Adaptor proteins mediate CXCR4 and PI4KA crosstalk in prostate cancer cells and the significance of PI4KA in bone tumor growth.

The chemokine receptor, CXCR4 signaling regulates cell growth, invasion, and metastasis to the bone-marrow niche in prostate cancer (PCa). Previously, we established that CXCR4 interacts with phosphatidylinositol 4-kinase IIIα (PI4KIIIα encoded by PI4KA) through its adaptor proteins and PI4KA overexpressed in the PCa metastasis. To further characterize how the CXCR4-PI4KIIIα axis promotes PCa metastasis, here we identify CXCR4 binds to PI4KIIIα adaptor proteins TTC7 and this interaction induce plasma membrane PI4P production in prostate cancer cells. Inhibiting PI4KIIIα or TTC7 reduces plasma membrane PI4P production, cellular invasion, and bone tumor growth. Using metastatic biopsy sequencing, we found PI4KA expression in tumors correlated with overall survival and contributes to immunosuppressive bone tumor microenvironment through preferentially enriching non-activated and immunosuppressive macrophage populations. Altogether we have characterized the chemokine signaling axis through CXCR4-PI4KIIIα interaction contributing to the growth of prostate cancer bone metastasis.

Paraventricular Nucleus V1a Receptor Knockdown Blunts Neurocardiovascular Responses to Acute Stress in Male Rats after Chronic Mild Unpredictable Stress.

Chronic stress and depression impart increased risk for adverse cardiovascular events. Autonomic dysregulation, particularly sympathoexcitation, has long been associated with poor cardiovascular outcomes. Vasopressin (AVP) receptors with the paraventricular nucleus (PVN), known as an integrating locus for hemodynamic and autonomic function, have been implicated in behavior and stress. The present studies were designed to test the hypothesis that knockdown of vasopressin V1aR within the PVN in male Sprague Dawley rats subjected to chronic mild unpredictable stress (CMS) would result in lower resting hemodynamics and renal sympathetic nerve activity (RSNA) and mitigate the responses to acute stressors. Male rats underwent CMS for 4 weeks; controls were housed in standard caging. Twenty days into the paradigm, the PVN was injected with either small interfering RNA (siRNA) directed against V1aR or scrambled RNA (scrRNA). Arterial pressure, heart rate and RSNA were ascertained by telemetry with the animals in their home cages. Pretreatment with siRNA to V1aR prevented the increase in arterial pressure to PVN microinjection with exogenous AVP. Basal mean arterial pressure (MAP) was significantly higher in scrRNA-treated but not in siRNA-treated CMS rats vs control rats. Paradoxically, basal RSNA was approximately two-fold higher in siRNA-treated CMS rats. Acute emotional stress delivered as 15-sec air-jet resulted in greater peak and duration of the MAP and RSNA responses in scrRNA-treated CMS rats vs control; siRNA treatment inhibited the responses. The 15-sec exposure to ammonia to test the nasopharyngeal reflex, whose circuitry does not include the PVN, produced similar increases in arterial pressure, heart rate, and RSNA in controls and both groups of CMS rats. Thus, CMS increases arterial pressure and predisposes to greater hemodynamic and RSNA responses to acute emotional stress. The higher basal RSNA in siRNA-treated rats may be due to functional and/or anatomical neuroplasticity occurring during more protracted inhibition of V1aR PVN signaling. Vasopressinergic signaling via V1aR in PVN modulates the cardiovascular and sympathetic responses to both the chronic and acute stress.

Telmisartan Facilitates the Anticancer Effects of CARP-1 Functional Mimetic and Sorafenib in Rociletinib Resistant Non-small Cell Lung Cancer.

BACKGROUND/AIM: Tyrosine kinase inhibitors (TKIs) are used for the treatment of both wild type and mutant non-small cell lung cancer (NSCLC); however, acquired resistance is a major clinical challenge. Herein, we aimed to investigate the effects of telmisartan (Tel), CFM 4.16 and sorafenib combination in rociletinib resistant NSCLC tumors. MATERIALS AND METHODS: 3D spheroid cultures and western blotting were used for evaluating cytotoxic effects and protein expression. An in vivo rociletinib resistant H1975 xenograft model of NSCLC was developed by subcutaneous injection of rociletinib resistant H1975 cells into nude mice. RESULTS: Tel, CFM 4.16 and sorafenib combination displayed superior anti-cancer effects in 3D spheroid cultures and a rociletinib resistant H1975 xenograft model of NSCLC by decreasing the protein expression of oncogenic and cancer stem cell markers (Nanog, Sox2 and Oct4). CONCLUSION: Tel facilitates effective penetration of CFM 4.16 and sorafenib in rociletinib resistant H1975 models of NSCLC.

Combined Ubisol-Q10 and Ashwagandha Root Extract Target Multiple Biochemical Mechanisms and Reduces Neurodegeneration in a Paraquat-Induced Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by progressive neurodegeneration in the substantia nigra (SN) region resulting in loss of movement coordination. Current therapies only provide symptomatic relief, and there is no agent to halt the progression of PD. Previously, Ubisol-Q10, a water-soluble formulation of coenzyme-Q10, and ethanolic root extract of ashwagandha (ASH) have been shown to inhibit PD pathology in rodent models when used alone. Here, we evaluated the neuroprotective efficacy of oral administration of ASH and Ubisol-Q10 alone and in combination in a paraquat-induced PD rat model. The combined treatment resulted in better-preserved neuron morphology compared to Ubsiol-Q10 or ASH alone. The combination treatment enhanced activation of pro-survival astroglia and inhibited pro-inflammatory microglia. While anti-oxidative effects were seen with both agents, Ubisol-Q10 activated autophagy, whereas ashwagandha showed a better anti-inflammatory response. Thus, the combined treatment caused inhibition of oxidative stress, autophagy activation, inhibition of pro-inflammatory microglia, and activation of pro-survival astroglia. Consequently, paraquat (PQ)-treated rats given the combination treatment in drinking water did not show motor impairment. Based on these interesting observations, the combined treatment containing two well-tolerated natural compounds could be a more effective strategy to halt the progression of PD.

Polysaccharide hydrogel based 3D printed tumor models for chemotherapeutic drug screening.

A series of stable and ready-to-use bioinks have been developed based on the xeno-free and tunable hydrogel (VitroGel) system. Cell laden scaffold fabrication with optimized polysaccharide-based inks demonstrated that Ink H4 and RGD modified Ink H4-RGD had excellent rheological properties. Both bioinks were printable with 25-40 kPa extrusion pressure, showed 90% cell viability, shear-thinning and rapid shear recovery properties making them feasible for extrusion bioprinting without UV curing or temperature adjustment. Ink H4-RGD showed printability between 20 and 37 °C and the scaffolds remained stable for 15 days at temperature of 37 °C. 3D printed non-small-cell lung cancer (NSCLC) patient derived xenograft cells (PDCs) showed rapid spheroid growth of size around 500 µm in diameter and tumor microenvironment formation within 7 days. IC50 values demonstrated higher resistance of 3D spheroids to docetaxel (DTX), doxorubicin (DOX) and erlotinib compared to 2D monolayers of NSCLC-PDX, wild type triple negative breast cancer (MDA-MB-231 WT) and lung adenocarcinoma (HCC-827) cells. Results of flow property, shape fidelity, scaffold stability and biocompatibility of H4-RGD suggest that this hydrogel could be considered for 3D cell bioprinting and also for in-vitro tumor microenvironment development for high throughput screening of various anti-cancer drugs.

Synergistic effects of methyl 2-cyano-3,11-dioxo-18beta-olean-1,-12-dien-30-oate and erlotinib on erlotinib-resistant non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is often characterized by an underlying mutation in the epidermal growth factor receptor (EGFR), contributing to aggressive metastatic disease. Methyl 2-cyano-3,11-dioxo-18beta-olean-1,12-dien-30-oate (CDODA-Me), a glycyrrhetinic acid derivative, reportedly improves the therapeutic response to erlotinib (ERL), an EGFR tyrosine kinase inhibitor. In the present study, we performed a series of studies to demonstrate the efficacy of CDODA-Me (2 µM) in sensitizing HCC827R (ERL-resistant) cells to ERL. Herein, we first established the selectivity of ERL-induced drug resistance in the HCC827R cells, which was sensitized when ERL was combined with CDODA-Me (2 µM), shifting the IC50 from 23.48 µM to 5.46 µM. Subsequently, whole transcriptomic microarray expression data demonstrated that the combination of ERL + CDODA-Me elicited 210 downregulated genes (0.44% of the whole transcriptome (WT)) and 174 upregulated genes (0.36% of the WT), of which approximately 80% were unique to the ERL + CDODA-Me group. Synergistic effects centered on losses to cell cycle progression transcripts, a reduction of minichromosome maintenance complex components (MCM2-7), all key components of the Cdc45·MCM2-7GINS (CMG) complex, and replicative helicases; these effects were tantamount to the upregulation of processes associated with the nuclear factor erythroid 2 like 2 translational response to oxidative stress, including sulfiredoxin 1, heme oxygenase 1, and stress-induced growth inhibitor 1. Collectively, these findings indicate that the synergistic therapeutic effects of ERL + CDODA-Me on resistant NSCLC cells are mediated via the inhibition of mitosis and induction of oxidative stress.

Role of nano-lipid formulation of CARP-1 mimetic, CFM-4.17 to improve systemic exposure and response in osimertinib resistant non-small cell lung cancer.

BACKGROUND: EGFR mutated NSCLCs have been shown to employ the use of CARP-1 in overriding the signaling inhibition of tyrosine kinase inhibitors (such as Osimertinib). CFM 4.17 is a CARP-1 inhibitor which has a promising role in overcoming Tyrosine Kinase Inhibitor (TKI) resistance when used as a pre-treatment through promoting apoptosis. Lack of solubility, hydrophobicity leading to poor systemic exposure are the limitations of CFM 4.17. This can be overcome by nano lipid-based formulation (NLPF) of CFM 4.17 which can enhance systemic exposure in preclinical animal models as well as improve therapeutic efficacy in drug-resistant cancer cell lines. METHODS: Molecular docking simulation studies were performed for CFM 4.17. CFM 4.17-NLPF was formulated by melt dispersion technique and optimized using a Box-Behnken designed surface response methodology approach using Design Expert and MATLAB. In vitro, CFM 4.17 release studies were performed in simulated gastric fluids (SGF-pH-1.2) and simulated intestinal fluids (SIF- pH-6.8). Cell viability assays were performed with HCC827 and H1975 Osimertinib resistant and non-resistant cells in 2D and 3D culture models of Non-small cell lung cancer to determine the effects of CFM 4.17 pre-treatment in Osimertinib response. In vivo pharmacokinetics in rats were performed measuring the effects of NLPF on CFM 4.17 to improve the systemic exposure. RESULTS: CFM 4.17 was well accommodated in the active pocket of the active site of human EGFR tyrosine kinase. CFM 4.17 NLPF was optimized with robust experimental design with particle size less than 300 nm and % entrapment efficiency of 92.3 ± 1.23. Sustained diffusion-based release of CFM 4.17 was observed from NLPF in SGF and SIFs with Peppas and Higuchi based release kinetics, respectively. CFM 4.17 pretreatment improved response by decreasing IC50 value by 2-fold when compared to single treatment Osimertinib in both 2D monolayer and 3D spheroid assays in HCC827 and H1975 Osimertinib resistant and non-resistant cells of Non-small cell lung cancer. There were no differences between CFM 4.17 NLPF and suspension in 2D monolayer culture pretreatments; however, The 3D culture assays showed that CFM 4.17 NLPF improved combination sensitivity. Pharmacokinetic analysis showed that CFM 4.17 NLPF displayed higher AUCtot (2.9-fold) and Cmax (1.18-fold) as compared to free CFM 4.17. In contrast, the animal groups administered CFM 4.17 NLPF showed a 4.73-fold (in half-life) and a 3.07-fold increase (in MRT) when compared to equivalent dosed suspension. CONCLUSION: We have successfully formulated CFM 4.17 NLPFs by robust RSM design approach displaying improved response through sensitizing cells to Osimertinib treatment as well as improving the oral bioavailability of CFM 4.17.

Antagonizing binding of cell cycle and apoptosis regulatory protein 1 (CARP-1) to the NEMO/IKKγ protein enhances the anticancer effect of chemotherapy.

NF-κB is a pro-inflammatory transcription factor that critically regulates immune responses and other distinct cellular pathways. However, many NF-κB-mediated pathways for cell survival and apoptosis signaling in cancer remain to be elucidated. Cell cycle and apoptosis regulatory protein 1 (CARP-1 or CCAR1) is a perinuclear phosphoprotein that regulates signaling induced by anticancer chemotherapy and growth factors. Although previous studies have reported that CARP-1 is a part of the NF-κB proteome, regulation of NF-κB signaling by CARP-1 and the molecular mechanism(s) involved are unclear. Here, we report that CARP-1 directly binds the NF-κB-activating kinase IκB kinase subunit γ (NEMO or NF-κB essential modulator) and regulates the chemotherapy-activated canonical NF-κB pathway. Importantly, blockade of NEMO-CARP-1 binding diminished NF-κB activation, indicated by reduced phosphorylation of its subunit p65/RelA by the chemotherapeutic agent adriamycin (ADR), but not NF-κB activation induced by tumor necrosis factor α (TNFα), interleukin (IL)-1ß, or epidermal growth factor. High-throughput screening of a chemical library yielded a small molecule inhibitor of NEMO-CARP-1 binding, termed selective NF-κB inhibitor 1 (SNI)-1). We noted that SNI-1 enhances chemotherapy-dependent growth inhibition of a variety of cancer cells, including human triple-negative breast cancer (TNBC) and patient-derived TNBC cells in vitro, and attenuates chemotherapy-induced secretion of the pro-inflammatory cytokines TNFα, IL-1ß, and IL-8. SNI-1 also enhanced ADR or cisplatin inhibition of murine TNBC tumors in vivo and reduced systemic levels of pro-inflammatory cytokines. We conclude that inhibition of NEMO-CARP-1 binding enhances responses of cancer cells to chemotherapy.

Author Correction: Characterization and printability of Sodium alginate -Gelatin hydrogel for bioprinting NSCLC co-culture.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Role of Self-Nanoemulsifying Drug Delivery Systems of CDODA-Me in Sensitizing Erlotinib-Resistant Non-Small Cell Lung Cancer.

We have investigated the effects of combination treatment involving ERL (erlotinib) with a glycyrrhetinic acid analog, CDODA-Me in overcoming ERL resistance, providing efforts to improve the oral bioavailability of this treatment using self-nanoemulsifying drug delivery systems (SNEDDS). A Qbd (quality-by-design) approach was used to prepare CDMS (CDODA-SNEDDS, 2 µΜ), which was characterized using surface response methodology to optimize drug content, particle size, and drug release. CDMS/ERL combinations showed synergism in wild-type and resistant H1975 and HCC827 cell lines with combination index values less than 1. Increased apoptosis, mitochondrial membrane potential depletion, and enhanced intracellular ROS levels were also observed in combination therapy. Western blot analysis showed that combination therapy inhibited phosphorylation of epidermal growth factor receptor (EGFR) (p < 0.01 in all cell lines) and Met receptor tyrosine kinase (MET) (p < 0.01 in all cell lines). In vivo, the relative bioavailability of CDMS increased significantly from 22.13 to 151.76 µg/mL compared to the dosing of oral suspension (dose equivalent). Our results demonstrate that combination therapy involving ERL and CDODA-Me overcomes resistance through dual inhibition of p-EGFR and p-MET leading to the induction of apoptosis, intracellular ROS accumulation, and decreased mitochondrial potential. Furthermore, CDMS improved the oral bioavailability of CDODA-Me.

Novel strategies to target chemoresistant triple-negative breast cancer.

Previous studies from our group and others have shown that current drug treatment(s) strategies eliminate bulk of tumor cells (non-CSCs) but it had a minimal effect on cancer stem cells (CSCs) leading to resistance and tumor recurrence. We studied the effects of CFM-4.16 (CARP-1 functional mimetic) and/or cisplatin on four Triple-negative breast cancer (TNBC) MDA-MB-468, MDA-MB-231, CRL-2335 and BR-1126, two cisplatin resistant CisR/MDA-231 and CisR/MDA-468 and cancer stem cells (CSCs) from resistant cell lines. TNBC cells treated with CFM-4.16 plus cisplatin inhibited the expression of FZD8, LRP6 and c-Myc and significantly enhanced cell death in all the cell lines by ~70%-80% compared with the control(s). When Cisplatin resistant CisR/MDA-231 and CisR/MDA-468 were treated with CFM-4.16 plus cisplatin, they also showed a reduction in FZD8 and LRP6 and increased apoptosis compared to control group. Similarly, CFM-4.16 plus cisplatin treatment reduced mammospheres formation abilities of CSCs by 80-90% compared to control group, increased PARP cleavage and apoptosis. Data shows CFM-4.16 plus cisplatin treatment significantly increased apoptosis/cell death in parental, cisplatin resistant and CSCs. Taken together the data suggests that FZD8-mediated Wnt-signaling plays a major role in mediating CSCs growth and resistance to chemotherapy and its inhibition enhances the chemotherapeutic response in TNBC.

Characterization and printability of Sodium alginate -Gelatin hydrogel for bioprinting NSCLC co-culture.

3D bioprinting improves orientation of in vitro tumor models by offering layer by layer positioning of cancer cells and cancer associated fibroblasts (CAFs) which can replicate tumor microenvironment. Aim of this study was to develop a sodium alginate -gelatin (SA-GL) hydrogel by optimizing rheological parameters to print non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) cells and lung CAFs co-cultures. SA-GL hydrogels were prepared, and rheological properties were evaluated. Both the cells were mixed with the hydrogel and printed using INKREDIBLE bioprinter. Hydrogels prepared with 3.25% and 3.5% (w/v) SA and 4% (w/v) GL showed higher printability and cell viability. A significant decline in viscosity with shear rate was observed in these hydrogels suggesting the shear thinning property of hydrogels. Spheroid size distribution after 15 days was in the diameter range of 50-1100 µm. Up-regulation of vimentin, α-SMA and loss of E-cadherin in co-culture spheroids confirmed cellular crosstalk. This study demonstrates that rheological optimization of SA-GL hydrogel enhances printability and viability of NSCLC PDX and CAF co-culture which allows 3D co-culture spheroid formation within the printed scaffold. Therefore, this model can be used for studying high throughput drug screening and other pre-clinical applications.

A H2AX⁻CARP-1 Interaction Regulates Apoptosis Signaling Following DNA Damage.

Cell Cycle and Apoptosis Regulatory Protein (CARP-1/CCAR1) is a peri-nuclear phosphoprotein that regulates apoptosis via chemotherapeutic Adriamycin (doxorubicin) and a novel class of CARP-1 functional mimetic (CFM) compounds. Although Adriamycin causes DNA damage, data from Comet assays revealed that CFM-4.16 also induced DNA damage. Phosphorylation of histone 2AX (γH2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Adriamycin or CFM-4.16 treatments inhibited cell growth and caused elevated CARP-1 and γH2AX in human breast (HBC) and cervical cancer (HeLa) cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and γH2AX occurred in cells undergoing apoptosis. Knock-down of CARP-1 diminished γH2AX, their co-localization, and apoptosis in CFM-4.16- or Adriamycin-treated cells. We found that CARP-1 directly binds with H2AX, and H2AX interacted with CARP-1, but not CARP-1 (Δ600⁻652) mutant. Moreover, cells expressing CARP-1 (Δ600⁻652) mutant were resistant to apoptosis, and had diminished levels of γH2AX, when compared with cells expressing wild-type CARP-1. Mutagenesis studies revealed that H2AX residues 1⁻35 harbored a CARP-1-binding epitope, while CARP-1 amino acids 636⁻650 contained an H2AX-interacting epitope. Surface plasmon resonance studies revealed that CARP-1 (636⁻650) peptide bound with H2AX (1⁻35) peptide with a dissociation constant (Kd) of 127 nM. Cells expressing enhanced GFP (EGFP)-tagged H2AX (1⁻35) peptide or EGFP-tagged CARP-1 (636⁻650) peptide were resistant to inhibition by Adriamycin or CFM-4.16. Treatment of cells with transactivator of transcription (TAT)-tagged CARP-1 (636⁻650) peptide resulted in a moderate, statistically significant abrogation of Adriamycin-induced growth inhibition of cancer cells. Our studies provide evidence for requirement of CARP-1 interaction with H2AX in apoptosis signaling by Adriamycin and CFM compounds.

AT1 receptors in the subfornical organ modulate arterial pressure and the baroreflex in two-kidney, one-clip hypertensive rats.

The subfornical organ (SFO), a forebrain circumventricular organ that lies outside the blood-brain barrier, has been implicated in arterial pressure and baroreflex responses to angiotensin II (ANG II). We tested whether pharmacological inhibition or selective silencing of SFO ANG II type 1 receptors (AT1R) of two-kidney, one-clip rats with elevated plasma ANG II decreases resting arterial pressure and renal sympathetic nerve activity (RSNA) and/or modulates arterial baroreflex responses of heart rate (HR) and RSNA. Male Sprague-Dawley rats underwent renal artery clipping [2-kidney, 1-clip (2K,1C)] or sham clipping (sham). After 6 wk, conscious rats instrumented with vascular catheters, renal nerve electrodes, and a cannula directed to the SFO were studied. In another set of experiments, rats were instrumented with hemodynamic and nerve radio transmitters and injected with scrambled RNA or silencing RNA targeted against AT1R. Mean arterial pressure (MAP) was significantly higher in 2K,1C rats. Acute SFO injection with the AT1R inhibitor losartan did not change MAP in sham or 2K,1C rats. Baroreflex curves of HR and RSNA were shifted rightward in 2K,1C rats. Losartan exerted no effect. SFO AT1R knockdown did not influence MAP in sham rats but decreased MAP in 2K,1C rats, despite no change in plasma ANG II or resting RSNA. AT1R knockdown prevented the reduction in maximum gain and slope of baroreflex responses of HR and RSNA; the reduced RSNA response to baroreceptor unloading was partially restored in 2K,1C rats. These findings show that AT1R activation within the SFO contributes to hypertension and baroreflex dysfunction in 2K,1C rats and highlight the temporal requirement for reversal of these effects.

Tumor hypoxia directed multimodal nanotherapy for overcoming drug resistance in renal cell carcinoma and reprogramming macrophages.

Drug resistance is one of the significant clinical burden in renal cell carcinoma (RCC). The development of drug resistance is attributed to many factors, including impairment of apoptosis, elevation of carbonic anhydrase IX (CA IX, a marker of tumor hypoxia), and infiltration of tumorigenic immune cells. To alleviate the drug resistance, we have used Sorafenib (Sor) in combination with tumor hypoxia directed nanoparticle (NP) loaded with a new class of apoptosis inducer, CFM 4.16 (C4.16), namely CA IX-C4.16. The NP is designed to selectively deliver the payload to the hypoxic tumor (core), provoke superior cell death in parental (WT) and Everolimus-resistant (Evr-res) RCC and selectively downmodulate tumorigenic M2-macrophage. Copper-free 'click' chemistry was utilized for conjugating SMA-TPGS with Acetazolamide (ATZ, a CA IX-specific targeting ligand). The NP was further tagged with a clinically approved NIR dye (S0456) for evaluating hypoxic tumor core penetration and organ distribution. Imaging of tumor spheroid treated with NIR dye-labeled CA IX-SMA-TPGS revealed remarkable tumor core penetration that was modulated by CA IX-mediated targeting in hypoxic-A498 RCC cells. The significant cell killing effect with synergistic combination index (CI) of CA IX-C4.16 and Sor treatment suggests efficient reversal of Evr-resistance in A498 cells. The CA IX directed nanoplatform in combination with Sor has shown multiple benefits in overcoming drug resistance through (i) inhibition of p-AKT, (ii) upregulation of tumoricidal M1 macrophages resulting in induction of caspase 3/7 mediated apoptosis of Evr-res A498 cells in macrophage-RCC co-culturing condition, (iii) significant in vitro and in vivo Evr-res A498 tumor growth inhibition as compared to individual therapy, and (iv) untraceable liver and kidney toxicity in mice. Near-infrared (NIR) imaging of CA IX-SMA-TPGS-S0456 in Evr-res A498 RCC model exhibited significant accumulation of CA IX-oligomer in tumor core with >3-fold higher tumor uptake as compared to control. In conclusion, this proof-of-concept study demonstrates versatile tumor hypoxia directed nanoplatform that can work in synergy with existing drugs for reversing drug-resistance in RCC accompanied with re-education of tumor-associated macrophages, that could be applied universally for several hypoxic tumors.

A CARP-1 functional mimetic compound is synergistic with BRAF-targeting in non-small cell lung cancers.

Non-small cell lung cancers (NSCLC) account for 85% of all lung cancers, and the epidermal growth factor receptor (EGFR) is highly expressed or activated in many NSCLC that permit use of EGFR tyrosine kinase inhibitors (TKIs) as frontline therapies. Resistance to EGFR TKIs eventually develops that necessitates development of improved and effective therapeutics. CARP-1/CCAR1 is an effector of apoptosis by Doxorubicin, Etoposide, or Gefitinib, while CARP-1 functional mimetic (CFM) compounds bind with CARP-1, and stimulate CARP-1 expression and apoptosis. To test whether CFMs would inhibit TKI-resistant NSCLCs, we first generated and characterized TKI-resistant NSCLC cells. The GI 50 dose of Erlotinib for parental and Erlotinib-resistant HCC827 cells was ∼0.1 µM and ≥15 µM, respectively. While Rociletinib or Ocimertinib inhibited the parental H1975 cells with GI 50 doses of ≤0.18 µM, the Ocimertinib-resistant pools of H1975 cells had a GI50 dose of ∼12 µM. The GI50 dose for Rociletinib-resistant H1975 sublines ranged from 4.5-8.0 µM. CFM-4 and its novel analog CFM-4.16 attenuated growth of the parental and TKI-resistant NSCLC cells. CFMs activated p38/JNKs, inhibited oncogenic cMet and Akt kinases, while CARP-1 depletion blocked NSCLC cell growth inhibition by CFM-4.16 or Erlotinib. CFM-4.16 was synergistic with B-Raf-targeting in NSCLC, triple-negative breast cancer, and renal cancer cells. A nano-lipid formulation (NLF) of CFM-4.16 in combination with Sorafenib elicited a superior growth inhibition of xenografted tumors derived from Rociletinib-resistant H1975 NSCLC cells in part by stimulating CARP-1 and apoptosis. These findings support therapeutic potential of CFM-4.16 together with B-Raf targeting in treatment of TKI-resistant NSCLCs.

SM22α suppresses cytokine-induced inflammation and the transcription of NF-κB inducing kinase (Nik) by modulating SRF transcriptional activity in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) phenotypic modulation is characterized by the downregulation of SMC actin cytoskeleton proteins. Our published study shows that depletion of SM22α (aka SM22, Transgelin, an actin cytoskeleton binding protein) promotes inflammation in SMCs by activating NF-κB signal pathways both in cultured VSMCs and in response to vascular injury. The goal of this study is to investigate the underlying molecular mechanisms whereby SM22 suppresses NF-κB signaling pathways under inflammatory condition. NF-κB inducing kinase (Nik, aka MAP3K14, activated by the LTßR) is a key upstream regulator of NF-κB signal pathways. Here, we show that SM22 overexpression suppresses the expression of NIK and its downstream NF-κB canonical and noncanonical signal pathways in a VSMC line treated with a LTßR agonist. SM22 regulates NIK expression at both transcriptional and the proteasome-mediated post-translational levels in VSMCs depending on the culture condition. By qPCR, chromatin immunoprecipitation and luciferase assays, we found that Nik is a transcription target of serum response factor (SRF). Although SM22 is known to be expressed in the cytoplasm, we found that SM22 is also expressed in the nucleus where SM22 interacts with SRF to inhibit the transcription of Nik and prototypical SRF regulated genes including c-fos and Egr3. Moreover, carotid injury increases NIK expression in Sm22-/- mice, which is partially relieved by adenovirally transduced SM22. These findings reveal for the first time that SM22 is expressed in the nucleus in addition to the cytoplasm of VSMCs to regulate the transcription of Nik and its downstream proinflammatory NF-kB signal pathways as a modulator of SRF during vascular inflammation.

A CARP-1 functional mimetic loaded vitamin E-TPGS micellar nano-formulation for inhibition of renal cell carcinoma.

Current treatments for Renal Cell Carcinoma (RCC) include a combination of surgery, targeted therapy, and immunotherapy. Emergence of resistant RCCs contributes to failure of drugs and poor prognosis, and thus warrants development of new and improved treatment options for RCCs. Here we generated and characterized RCC cells that are resistant to Everolimus, a frontline mToR-targeted therapy, and tested whether our novel class of CARP-1 functional mimetic (CFM) compounds inhibit parental and Everolimus-resistant RCC cells. CFMs inhibited RCC cell viability in a dose-dependent manner that was comparable to Everolimus treatments. The GI50 dose of Everolimus for parental A498 cells was ∼1.2µM while it was <0.02µM for the parental UOK262 and UOK268 cells. The GI50 dose for Everolimus-resistant A498, UOK262, and UOK268 cells were ≥10.0µM, 1.8-7.0µM, and 7.0-≥10.0µM, respectively. CFM-4 and its novel analog CFM-4.16 inhibited viabilities of Everolimus resistant RCC cells albeit CFM-4.16 was more effective than CFM-4. CFM-dependent loss of RCC cell viabilities was due in part to reduced cyclin B1 levels, activation of pro-apoptotic, stress-activated protein kinases (SAPKs), and apoptosis. CFM-4.16 suppressed growth of resistant RCC cells in three-dimensional suspension cultures. However, CFMs are hydrophobic and their intravenous administration and dose escalation for in-vivo studies remain challenging. In this study, we encapsulated CFM-4.16 in Vitamin-E TPGS-based- nanomicelles that resulted in its water-soluble formulation with higher CFM-4.16 loading (30% w/w). This CFM-4.16 formulation inhibited viability of parental and Everolimus-resistant RCC cells in vitro, and suppressed growth of parental A498 RCC-cell-derived xenografts in part by stimulating apoptosis. These findings portent promising therapeutic potential of CFM-4.16 for treatment of RCCs.

Myc mediates cancer stem-like cells and EMT changes in triple negative breast cancers cells.

Women with triple negative breast cancer (TNBC) have poor prognosis compared to other breast cancer subtypes. There were several reports indicating racial disparity in breast cancer outcomes between African American (AA) and European American (EA) women. For example, the mortality rates of AA breast cancer patients were three times higher than of EA patients, even though, the incidence is lower in AA women. Our in vitro studies indicate that cancer stem-like cells (CSCs) derived from AA TNBC cell lines have significantly higher self-renewal potential (mammosphere formation) than CSCs derived from EA cell lines. TNBC tumors express high levels of Myc compared to luminal A or HER2 expressing breast cancers. We studied the effects of c-Myc overexpression on CSCs and chemotherapy in AA, and EA derived TNBC cell line(s). Overexpression of c-Myc in AA derived MDA-MB-468 (Myc/MDA-468) cells resulted in a significant increase in CSCs and with minimal changes in epithelial-to-mesenchymal transition (EMT) compared to the control group. In contrast, overexpression of c-Myc in EA derived MDA-MB-231(Myc/MDA-231) cells led to increased epithelial-to-mesenchymal transition (EMT), with a minimal increase in CSCs compared to the control group. Myc/MDA-468 cells were resistant to standard chemotherapeutic treatments such as iniparib (PARP inhibitor) plus cisplatin, / iniparib, cisplatin, paclitaxel and docetaxel. However, Myc/MDA-231 cells, which showed EMT changes responded to iniparib with cisplatin, but were resistant to other drugs, such as iniparib, cisplatin, paclitaxel and docetaxel. Collectively, our results indicate that intrinsic differences in the tumor biology may contribute to the breast cancer disparities.

Di-Block PLCL and Tri-Block PLCLG Matrix Polymeric Nanoparticles Enhanced the Anticancer Activity of Loaded 5-Fluorouracil.

In the current study, 5-FU-loaded nanoparticles (NPs) were prepared using polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), di-block poly lactide-co-caprolactone (PLCL) and tri-block poly L-lactide-co-caprolactone-co-glycolide (PLCLG). The influence of these polymers on the particle sizes, morphology, drug loading, and in vitro drug release was investigated. The anticancer activity was assessed utilizing MTT assay in three human cancer cell lines of different tissue origin; brain (Daoy), liver (HepG2), and colorectal (HT29) using suitable negative and positive controls. The prepared NPs showed a uniform spherical shape with an average size range of 193.5± 6.3 to 303.5± 3.3 nm with negative zeta potential. The entrapment efficiency achieved with F4-F6 (block copolymer NPs) was 78-79% and significantly higher compared with F1 PLGA (31%) and F2; PCL (37%). An initial rapid 5-FU release followed by a slow release ranging from 35% to 81% after 72 h was observed. All the prepared NPs formulations showed enhancement in the cytotoxicity of 5-FU towards all the three cancer cell lines. Generally, block copolymer NPs (F4-F6) showed higher % cell death over PLGA (F1) and PCL (F2) NPs after 48 and 72 h incubation in the case of HepG2 and HT-29. The incorporation of PEG with the tri-block (F6) caused a significant increase in the cytotoxicity of NPs in all of the three cancer cell lines. Block copolymer-based NPs can be considered as promising carriers for enhancing the efficacy of 5-FU in cancer therapy.