Modulation of the tumor microenvironment and mechanism of immunotherapy-based drug resistance in breast cancer.

Breast cancer, the most frequent female malignancy, is often curable when detected at an early stage. The treatment of metastatic breast cancer is more challenging and may be unresponsive to conventional therapy. Immunotherapy is crucial for treating metastatic breast cancer, but its resistance is a major limitation. The tumor microenvironment (TME) is vital in modulating the immunotherapy response. Various tumor microenvironmental components, such as cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs), are involved in TME modulation to cause immunotherapy resistance. This review highlights the role of stromal cells in modulating the breast tumor microenvironment, including the involvement of CAF-TAM interaction, alteration of tumor metabolism leading to immunotherapy failure, and other latest strategies, including high throughput genomic screening, single-cell and spatial omics techniques for identifying tumor immune genes regulating immunotherapy response. This review emphasizes the therapeutic approach to overcome breast cancer immune resistance through CAF reprogramming, modulation of TAM polarization, tumor metabolism, and genomic alterations.

Poly(d,l-lactide-co-glycolide) Surface-Anchored Biotin-Loaded Irinotecan Nanoparticles for Active Targeting of Colon Cancer.

A poly(d,l-lactide-co-glycolide) (PLGA) copolymer was synthesized using the ring-opening polymerization of d,l-lactide and glycolide monomers in the presence of zinc proline complex in bulk through the green route and was well characterized using attenuated total reflectance-Fourier transform infrared, 1H and 13C nuclear magnetic resonance, gel permeation chromatography, differential scanning calorimetry, X-ray diffraction, matrix-assisted laser desorption/ionization time-of-flight, etc. Furthermore, PLGA-conjugated biotin (PLGA-B) was synthesized using the synthesized PLGA and was employed to fabricate nanoparticles for irinotecan (Ir) delivery. These nanoparticles (PLGA-NP-Ir and PLGA-B-NP-Ir) were tested for physicochemical and biological characteristics. PLGA-B-NP-Ir exhibited a stronger cellular uptake and anticancer activity as compared to PLGA-NP-Ir in CT-26 cancer cells (log p < 0.05). The accumulation and retention of fluorescence-labeled nanoparticles were observed to be better in CT-26-inoculated solid tumors in Balb/c mice. The PLGA-B-NP-Ir-treated group inhibited tumor growth significantly more (log p < 0.001) than the untreated control, PLGA-NP-Ir, and Ir-treated groups. Furthermore, no body weight loss, hematological, and blood biochemical tests demonstrated the nanocarriers' nontoxic nature. This work presents the use of safe PLGA and the demonstration of a proof-of-concept of biotin surface attached PLGA nanoparticle-mediated active targeted Ir administration to combat colon cancer. To treat colon cancer, PLGA-B-NP-Ir performed better due to specific active tumor targeting and greater cellular uptake due to biotin.

Therapeutic implications of cancer stem cells in prostate cancer.

Prostate cancer, one of the most frequently occurring cancers in men, is a heterogeneous disease involving multiple cell types within tumors. This tumor heterogeneity at least partly results from genomic instability leading to sub-clonal cellular differentiation. The differentiated cell populations originate from a small subset of cells with tumor-initiating and stem-like properties. These cells, termed prostate cancer stem cells (PCSCs), play crucial roles in disease progression, drug resistance, and relapse. This review discusses the origin, hierarchy, and plasticity of PCSCs; methods for isolation and enrichment of PCSCs; and various cellular and metabolic signaling pathways involved in PCSC induction and maintenance, as well as therapeutic targeting.

AKR1B1 inhibition using NARI-29-an Epalrestat analogue-alleviates Doxorubicin-induced cardiotoxicity via modulating Calcium/CaMKII/MuRF-1 axis.

The clinical use of doxorubicin (Dox) is narrowed due to its carbonyl reduction to doxorubicinol (Doxol) implicating resistance and cardiotoxicity. Hence, in the present study we have evaluated the cardioprotective effect of AKR1B1 (or aldose reductase, AR) inhibitor NARI-29 (epalrestat (EPS) analogue) and its effect in the Dox-modulated calcium/CaMKII/MuRF1 axis. Initially, the breast cancer patient survival associated with AKR1B1 expression was calculated using Kaplan Meier-plotter (KM-plotter). Further, breast cancer, cardiomyoblast (H9c2), and macrophage (RAW 264.7) cell lines were used to establish the in vitro combination effect of NARI-29 and Dox. To develop the cardiotoxicity model, mice were given Dox 2.5 mg/kg (i.p.), biweekly. The effect of AKR1B1 inhibition using NARI-29 on molecular and cardiac functional changes was measured using echocardiography, fluorescence-imaging, ELISA, immunoblotting, flowcytometry, High-Performance Liquid Chromatography with Fluorescence Detection (HPLC-FD) and cytokine-bead array methods. The bioinformatics data suggested that a high expression of AKR1B1 is associated with significantly low survival of breast cancer patients undergoing chemotherapy; hence, it could be a target for chemo-sensitization and chemo-prevention. Further, in vitro studies showed that AKR1B1 inhibition with NARI-29 has increased the accumulation and sensitized Dox to breast cancer cell lines. However, treatment with NARI-29 has alleviated the Dox-induced toxicity to cardiomyocytes and decreased the secretion of inflammatory cytokines from RAW 264.7 cells. In vivo studies revealed that the NARI-29 (25 and 50 mg/kg) has prevented the functional, histological, biochemical, and molecular alterations induced by Dox treatment. Moreover, we have shown that NARI-29 has prevented the carbonyl reduction of Dox to Doxol in the mouse heart, which reduced the calcium overload, prevented phosphorylation of CaMKII, and reduced the expression of MuRF1 to protect from cardiac injury and apoptosis. Hence in conclusion, AKR1B1 inhibitor NARI-29 could be used as an adjuvant therapeutic agent with Dox to prevent cardiotoxicity and synergize anti-breast cancer activity.

Hybrid Inorganic Complexes as Cancer Therapeutic Agents: In-vitro Validation.

A series of novel mixed transition metal-Magnesium tartarate complexes of general formulation [MMg(C4H4O6)2 .xH2O] (where M = Mn, Fe, Co, Ni, Cu and Zn) is prepared with bidentate tartarate ligand. The synthesized complexes (C1 to C6) are characterized by various analytical techniques such as Elemental analysis, Thermo gravimetric analysis, FT-IR Spectroscopy, X-ray Diffraction, Magnetic susceptibility study etc. All complexes exhibit the composition MMgL2 where M = Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II) and L = bidentate tartarate ligand. Analytical data reveals all complexes possesses 1:1 (metal: ligand) ratio. FT-IR spectral study shows that bidentate tartarate ligand coordinate with metal ion in a bidentate manner through two oxygen atoms. Thermo gravimetric analysis of all complexes shows that degradation curves of complexes agrees with recommended formulae of the complexes. X-ray diffraction technique suggests that all complexes (C1 to C6) are polycrystalline in nature. All newly synthesized metal tartarate complexes and ligand were screened in vitro for their anticancer activity against human breast cancer (MDA-MB-231) cell line. The bioassays of all these complexes showed C3 (Co) and C5 (Cu) Mg-tartarate complexes contains maximum antiproliferative activity at 200 µg/ml concentration on MDA-MB-231 cells as compared to other complexes. MDA-MB-231 cells treated with C3 (Co) and C5 (Cu) Mg-tartarate complexes also showed inhibition in cell migration.

Prosopis juliflora (Sw.) DC phytochemicals induce apoptosis and inhibit cell proliferation signaling pathways, EMT, migration, invasion, angiogenesis and stem cell markers in melanoma cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Prosopis juliflora (Sw.), DC is a xerophytic plant species that extensively grow in Asia, Africa, Australia, and Brazil. From ancient time P. juliflora is being utilized in various folk remedies for example in wound healing, fever, inflammation, measles, excrescences, diarrhea and dysentery. Traditionally, gum, paste, and smoke obtained from the leaves and pods are applied for anticancer, antidiabetic, anti-inflammatory, and antimicrobial purposes. AIM OF THE STUDY: Our previous studies have demonstrated the promising potential of Prosopis Juliflora leaves methanol extract (PJLME) against breast cancer, and suggested its possible integration as a complementary medicine for the effective management of breast cancer. However, evidence against how PJLME mechanistically target the cancer proliferative pathways and other targets is poorly understood. The basic aim of the present study was to understand the anti-melanoma potential of PJLME against B16f10 cells with possible mechanisms of action. MATERIALS AND METHODS: MTT assay was used to determine cell viability. Wound and transwell migration assay was performed to check migration potential of cells after PJLME treatment, while clonogenic assay was carried out to understand its colony inhibition actvity. Flow cytometry was used to perform annexin V/PI assay (apoptosis assay), ROS assay, cell cycle analysis. In-vitro angiogenesis assay was performed to check formation of capillary like vascular structure after PJLME treatment. Apoptotic genes, signaling pathways markers, EMT markers and stem cell markers were determined by western blotting. In-vivo BALB/C mice xenograft model study was performed to check the effect of PJLME on in-vivo melanoma tumor growth. RESULTS: The experimental outcome of the present study has clearly demonstrated the inhibition of growth, migration, invasion, colony formation and apoptosis inducing potential of PJLME against mouse melanoma cancer cells. Treatment of B16F10 melanoma cells with PJLME resulted in arrest of cell cycle at G0/G1 phase. Annexin V-FITC/PI assay confirmed the apoptosis inducing potential of PJLME in B16F10 and A375 melanoma cells. Furthermore, Western blot experiments confirmed that the treatment of PJLME downregulates the expression of anti-apoptotic gene like Bcl2 and increase the expression profile of pro-apoptotic genes like Bax, Bad, and Bak in B16F10 melanoma cells. HUVEC (Human umbilical vein endothelial cells) tube formation assay clearly demonstrated the anti-angiogenic potential of PJLME. The study also revealed that PJLME has potential to inhibit the Akt and Erk signaling pathways which are participating in cancer cell proliferation, migration, invasion etc. The outcome of qRT-PCR and immunoblotting analysis clearly unveiled that PJLME treatment leads to downregulation of epithelial-mesenchymal transition (EMT) as well as stem cell markers. Finally, the in-vivo animal xenograft model study also revealed the anti-melanoma potential of PJLME by significantly inhibiting the B16F10 melanoma tumor growth in BALB/c mice model. The LC-ESI-MS/MS analysis of PJLME showed the presence of variety of bioactive molecules associated with anticancer effects. CONCLUSION: The outcome of the present investigation clearly demonstrated the anti-melanoma potential of PJLME against B16f10 melanoma cells. PJLME can be explored as an adjuvant or complementary therapy against melanoma cancer, however further studies are required to understand the clinical efficacy of PJLME. Nevertheless, it can be further explored as a promising resource for identification of novel anticancer candidate drug.

Development and characterization of a patient­derived orthotopic xenograft of therapy­resistant breast cancer.

Numerous years of cell line­based studies have enhanced the current understanding of cancer and its treatment. However, limited success has been achieved in treating hormone receptor­positive, HER2­negative metastatic breast cancers that are refractory to treatment. The majority of cancer cell lines are unsuitable for use as pre­clinical models that mimic this critical and often fatal clinical type, since they are derived from treatment­naive or non­metastatic breast cancer cases. The aim of the present study was to develop and characterize patient­derived orthotopic xenografts (PDOXs) from patients with endocrine hormone receptor­positive, HER2­negative metastatic breast cancer who had relapsed on therapy. A patient who progressed on endocrine hormone therapy provided her tumor via a biobank. This tumor was implanted in mice. It was then serially passaged by implanting PDOX tumor fragments into another set of mice to develop further generations of PDOXs. These tissues were characterized using various histological and biochemical techniques. Histological, immunofluorescence and western blot analyses indicated that the PDOX tumors retained a similar morphology, histology and subtype­specific molecular features to that of the patient's tumor. The present study successfully established PDOXs of hormone­resistant breast cancer and characterized them in comparison with those derived from the original breast cancer tissue of the patient. The data highlight the reliability and usefulness of PDOX models for studies of biomarker discovery and preclinical drug screening. The present study was registered with the clinical trial registry of India (CTRI; registration no. CTRI/2017/11/010553; registered on 17/11/2017).

Antiproliferative and apoptotic potential of methotrexate lipid nanoparticles in a murine breast cancer model.

Aim: To evaluate the efficacy of novel methotrexate-loaded nanoparticles (MTX-NPs) in vitro and in vivo in the treatment of breast cancer. Materials & methods: MTX-NPs were tested for cellular uptake, cell viability, cell cycle, cellular wound migration and changes in tumor volume using characterized NPs. Results: The solid lipid NPs (SLNPs) showed strong cellular uptake, increased apoptosis, controlled cytotoxicity at lower IC50 of methotrexate and a sizable reduction in tumor burden. Conclusion: MTX-NP oral formulation can be a promising candidate in breast cancer treatment with improved cellular uptake and in vivo efficacy.

CRISPR based therapeutics: a new paradigm in cancer precision medicine.

BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems are the latest addition to the plethora of gene-editing tools. These systems have been repurposed from their natural counterparts by means of both guide RNA and Cas nuclease engineering. These RNA-guided systems offer greater programmability and multiplexing capacity than previous generation gene editing tools based on zinc finger nucleases and transcription activator like effector nucleases. CRISPR-Cas systems show great promise for individualization of cancer precision medicine. MAIN BODY: The biology of Cas nucleases and dead Cas based systems relevant for in vivo gene therapy applications has been discussed. The CRISPR knockout, CRISPR activation and CRISPR interference based genetic screens which offer opportunity to assess functions of thousands of genes in massively parallel assays have been also highlighted. Single and combinatorial gene knockout screens lead to identification of drug targets and synthetic lethal genetic interactions across different cancer phenotypes. There are different viral and non-viral (nanoformulation based) modalities that can carry CRISPR-Cas components to different target organs in vivo. CONCLUSION: The latest developments in the field in terms of optimization of performance of the CRISPR-Cas elements should fuel greater application of the latter in the realm of precision medicine. Lastly, how the already available knowledge can help in furtherance of use of CRISPR based tools in personalized medicine has been discussed.

Tumor-associated macrophage derived IL-6 enriches cancer stem cell population and promotes breast tumor progression via Stat-3 pathway.

BACKGROUND: Cancer stem cells (CSCs) play crucial role in tumor progression, drug resistance and relapse in various cancers. CSC niche is comprised of various stromal cell types including Tumor-associated macrophages (TAMs). Extrinsic ques derived from these cells help in maintenance of CSC phenotype. TAMs have versatile roles in tumor progression however their function in enrichment of CSC is poorly explored. METHODS: Mouse macrophages (RAW264.7) cells were activated by interaction with conditioned media (CM) of murine breast cancer cells (4T1) into TAMs and the effect of activated macrophage (TAM) derived factors was examined on enrichment of cancer stem cells (CSCs) and tumor growth using in vitro and in vivo models. RESULTS: In this study, we report that macrophages upon interaction with breast cancer cells activate tumor promoting function and exhibit differential expression of various proteins as shown by secretome analysis using proteomics studies. Based on secretome data, we found that Interleukin-6 (IL-6) is one of the up-regulated genes expressed in activated macrophages. Further, we confirm that TAMs produce high levels of IL-6 and breast cancer cell derived factors induce IL-6 production in activated macrophages via p38-MAPK pathway. Furthermore, we demonstrate that tumor activated macrophages induce enrichment of CSCs and expression of CSC specific transcription factors such as Sox-2, Oct-3/4 and Nanog in breast cancer cells. We further prove that TAM derived IL-6 plays a key role in TAM mediated CSC enrichment through activation of Signal transducer and activator of transcription 3 (STAT-3) signaling. TAM derived IL-6 influences breast cancer cell migration and angiogenesis. Moreover, our in vivo findings indicated that TAM derived IL-6 induces CSC population and resulting tumor growth in breast cancer. CONCLUSION: These finding provide evidence that TAM derived IL-6 plays a major role in CSC enrichment and tumor progression in breast cancer and IL-6 and its regulated signalling network may act as potential therapeutic target for management of breast cancer.

Herbal medicine AnoSpray suppresses proinflammatory cytokines COX-2 and RANTES in the management of hemorrhoids, acute anal fissures and perineal wounds.

Hemorrhoids, anal fistula and fissure are common anorectal complications. Anorectal diseases are associated with severe pain, inflammation, swelling, itching and bleeding. These diseases may be managed with different medical treatments or surgical procedures, depending on their severity. Surgical procedures, however, are highly invasive and are associated with higher costs and the possibility of recurrence. In addition, surgical removal of fistula-in-ano leads to the formation of perineal wounds. Therefore, developing therapeutic interventions that are effective in alleviating inflammation and pain are desirable for the effective management of anorectal diseases. Herbal compounds have previously been indicated to suppress inflammation and pain in different pathological conditions. The aim of the present study was to examine the effects elicited by a polyherbal formulation, AnoSpray®, on the migration of inflammatory cells and on the expression of inflammatory cytokines in anorectal diseases. The effect of AnoSpray on cell viability and migration was studied using MTT and wound-migration assays, respectively. Furthermore, the effects of AnoSpray on the expression of the inflammatory cytokines regulated upon activation, normal T cell expressed and presumably secreted (RANTES) and VEGF, as well as on cyclooxygenase-2 (COX)-2, were investigated using western blot analysis. The expression of RANTES and COX-2 in human hemorrhoid specimens was also analyzed to corroborate the in vitro findings. The results obtained revealed that AnoSpray did not exhibit any cytotoxic effects; however, it did lead to a significant suppression in the migration of RAW 264.7 and BJ cells. Furthermore, the results suggested that AnoSpray suppressed the expression of the inflammatory cytokines RANTES and VEGF, and also the expression of COX-2. In addition, RANTES and COX-2 were significantly downregulated in the clinical specimens of AnoSpray-treated hemorrhoids compared with the controls. Taken together, the results of the present study suggested that AnoSpray may be a potential therapeutic agent in the treatment of bleeding hemorrhoids, anal fissures and perineal wounds.

Osteopontin Signaling in Shaping Tumor Microenvironment Conducive to Malignant Progression.

Context-dependent reciprocal crosstalk between cancer and surrounding stromal cells in the tumor microenvironment is imperative for the regulation of various hallmarks of cancer. A myriad of growth factors, chemokines, and their receptors aids in the interaction between cancer cells and tumor microenvironmental components. Osteopontin is a chemokine-like protein, overexpressed in different types of cancers. Osteopontin plays a crucial role in orchestrating dialogue between cancer and stromal cells. Osteopontin, in tumor microenvironment, is produced in tumor as well as stromal cells. Tumor-derived osteopontin regulates proliferation, migration, activation, and differentiation of different types of stromal cells. Osteopontin secreted from tumor cells regulates the generation of cancer-associated fibroblasts from resident fibroblasts and mesenchymal stem cells. Osteopontin also shapes immunosuppressive tumor microenvironment by controlling regulatory T cells and tumor-associated macrophages. Moreover, secretion of osteopontin from tumor stroma has been highly documented. Stromal cell-derived osteopontin induces epithelial-to-mesenchymal transition, angiogenesis, metastasis, and cancer stem cell enrichment. Tumor- or stroma-derived osteopontin mainly functions through binding with cell surface receptors, integrins and CD44, and activates downstream signaling events like PI-3 kinase/Akt and MAPK pathways. Presumably, disrupting the communication between the tumor cells and surrounding microenvironment by targeting osteopontin-regulated signaling using specific antibodies, small-molecule inhibitors, and chemotherapeutic agents is a novel therapeutic strategy for clinical management of cancer.

MiRNA-146a/AKT/ß-Catenin Activation Regulates Cancer Stem Cell Phenotype in Oral Squamous Cell Carcinoma by Targeting CD24.

CD44highCD24low population has been previously reported as cancer stem cells (CSCs) in Oral Squamous Cell Carcinoma (OSCC). Increasing evidence suggests potential involvement of microRNA (miRNA) network in modulation of CSC properties. MiRNAs have thus emerged as crucial players in tumor development and maintenance. However, their role in maintenance of OSCC stem cells remains unclear. Here we report an elevated expression of miR-146a in the CD44highCD24low population within OSCC cells and primary HNSCC tumors. Moreover, over-expression of miR-146a results in enhanced stemness phenotype by augmenting the CD44highCD24low population. We demonstrate that miR-146a stabilizes ß-catenin with concomitant loss of E-cadherin and CD24. Interestingly, CD24 is identified as a novel functional target of miR-146a and ectopic expression of CD24 abrogates miR-146a driven potential CSC phenotype. Mechanistic analysis reveals that higher CD24 levels inhibit AKT phosphorylation leading to ß-catenin degradation. Using stably expressing miR-146a/CD24 OSCC cell lines, we also validate that the miR-146a/CD24/AKT loop significantly alters tumorigenic ability in vivo. Furthermore, we confirmed that ß-catenin trans-activates miR-146a, thereby forming a positive feedback loop contributing to stem cell maintenance. Collectively, our study demonstrates that miR-146a regulates CSCs in OSCC through CD24-AKT-ß-catenin axis.

SMAR1 suppresses the cancer stem cell population via hTERT repression in colorectal cancer cells.

One of the hallmarks of a cancer cell is the ability for indefinite proliferation leading to the immortalization of the cell. Activation of several signaling pathways leads to the immortalization of cancer cells via the reactivation of enzyme telomerase (hTERT). hTERT is active in germ cells, stem cells and also cancer cells. An earlier report from our lab suggests that SMAR1, a tumor suppressor protein, is significantly downregulated in the higher grades of colorectal cancers. Our study identifies SMAR1 as a transcriptional repressor of hTERT. We find that SMAR1 interacts with HDAC1/mSin3a co-repressor complex at the hTERT promoter and brings about HDAC1-mediated transcriptional repression of the promoter. Most solid tumors including colorectal cancer reactivate hTERT expression as it confers several advantages to the cancer cells like increased proliferation and angiogenesis. One of these non-canonical functions of hTERT is inducing the pool of cancer stem cell population. We find that in the CD133HighCD44High cancer stem cells population, SMAR1 expression is highly diminished leading to elevated hTERT expression. We also find that knockdown of SMAR1 promotes total CD133+CD44+ population and impart enhanced sphere-forming ability to the colorectal cancer cells. SMAR1 also inhibits invasion and metastasis in colorectal cancer cell lines via repression of hTERT. Our study provides evidence that downregulation of SMAR1 causes activation of hTERT leading to an increase in the cancer stem cell phenotype in colorectal cancer cells.

Polyherbal formulation Anoac­H suppresses the expression of RANTES and VEGF for the management of bleeding hemorrhoids and fistula.

Hemorrhoids and fistula are considered the most common anorectal conditions in the general population. These conditions affect the quality of a patient's life by causing pain and bleeding during defecation or even in the resting state. Lower grades of hemorrhoids may be controlled by traditional measures. However, surgery is an effective treatment option in recurrent­lower and higher­grade hemorrhoids. Surgical procedures are associated with various complications, including pain and delayed wound healing. Recurrence of hemorrhoids is also a major concern in the post­operative period. An anal fistula is the connection between the anus and the skin and causes severe pain, swelling, as well as blood and pus discharge. Fistula has serious social and economic consequences. Hence, it is important to understand the pathophysiology and molecular pathology of hemorrhoids and fistula, to identify the molecular targets and to develop pharmacological­interventions. In a previous study by our group, the polyherbal formulation Anoac­H was developed for the treatment of different stages of hemorrhoids and fistula, and it was demonstrated that Anoac­H is an effective formulation for treating hemorrhoids. However, the molecular mode of action of Anoac­H on hemorrhoids and fistula had remained elusive. In the present study, it was determined that this formulation reduces the migration of mesenchymal (fibroblasts) and immune (RAW 264.7) cells without affecting their viability. It was also observed that Anoac­H suppresses the expression of regulated upon activation, normal T cell expressed and presumably secreted (RANTES) and VEGF in fibroblasts and macrophages. Inflammation and elevated expression of RANTES and VEGF were observed in hemorrhoids and fistula. However, inflammation, as well as the expression of RANTES and VEGF, were significantly reduced in treated human hemorrhoid and fistula tissues as compared to untreated ones, confirming the in vitro results.

Induction of monoamine oxidase A-mediated oxidative stress and impairment of NRF2-antioxidant defence response by polyphenol-rich fraction of Bergenia ligulata sensitizes prostate cancer cells in vitro and in vivo.

Prostate cancer (PCa) is a major cause of mortality and morbidity in men. Available therapies yield limited outcome. We explored anti-PCa activity in a polyphenol-rich fraction of Bergenia ligulata (PFBL), a plant used in Indian traditional and folk medicine for its anti-inflammatory and antineoplastic properties. PFBL constituted of about fifteen different compounds as per LCMS analysis induced apoptotic death in both androgen-dependent LNCaP and androgen-refractory PC3 and DU145 cells with little effect on NKE and WI38 cells. Further investigation revealed that PFBL mediates its function through upregulating ROS production by enhanced catalytic activity of Monoamine oxidase A (MAO-A). Notably, the differential inactivation of NRF2-antioxidant response pathway by PFBL resulted in death in PC3 versus NKE cells involving GSK-3ß activity facilitated by AKT inhibition. PFBL efficiently reduced the PC3-tumor xenograft in NOD-SCID mice alone and in synergy with Paclitaxel. Tumor tissues in PFBL-treated mice showed upregulation of similar mechanism of cell death as observed in isolated PC3 cells i.e., elevation of MAO-A catalytic activity, ROS production accompanied by activation of ß-TrCP-GSK-3ß axis of NRF2 degradation. Blood counts, liver, and splenocyte sensitivity analyses justified the PFBL safety in the healthy mice. To our knowledge this is the first report of an activity that crippled NRF2 activation both in vitro and in vivo in response to MAO-A activation. Results of this study suggest the development of a novel treatment protocol utilizing PFBL to improve therapeutic outcome for patients with aggressive PCa which claims hundreds of thousands of lives each year.

Structural Constraint of Osteopontin Facilitates Efficient Binding to CD44.

Since the original description in 1996, the interaction between the cytokine osteopontin (OPN) and the homing receptor CD44 has been extensively studied in cancer, inflammation, bone remodeling, and various other conditions. Alternative splicing and extensive posttranslational modifications by both binding partners, as well as the possibility for lateral recruitment of additional membrane receptors or soluble co-ligands into a complex have left the exact molecular requirements for high-affinity OPN-CD44 binding unresolved. We now report that there is a moderate engagement between the unmodified molecules, which results in curved double-reciprocal plots for OPN titration, suggesting the existence of two binding sites or two binding conformations. Structural constraint of OPN, by immobilization or by addition of heparin, is required for its strong ligation of CD44. Prior literature provides evidence that heparin binding to OPN prompts the unfolding of a core element in the protein. This conformational adjustment may be essential for efficient CD44 interaction. The integrin α9ß1 seems to compete with the OPN-CD44 engagement, while the integrin αVß3 reflects additive binding, suggesting that the CD44 contact sites on OPN are downstream of the RGD motif but overlap with the SVVYGLR domain. Hyaluronate has no effect, placing the relevant domain on CD44 downstream of the N-terminus.

Tumor suppressor SMAR1 regulates PKM alternative splicing by HDAC6-mediated deacetylation of PTBP1.

BACKGROUND: Highly proliferating cancer cells exhibit the Warburg effect by regulation of PKM alternative splicing and promoting the expression of PKM2. Majority of the alternative splicing events are known to occur in the nuclear matrix where various MARBPs actively participate in the alternative splicing events. SMAR1, being a MARBP and an important tumor suppressor, is known to regulate the splicing of various cancer-associated genes. This study focuses on the regulation of PKM alternative splicing and inhibition of the Warburg effect by SMAR1. METHODS: Immunohistochemistry was performed in breast cancer patient samples to establish the correlation between SMAR1 and PKM isoform expression. Further, expression of PKM isoforms upon modulation in SMAR1 expression in breast cancer cell lines was quantified by qRT-PCR and western blot. The acetylation status of PTBP1 was estimated by immunoprecipitation along with its enrichment on PKM pre-mRNA by CLIP in SMAR1 knockdown conditions. The role of SMAR1 in tumor metabolism and tumorigenesis was explored by in vitro enzymatic assays and functional assays upon SMAR1 knockdown. Besides, in vivo tumor formation by injecting adeno-SMAR1-transduced MDA-MB-231 cells in NOD/SCID mice was performed. RESULTS: The expression profile of SMAR1 and PKM isoforms in breast cancer patients revealed that SMAR1 has an inverse correlation with PKM2 and a positive correlation with PKM1. Further quantitative PKM isoform expression upon modulation in SMAR1 expression also reflects that SMAR1 promotes the expression of PKM1 over tumorigenic isoform PKM2. SMAR1 deacetylates PTBP1 via recruitment of HDAC6 resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. SMAR1 inhibits the Warburg effect, tumorigenic potential of cancer cells, and in vivo tumor generation in a PKM2-dependent manner. CONCLUSIONS: SMAR1 regulates PKM alternative splicing by causing HDAC6-dependent deacetylation of PTBP1, resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. Additionally, SMAR1 suppresses glucose utilization and lactate production via repression of PKM2 expression. This suggests that tumor suppressor SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells via regulation of PKM alternative splicing.

Tumor-derived osteopontin drives the resident fibroblast to myofibroblast differentiation through Twist1 to promote breast cancer progression.

Tumor-stroma interactions are important determinants for the disease course in cancer. While stromal influence has been known to often play a tumor-promoting role, incomplete mechanistic insight into this phenomenon has prevented its therapeutic targeting. Stromal fibroblasts can be activated by tumor cells to differentiate into cancer-associated fibroblasts (CAFs), that exhibit the traits of myofibroblasts, and in turn, they increase cancer aggressiveness. Here, we report the crosstalk between the cancer cells and stromal fibroblasts that leads to tumor progression. The process is initiated by secretion of a chemokine like protein, osteopontin (OPN) from the cancer cells that differentiates the fibroblasts to myofibroblasts. Tumor-derived OPN achieves this transition by engaging CD44 and αvß3 integrins on the fibroblast surface, which mediates signaling via Akt and ERK to induce Twist1-dependent gene expression. The OPN-driven CAFs then secrete CXCL12, which in turn triggers epithelial to mesenchymal transition (EMT) in the tumor cells. OPN, produced by the cancer cells, and CXCL12, secreted by activated fibroblasts, are necessary and sufficient to perpetuate the crosstalk. Knocking out OPN in carcinogen-induced mammary tumors or knocking down OPN in cancer cells and fibroblast co-implanted xenografts abrogates myofibroblast differentiation, Twist1, and CXCL12 expression. OPN expression is correlated with CAF-specific gene signature as shown by breast tumor tissue microarray consisting of 100 patient specimens. Bioinformatics analyses have confirmed that the expression of OPN is significantly correlated with the expression of myofibroblast-specific markers as demonstrated in human breast carcinoma dataset of 2509 patients. Our findings describe OPN and CXCL12 act as compelling targets to curb the tumor-promoting features of the stromal components and further suggested that OPN-regulated CXCL12 network might act as potential therapeutic target for the management of CAF-mediated breast cancer progression.