cancer (TNBC) cells. We demonstrate that LASP1 co-immunoprecipitates with Ago2 endogenously in a CXCL12-dependent manner, with further confirmation of this interaction by proximity ligation assay. Furthermore, this association is specific to CXCR4 as it can be abrogated by the CXCR4 antagonist, AMD3465. By GST-pulldown approach, we identify that LASP1 directly binds to Ago2 through its LIM and SH3 domains, and that this binding is dictated by the S146 and Y171 phosphorylation sites of LASP1. Additionally, the phosphorylation status of LASP1 affected tumor suppressor microRNA (miRNA) Let-7a-guided Ago2 activity. Levels of several endogenous targets of Let-7a were found to be altered including C-C chemokine receptor type 7 (CCR7), which is another critical chemokine receptor involved in metastasis to lymph nodes. Our results suggest a novel role for the LASP1-Ago2 module in shaping the RNAi landscape, functionally impacting the invasive ability of cancer cells.

Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer.

The CXCL12-CXCR4 axis plays a vital role in many steps of breast cancer metastasis, but the molecular mechanisms have not been fully elucidated. We previously reported that activation of CXCR4 by CXCL12 promotes the nuclear localization of LASP1 (LIM and SH3 protein 1). The nuclear LASP1 then interacts with Snail1 in triple-negative breast cancer (TNBC) cell lines. In this study, we report that the nuclear accumulation and retention of Snail1 was dependent on an increase in nuclear LASP1 levels driven by active CXCR4. The CXCR4-LASP1 axis may directly regulate the stabilization of nuclear Snail1, by upregulating nuclear levels of pS473-Akt, pS9-GSK-3ß, A20, and LSD1. Furthermore, the activation of CXCR4 induced association of LASP1 with Snail1, A20, GSK-3ß, and LSD1 endogenously. Thus, nuclear LASP1 may also regulate protein-protein interactions that facilitate the stability of Snail1. Genetic ablation of LASP1 resulted in the mislocalization of nuclear Snail1, loss of the ability of TNBC cells to invade Matrigel and a dysregulated expression of both epithelial and mesenchymal markers, including an increased expression of ALDH1A1, a marker for epithelial breast cancer stem-like cells. Our findings reveal a novel role for the CXCR4-LASP1 axis in facilitating the stability of nuclear localized Snail1.

Targeting of the Eukaryotic Translation Initiation Factor 4A Against Breast Cancer Stemness.

Breast cancer stem cells (BCSCs) are intrinsically chemoresistant and capable of self-renewal. Following chemotherapy, patients can develop minimal residual disease due to BCSCs which can repopulate into a relapsed tumor. Therefore, it is imperative to co-target BCSCs along with the bulk tumor cells to achieve therapeutic success and prevent recurrence. So, it is vital to identify actionable molecular targets against both BCSCs and bulk tumor cells. Previous findings from our lab and others have demonstrated that inhibition of the emerging drug target eIF4A with Rocaglamide A (RocA) was efficacious against triple-negative breast cancer cells (TNBC). RocA specifically targets the pool of eIF4A bound to the oncogenic mRNAs that requires its helicase activity for their translation. This property enables specific targeting of tumor cells. The efficacy of RocA against BCSCs is unknown. In this study, we postulated that eIF4A could be a vulnerable node in BCSCs. In order to test this, we generated a paclitaxel-resistant TNBC cell line which demonstrated an elevated level of eIF4A along with increased levels of cancer stemness markers (ALDH activity and CD44), pluripotency transcription factors (SOX2, OCT4, and NANOG) and drug transporters (ABCB1, ABCG2, and ABCC1). Furthermore, genetic ablation of eIF4A resulted in reduced expression of ALDH1A1, pluripotency transcription factors and drug transporters. This pointed out that eIF4A is likely associated with selected set of proteins that are critical to BCSCs, and hence targeting eIF4A may eliminate BCSCs. Therefore, we isolated BCSCs from two TNBC cell lines: MDA-Bone-Un and SUM-159PT. Following RocA treatment, the self-renewal ability of the BCSCs was significantly reduced as determined by the efficiency of the formation of primary and secondary mammospheres. This was accompanied by a reduction in the levels of NANOG, OCT4, and drug transporters. Exposure to RocA also induced cell death of the BCSCs as evaluated by DRAQ7 and cell viability assays. RocA treatment induced apoptosis with increased levels of cleaved caspase-3. Overall, we identified that RocA is effective in targeting BCSCs, and eIF4A is an actionable molecular target in both BCSCs and bulk tumor cells. Therefore, anti-eIF4A inhibitors could potentially be combined synergistically with existing chemo-, radio- and/or immunotherapies.

Novel and Alternative Targets Against Breast Cancer Stemness to Combat Chemoresistance.

Breast cancer stem cells (BCSCs) play a vital role in tumor progression and metastasis. They are heterogeneous and inherently radio- and chemoresistant. They have the ability to self-renew and differentiate into non-BCSCs. These determinants of BCSCs including the plasticity between the mesenchymal and epithelial phenotypes often leads to minimal residual disease (MRD), tumor relapse, and therapy failure. By studying the resistance mechanisms in BCSCs, a combinatorial therapy can be formulated to co-target BCSCs and bulk tumor cells. This review addresses breast cancer stemness and molecular underpinnings of how the cancer stemness can lead to pharmacological resistance. This might occur through rewiring of signaling pathways and modulated expression of various targets that support survival and self-renewal, clonogenicity, and multi-lineage differentiation into heterogeneous bulk tumor cells following chemotherapy. We explore emerging novel and alternative molecular targets against BC stemness and chemoresistance involving survival, drug efflux, metabolism, proliferation, cell migration, invasion, and metastasis. Strategic targeting of such vulnerabilities in BCSCs may overcome the chemoresistance and increase the longevity of the metastatic breast cancer patients.

The CXCR4-LASP1-eIF4F Axis Promotes Translation of Oncogenic Proteins in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) remains clinically challenging as effective targeted therapies are lacking. In addition, patient mortality mainly results from the metastasized lesions. CXCR4 has been identified to be one of the major chemokine receptors involved in breast cancer metastasis. Previously, our lab had identified LIM and SH3 Protein 1 (LASP1) to be a key mediator in CXCR4-driven invasion. To further investigate the role of LASP1 in this process, a proteomic screen was employed and identified a novel protein-protein interaction between LASP1 and components of eukaryotic initiation 4F complex (eIF4F). We hypothesized that activation of the CXCR4-LASP1-eIF4F axis may contribute to the preferential translation of oncogenic mRNAs leading to breast cancer progression and metastasis. To test this hypothesis, we first confirmed that the gene expression of CXCR4, LASP1, and eIF4A are upregulated in invasive breast cancer. Moreover, we demonstrate that LASP1 associated with eIF4A in a CXCL12-dependent manner via a proximity ligation assay. We then confirmed this finding, and the association of LASP1 with eIF4B via co-immunoprecipitation assays. Furthermore, we show that LASP1 can interact with eIF4A and eIF4B through a GST-pulldown approach. Activation of CXCR4 signaling increased the translation of oncoproteins downstream of eIF4A. Interestingly, genetic silencing of LASP1 interrupted the ability of eIF4A to translate oncogenic mRNAs into oncoproteins. This impaired ability of eIF4A was confirmed by a previously established 5'UTR luciferase reporter assay. Finally, lack of LASP1 sensitizes 231S cells to pharmacological inhibition of eIF4A by Rocaglamide A as evident through BIRC5 expression. Overall, our work identified the CXCR4-LASP1 axis to be a novel mediator in oncogenic protein translation. Thus, our axis of study represents a potential target for future TNBC therapies.

Effect of sodium salt of Butrin, a novel compound isolated from Butea monosperma flowers on suppressing the expression of SIRT1 and Aurora B kinase-mediated apoptosis in colorectal cancer cells.

The infrequent manifestation of SIRT1 and Aurora B kinase has shown to play a pivotal role in colorectal cancer (CRC) progression by regulating Wnt signaling pathway. The present study investigates the signaling events that regulate the modulation of SIRT1 and Aurora B kinase expression and it's mediated cell proliferation in SW480 human primary adenocarcinoma CRC cells using Butea monosperma floral compounds (BMFC). In this, cell viability, mitochondrial mediated apoptosis, cell cycle arrest and inhibition of Wnt pathway were examined. Interestingly, the active novel compound, sodium salt of butrin, from BMFC significantly enhances the apoptosis activity, where SIRT1 and Aurora B kinase were ectopically overexpressed in CRC cells. Moreover, mRNA and protein expressions analysis indicates that the expression of GSK-3ß, ß-catenin, cyclin D1, pAKT, TGF-3ß, SIRT1 and Aurora B kinase were down regulated in BMFC treated cells. These findings provide valuable information that the active BMFC having great impact on SIRT1 and Aurora B kinase mediated Wnt signaling down regulation in SW480 CRC cells.

Targeting the interaction of Aurora kinases and SIRT1 mediated by Wnt signaling pathway in colorectal cancer: A critical review.

The Aurora kinases belong to the family of serine/threonine kinase, a central regulator of mitosis and their expression increased during G2/M phase. It is classified into Aurora A, B and C, each has distinct roles in cellular processes, which includes regulation of spindle assembly, function of centrosomes, cytoskeleton and cytokinesis. During cancer growth, their rapid increase makes most attractive marker for cancer treatment at present. However Aurora A kinase is known to be a marker for cancer therapy, the most important serine/threonine kinase of Aurora B kinase involvement in cancer is still inadequate. Subsequently, the recent findings revealed that the class III histone deacetylase of SIRT1 is a key regulator to activate Aurora kinases from S phase damaged DNA through Wnt signaling pathway. Even if both Aurora A kinase and SIRT1 serve as a marker for cancer therapy, the present review reveals it is interaction in Wnt signaling pathway that solely for colorectal cancer.

Isocoreopsin: An active constituent of n-butanol extract of Butea monosperma flowers against colorectal cancer (CRC).

The herb Butea monosperma constitutes several human health beneficial components, which are mostly studied for their anticancer effects. In this study, the activity of n-butanol fractions of B. monosperma floral extract was examined on inhibiting aberrant crypt foci (ACF) formation in azoxymethane induced Wistar albino rats. The n-butanol extracts (150 mg/kg) decreased the ACF formation (per rat) by 92% and 78% in short- and long-term in vivo treatments, respectively. All the compounds in the n-butanol extract were isolated and purified using column and reverse-phase high pressure liquid chromatography (HPLC). Their structures were characterized using UV-visible spectroscopy, nuclear magnetic resonance (NMR) and electrospray-ionisation mass spectrometry (ESI-MS) to determine important flavonoids, namely isocoreopsin, butrin and isobutrin. These compounds were studied for their free radical scavenging and anticancer activities. The compound isocoreopsin showed significantly greater efficacy in cell death on human colon and liver cancer cell lines (50 µg/mL in HT-29 and 100 µg/mL in HepG2) than butrin (100 µg/mL in HT-29 and 500 µg/mL in HepG2) and isobutrin (80 µg/mL in HT-29 and 150 µg/mL in HepG2). These results suggest that isocoreopsin, butrin and isobutrin are the important key compounds for the chemoprevention of colon cancer and isocoreopsin can be considered as a promising novel drug.