Unique sphingolipid signature identifies luminal and triple-negative breast cancer subtypes.

Breast cancer (luminal and triple-negative breast cancer [TNBC]) is the most common cancer among women in India and worldwide. Altered sphingolipid levels have emerged as a common phenomenon during cancer progression. However, these alterations are yet to be translated into robust diagnostic and prognostic markers for cancer. Here, we present the quantified sphingolipids of tumor and adjacent-normal tissues from patients of luminal (n = 70) and TNBC (n = 42) subtype from an Indian cohort using targeted liquid chromatography mass spectrometry. We recorded unique sphingolipid profiles that distinguished luminal and TNBC tumors in comparison to adjacent normal tissue by six-sphingolipid signatures. Moreover, systematic comparison of the profiles of luminal and TNBC tumors provided a unique five-sphingolipid signature distinguishing the two subtypes. We further identified key sphingolipids that can stratify grade II and grade III tumors of luminal and TNBC subtype as well as their lymphovascular invasion status. Therefore, we provide the right evidence to develop these candidate sphingolipids as widely acceptable marker/s capable of diagnosing luminal vs TNBC subtype of breast cancer, and predicting the disease severity by identifying the tumor grade.

A localized hydrogel-mediated chemotherapy causes immunogenic cell death via activation of ceramide-mediated unfolded protein response.

Treatment of triple-negative breast cancer (TNBC) is challenging because of its "COLD" tumor immunosuppressive microenvironment (TIME). Here, we present a hydrogel-mediated localized delivery of a combination of docetaxel (DTX) and carboplatin (CPT) (called DTX-CPT-Gel therapy) that ensured enhanced anticancer effect and tumor regression on multiple murine syngeneic and xenograft tumor models. DTX-CPT-Gel therapy modulated the TIME by an increase of antitumorigenic M1 macrophages, attenuation of myeloid-derived suppressor cells, and increase of granzyme B+CD8+ T cells. DTX-CPT-Gel therapy elevated ceramide levels in tumor tissues that activated the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-mediated unfolded protein response (UPR). This UPR-mediated activation of apoptotic cell death led to release of damage-associated molecular patterns, thereby activating the immunogenic cell death that could even clear the metastatic tumors. This study provides a promising hydrogel-mediated platform for DTX-CPT therapy that induces tumor regression and effective immune modulation and, therefore, can be explored further for treatment of TNBC.

Ceramide Kinase (CERK) Emerges as a Common Therapeutic Target for Triple Positive and Triple Negative Breast Cancer Cells.

Sphingolipids are key signaling biomolecules that play a distinct role in cell proliferation, migration, invasion, drug resistance, metastasis, and apoptosis. Triple-negative (ER-PR-HER2-) and triple-positive (ER+PR+HER2+) breast cancer (called TNBC and TPBC, respectively) subtypes reveal distinct phenotypic characteristics and responses to therapy. Here, we present the sphingolipid profiles of BT-474 and MDA-MB-231 breast cancer cell lines representing the TPBC and TNBC subtypes. We correlated the level of different classes of sphingolipids and the expression of their corresponding metabolizing enzymes with the cell proliferation and cell migration properties of BT-474 and MDA-MB-231 cells. Our results showed that each cell type exhibits a unique sphingolipid profile, and common enzymes such as ceramide kinase (CERK, responsible for the synthesis of ceramide-1-phosphates) are deregulated in these cell types. We showed that siRNA/small molecule-mediated inhibition of CERK can alleviate cell proliferation in BT-474 and MDA-MB-231 cells, and cell migration in MDA-MB-231 cells. We further demonstrated that nanoparticle-mediated delivery of CERK siRNA and hydrogel-mediated sustained delivery of CERK inhibitor to the tumor site can inhibit tumor progression in BT-474 and MDA-MB-231 tumor models. In summary, distinct sphingolipid profiles of TPBC and TNBC representing cell lines provide potential therapeutic targets such as CERK, and nanoparticle/hydrogel mediated pharmacological manipulations of such targets can be explored for future cancer therapeutics.

Targeting Vancomycin-Resistant Enterococci (VRE) Infections and Van Operon-Mediated Drug Resistance Using Dimeric Cholic Acid-Peptide Conjugates.

Emergence of vancomycin resistance in Gram-positive bacteria and the prevalence of vancomycin-resistant Enterococci (VRE) infections are highly alarming as very limited antibiotic options are available against VRE infections. Here, we present the synthesis of cholic acid-derived dimeric amphiphiles where two cholic acid moieties are tethered through carboxyl terminals using different alkylene spacers. Our investigations revealed that dimer 5 possessing a propylene spacer and glycine-valine peptides tethered on hydroxyl groups is the most effective antimicrobial against VRE. Dimer 5 can permeabilize bacterial membranes, generate reactive oxygen species, and clear preformed biofilms. We further demonstrate that dimer 5 downregulates vancomycin-mediated transcriptional activation of the vanHAX gene cluster and does not allow VSE to develop vancomycin resistance until 100 generations. Therefore, this study, for the first time, presents a bacterial membrane-targeting amphiphile that can mitigate VRE infections and inhibit the emergence of vancomycin resistance.

Nonimmunogenic Hydrogel-Mediated Delivery of Antibiotics Outperforms Clinically Used Formulations in Mitigating Wound Infections.

Treatment of chronic wound infections caused by Gram-positive bacteria such as Staphylococcus aureus is highly challenging due to the low efficacy of existing formulations, thereby leading to drug resistance. Herein, we present the synthesis of a nonimmunogenic cholic acid-glycine-glycine conjugate (A6) that self-assembles into a supramolecular viscoelastic hydrogel (A6 gel) suitable for topical applications. The A6 hydrogel can entrap different antibiotics with high efficacy without compromising its viscoelastic behavior. Activities against different bacterial species using a disc diffusion assay demonstrated the antimicrobial effect of the ciprofloxacin-loaded A6 hydrogel (CPF-Gel). Immune profiling and gene expression studies after the application of the A6 gel to mice confirmed its nonimmunogenic nature to host tissues. We further demonstrated that topical application of CPF-Gel clears S. aureus-mediated wound infections more effectively than clinically used formulations. Therefore, cholic acid-derived hydrogels are an efficacious matrix for topical delivery of antibiotics and should be explored further.

Alternative splicing of ceramide synthase 2 alters levels of specific ceramides and modulates cancer cell proliferation and migration in Luminal B breast cancer subtype.

Global dysregulation of RNA splicing and imbalanced sphingolipid metabolism has emerged as promoters of cancer cell transformation. Here, we present specific signature of alternative splicing (AS) events of sphingolipid genes for each breast cancer subtype from the TCGA-BRCA dataset. We show that ceramide synthase 2 (CERS2) undergoes a unique cassette exon event specifically in Luminal B subtype tumors. We validated this exon 8 skipping event in Luminal B cancer cells compared to normal epithelial cells, and in patient-derived tumor tissues compared to matched normal tissues. Differential AS-based survival analysis shows that this AS event of CERS2 is a poor prognostic factor for Luminal B patients. As Exon 8 corresponds to catalytic Lag1p domain, overexpression of AS transcript of CERS2 in Luminal B cancer cells leads to a reduction in the level of very-long-chain ceramides compared to overexpression of protein-coding (PC) transcript of CERS2. We further demonstrate that this AS event-mediated decrease of very-long-chain ceramides leads to enhanced cancer cell proliferation and migration. Therefore, our results show subtype-specific AS of sphingolipid genes as a regulatory mechanism that deregulates sphingolipids like ceramides in breast tumors, and can be explored further as a suitable therapeutic target.

Identification and molecular characterization of Bean yellow mosaic virus infecting French bean in Himachal Pradesh.

French bean (Phaseolus vulgaris L.), is one of the most widely grown vegetable crop. Disease samples showing yellow mosaic symptoms on leaves and pods were collected from Himachal Pradesh and inoculated on common bean cv. Jawala through sap inoculation. The virus successfully transmitted by mechanical inoculation produced yellow mosaic, leaf distortion, curling, wrinkling of leaves followed by stunting of plants. The identity of the virus as Bean yellow mosaic virus (BYMV) was established through Double antibody sandwich-enzyme linked immunosorbent assay, multiple sequence alignment and phylogenetic analysis of the coat protein gene sequence amplified by reverse transcription-polymerase chain reaction. The cp gene contained 819 nucleotides potentially coding for 273 amino acids. The sequence showed 83-99 % nucleotide and 89-99 % amino acid sequence identities with other BYMV isolates/strains and shared maximum identity with BYMV strain reported from Gladiolus sp. in Japan. This study constitutes the first report of BYMV occurrence on P. vulgaris in Himachal Pradesh.