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.

Bile Acid Tethered Docetaxel-Based Nanomicelles Mitigate Tumor Progression through Epigenetic Changes.

In this study, we describe the engineering of sub-100 nm nanomicelles (DTX-PC NMs) derived from phosphocholine derivative of docetaxel (DTX)-conjugated lithocholic acid (DTX-PC) and poly(ethylene glycol)-tethered lithocholic acid. Administration of DTX-PC NMs decelerate tumor progression and increase the mice survivability compared to Taxotere (DTX-TS), the FDA-approved formulation of DTX. Unlike DTX-TS, DTX-PC NMs do not cause any systemic toxicity and slow the decay rate of plasma DTX concentration in rodents and non-rodent species including non-human primates. We further demonstrate that DTX-PC NMs target demethylation of CpG islands of Sparcl1 (a tumor suppressor gene) by suppressing DNA methyltransferase activity and increase the expression of Sparcl1 that leads to tumor regression. Therefore, this unique system has the potential to improve the quality of life in cancer patients and can be translated as a next-generation chemotherapeutic.

Tethering of Chemotherapeutic Drug/Imaging Agent to Bile Acid-Phospholipid Increases the Efficacy and Bioavailability with Reduced Hepatotoxicity.

Weakly basic drugs display poor solubility and tend to precipitate in the stomach's acidic environment causing reduced oral bioavailability. Tracing of these orally delivered therapeutic agents using molecular probes is challenged due to their poor absorption in the gastrointestinal tract (GIT). Therefore, we designed a gastric pH stable bile acid derived amphiphile where Tamoxifen (as a model anticancer drug) is conjugated to lithocholic acid derived phospholipid (LCA-Tam-PC). In vitro studies suggested the selective nature of LCA-Tam-PC for cancer cells over normal cells as compared to the parent drug. Fluorescent labeled version of the conjugate (LCA-Tam-NBD-PC) displayed an increased intracellular uptake compared to Tamoxifen. We then investigated the antitumor potential, toxicity, and median survival in 4T1 tumor bearing BALB/c mice upon LCA-Tam-PC treatment. Our studies confirmed a significant reduction in the tumor volume, tumor weight, and reduced hepatotoxicity with a significant increase in median survival on LCA-Tam-PC treatment as compared to the parent drug. Pharmacokinetic and biodistribution studies using LCA-Tam-NBD-PC witnessed the enhanced gut absorption, blood circulation, and tumor site accumulation of phospholipid-drug conjugate leading to improved antitumor activity. Therefore, our studies revealed that conjugation of chemotherapeutic/imaging agents to bile acid phospholipid can provide a new platform for oral delivery and tracing of chemotherapeutic drugs.

Insights into molecular mechanisms of chemotherapy resistance in cancer.

Cancer heterogeneity poses a significant hurdle to the successful treatment of the disease, and is being influenced by genetic inheritance, cellular and tissue biology, disease development, and response to therapy. While chemotherapeutic drugs have demonstrated effectiveness, their efficacy is impeded by challenges such as presence of resilient cancer stem cells, absence of specific biomarkers, and development of drug resistance. Often chemotherapy leads to a myriad of epigenetic, transcriptional and post-transcriptional alterations in gene expression as well as changes in protein expression, thereby leading to massive metabolic reprogramming. This review seeks to provide a detailed account of various transcriptional regulations, proteomic changes, and metabolic reprogramming in various cancer models in response to three primary chemotherapeutic interventions, docetaxel, carboplatin, and doxorubicin. Discussing the molecular targets of some of these regulatory events and highlighting their contribution in sensitivity to chemotherapy will provide insights into drug resistance mechanisms and uncover novel perspectives in cancer treatment.

Engineered nanomicelles inhibit the tumour progression via abrogating the prostaglandin-mediated immunosuppression.

Cancer treatment is challenged due to immunosuppressive inflammatory tumour microenvironment (TME) caused by infiltration of tumour-promoting and inhibition of tumour-inhibiting immune cells. Here, we report the engineering of chimeric nanomicelles (NMs) targeting the cell proliferation using docetaxel (DTX) and inflammation using dexamethasone (DEX) that alters the immunosuppressive TME. We show that a combination of phospholipid-DTX conjugate and PEGylated-lipid-DEX conjugate can self-assemble to form sub-100 nm chimeric NMs (DTX-DEX NMs). Anti-cancer activities against syngeneic and xenograft mouse models showed that the DTX-DEX NMs are more effective in tumour regression, enhance the survival of mice over other treatment modes, and alter the tumour stroma. DTX-DEX NMs cause a significant reduction in myeloid-derived suppressor cells, alter the polarization of macrophages, and enhance the accumulation of cytotoxic CD4+ and CD8+ T cells in tumour tissues, along with alterations in cytokine expression. We further demonstrated that these DTX-DEX NMs inhibit the synthesis of prostaglandins, especially PGE2, by targeting the cyclooxygenase 2 that is partly responsible for immunosuppressive TME. Therefore, this study presents, for the first time, the engineering of lithocholic acid-derived chimeric NMs that affect the prostaglandin pathway, alter the TME, and mitigate tumour progression with enhanced mice survival.

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.

Hypercalcaemic encephalopathy as a presenting manifestation of sarcoidosis.

A 66-year-old woman presented to us with features of encephalopathy with asterixis, preceded by unsteadiness of gait and behavioural abnormalities. On subsequent investigations, hypercalcaemic crisis and compromised renal function were noted. Stepwise approach to determine the cause behind hypercalcaemia with compromised renal function revealed underlying granulomatous disease (sarcoidosis). Later, development of maculopapular rash and subsequent biopsy from the lesion confirmed the diagnosis of sarcoidosis. Her clinical and biochemical parameters improved considerably on initiation of conservative pharmacological therapy.

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.

A Localized Chimeric Hydrogel Therapy Combats Tumor Progression through Alteration of Sphingolipid Metabolism.

Rapid proliferation of cancer cells assisted by endothelial cell-mediated angiogenesis and acquired inflammation at the tumor microenvironment (TME) lowers the success rate of chemotherapeutic regimens. Therefore, targeting these processes using localized delivery of a minimally toxic drug combination may be a promising strategy. Here, we present engineering of a biocompatible self-assembled lithocholic acid-dipeptide derived hydrogel (TRI-Gel) that can maintain sustained delivery of antiproliferating doxorubicin, antiangiogenic combretastatin-A4 and anti-inflammatory dexamethasone. Application of TRI-Gel therapy to a murine tumor model promotes enhanced apoptosis with a concurrent reduction in angiogenesis and inflammation, leading to effective abrogation of tumor proliferation and increased median survival with reduced drug resistance. In-depth RNA-sequencing analysis showed that TRI-Gel therapy induced transcriptome-wide alternative splicing of many genes responsible for oncogenic transformation including sphingolipid genes. We demonstrate that TRI-Gel therapy targets the reversal of a unique intron retention event in ß-glucocerebrosidase 1 (Gba1), thereby increasing the availability of functional Gba1 protein. An enhanced Gba1 activity elevates ceramide levels responsible for apoptosis and decreases glucosylceramides to overcome drug resistance. Therefore, TRI-Gel therapy provides a unique system that affects the TME via post-transcriptional modulations of sphingolipid metabolic genes, thereby opening a new and rational approach to cancer therapy.

Injectable, Self-Healing Chimeric Catechol-Fe(III) Hydrogel for Localized Combination Cancer Therapy.

Conventional intravenous or oral administration of a combination of chemotherapeutics displays poor bioavailability and induces undesirable systemic toxicity. Therefore, localized delivery of such chemotherapeutic combinations using polymeric hydrogels is expected to help in enhancing drug efficacy and reducing systemic toxicity. In this manuscript, we have utilized a chitosan-catechol based hydrogel (CAT-Gel) assembled through catechol-Fe(III) coordinative interactions for localized combination therapy in murine lung and breast cancer models. CAT-Gel offers a unique blend of material properties such as injectability and self-healing along with useful biological attributes like their noncytotoxic and nonhemolytic nature. The amphipathic nature of this hydrogel enabled us to incorporate a recipe of hydrophilic doxorubicin hydrochloride (DOX) and hydrophobic docetaxel (DTX) anticancer drugs. Rheology studies confirmed the self-healing nature of this chimeric hydrogel even after drug loading. CAT-Gel was retained for more than 40 days in mice upon subcutaneous injection. The sequential and sustained release of the entrapped DOX and DTX from the hydrogel resulted in synergistic therapeutic effect with increased median survival against murine lung and breast cancer models. Therefore, CAT-Gel provides a new coordinatively assembled biocompatible scaffold for localized delivery of chemotherapeutic drugs.