Suparna Sengupta

α-Fodrin, a known scaffolding protein for cytoskeleton stabilization, performs various functions including cell adhesion, cell motility, DNA repair and apoptosis. Based on our previous results revealing its role in mitosis in glioblastoma, we have examined its effect in pancreatic cancer, which is often linked to mitotic aberrations including aneuploidy and chromosome instability. Here, we show that the expression of α-Fodrin increases in pancreatic adenocarcinoma tissues compared to its normal counterpart, suggesting its tumor promoting role. shRNA-mediated knock-down of α-Fodrin significantly reduces the xenograft growth in immunocompromised mice underscoring the importance of α-Fodrin in tumor progression. CENP-E (centromere-associated protein E) is a motor protein essential for chromosomal alignment and segregation during mitosis. We have found that α-Fodrin interacts with CENP-E to recruit it to the kinetochore and depletion of α-Fodrin has a crucial role in controlling aneuploidy. As these mitotic defects can lead to apoptosis, we have further evaluated the activation of possible upstream pathways. Paclitaxel, a chemotherapeutic agent that stabilizes microtubules, disrupts mitosis and induces apoptosis. We found that Paclitaxel triggered stronger activation of JNK, ERK, and P38 MAPKs, altered BCL2/BAX ratios, cytochrome C release causing increased apoptosis in α-Fodrin knockdown cells compared to cells with wild-type α-Fodrin. This enhanced sensitivity to paclitaxel is consistent with improved survival in pancreatic cancer patients with low α-Fodrin (SPTAN1) and low CENP-E expression compared to poor prognosis with high expressions of both the genes. Taken together, this study provides the molecular mechanism by which α-Fodrin - CENP-E axis regulates pancreatic cancer progression and drug response.

The regulation and recruitment of γ-TuRCs, the prime nucleator of microtubules, to the centrosome are still thrust areas of research. The interaction of fodrin, a sub-plasmalemmal cytoskeletal protein, with γ-tubulin is a new area of interest. To understand the cellular significance of this interaction, we show that depletion of α-fodrin brings in a significant reduction of γ-tubulin in neural cell centrosomes making it functionally under-efficient. This causes a loss of nucleation ability that cannot efficiently form microtubules in interphase cells and astral microtubules in mitosis. Fluorescence Recovery after Photobleaching (FRAP) experiment implies that α-fodrin is important in the recruitment of γ-tubulin to the centrosome resulting in the aforementioned effects. Further, our experiments indicate that the interaction of α-fodrin with certain pericentriolar matrix proteins such as Pericentrin and CDK5RAP2 are crucial for the recruitment of γ-tubulin to the centrosome. Earlier we reported that α-fodrin limits the nucleation potential of γ-TuRC. In that context, this study suggests that α-fodrin is a γ-tubulin recruiting protein to the centrosome thus preventing cytoplasmic microtubule nucleation and thereby compartmentalizing the process to the centrosome for maximum efficiency. Summary statementα-fodrin is a γ-tubulin interacting protein that controls the process of γ-tubulin recruitment to the centrosome and thereby regulates the microtubule nucleation capacity spatially and temporally.

Although the role of microtubule dynamics in cancer progression is well-established, the roles of tubulin isotypes, their cargos and their specific function in the induction and sustenance of cancer stem cells (CSCs) were poorly explored. But emerging reports urge to focus on the transport function of tubulin isotypes in defining orchestrated expression of functionally critical molecules in establishing a stem cell niche, which is the key for CSC regulation. In this review, we summarize the role of specific tubulin isotypes in the transport of functional molecules that regulate metabolic reprogramming, which leads to the induction of CSCs and immune evasion. Recently, the surface expression of GLUT1 and GRP78 as well as voltage-dependent anion channel (VDAC) permeability, regulated by specific isotypes of β-tubulins have been shown to impart CSC properties to cancer cells, by implementing a metabolic reprogramming. Moreover, βIVb tubulin is shown to be critical in modulating EphrinB1signaling to sustain CSCs in oral carcinoma. These tubulin-interacting molecules, Ephrins, GLUT1 and GRP78, are also important regulators of immune evasion, by evoking PD-L1 mediated T-cell suppression. Thus, the recent advances in the field implicate that tubulins play a role in the controlled transport of molecules involved in CSC niche. The indication of tubulin isotypes in the regulation of CSCs offers a strategy to specifically target those tubulin isotypes to eliminate CSCs, rather than the general inhibition of microtubules, which usually leads to therapy resistance.

Exosomes are cell-secreted vesicles secreted by a majority of cells and, hence, populating most of the biological fluids, namely blood, tears, sweat, swab, urine, breast milk, etc. They vary vastly in size and density and are influenced by age, gender and diseases. The composition of exosomes includes lipids, DNA, proteins, and coding and noncoding RNA. There is a significant interest in selectively isolating small exosomes (≤50 nm) from human serum to investigate their role in different diseases and regeneration. However, current techniques for small exosome isolation/purification are time-consuming and highly instrument-dependent, with limited specificity and recovery. Thus, rapid and efficient methods to isolate them from bio fluids are strongly needed for both basic research and clinical applications. In the present work, we explored the application of a bench-top centrifuge for isolating mostly the small exosomes (≤50 nm). This can be achieved at low g-force by adding additional weight to the exosomes by conjugating them with citrate-capped gold nanoparticles (CGNP). CGNPs were functionalized with polyethylene glycol (PEG) to form PEGylated GNP (PGNP). EDC/SNHS chemistry is used to activate the –COOH group of the PEG to make it suitable for conjugation with antibodies corresponding to exosomal surface proteins. These antibody-conjugated PGNPs were incubated with the serum to form PGNP-exosome complexes which were separated directly by centrifugation at a low g-force of 7000× g. This makes this technique efficient compared to that of standard ultracentrifugation exosome isolation (which uses approximately 100,000× g). Using the technique, the exosome isolation from serum was achieved successfully in less than two hours. The purification of small exosomes, characterized by the presence of CD63, CD9 and CD81, and sized between 20 nm to 50 nm, was confirmed by western blot, dynamic light scattering (DLS), transmission electron microscopy (TEM) and nanoparticle tracking analyser (NTA).

Recent advancements in cancer research have shown that cancer stem cell (CSC) niche is a crucial factor modulating tumor progression and treatment outcomes. It sustains CSCs by orchestrated regulation of several cytokines, growth factors, and signaling pathways. Although the features defining adult stem cell niches are well-explored, the CSC niche is poorly characterized. Since membrane trafficking proteins have been shown to be essential for the localization of critical proteins supporting CSCs, we investigated the role of TUBB4B, a probable membrane trafficking protein that was found to be overexpressed in the membranes of stem cell enriched cultures, in sustaining CSCs in oral cancer. Here, we show that the knockdown of TUBB4B downregulates the expression of pluripotency markers, depletes ALDH1A1⁺ population, decreases in vitro sphere formation, and diminishes the tumor initiation potential in vivo. As TUBB4B is not known to have any role in transcriptional regulation nor cell signaling, we suspected that its membrane trafficking function plays a role in constituting a CSC niche. The pattern of its expression in tissue sections, forming a gradient in and around the CSCs, reinforced the notion. Later, we explored its possible cooperation with a signaling protein, Ephrin-B1, the abrogation of which reduces the self-renewal of oral cancer stem cells. Expression and survival analyses based on the TCGA dataset of head and neck squamous cell carcinoma (HNSCC) samples indicated that the functional cooperation of TUBB4 and EFNB1 results in a poor prognosis. We also show that TUBB4B and Ephrin-B1 cohabit in the CSC niche. Moreover, depletion of TUBB4B downregulates the membrane expression of Ephrin-B1 and reduces the CSC population. Our results imply that the dynamics of TUBB4B is decisive for the surface localization of proteins, like Ephrin-B1, that sustain CSCs by their concerted signaling.

Cortical cytoskeletal proteins are significant in controlling various cellular mechanisms such as migration, cell adhesion, intercellular attachment, cellular signaling, exo- and endocytosis and plasma membrane integrity, stability and flexibility. Our earlier studies involving in vitro and ex vivo approaches led us to identify certain undiscovered characteristics of α-fodrin, a prominent cortical protein. The conventional functions attributed to this protein mainly support the plasma membrane. In the present study, we utilized a global protein expression analysis approach to detect underexplored functions of this protein. We report that downregulation of α-fodrin in glioblastoma cells, U-251 MG, results in upregulation of genes affecting the regulation of the cytoskeleton, cell cycle and apoptosis. Interestingly, certain key microtubule kinesins such as KIF23, KIF2B and KIF3C are downregulated upon α-fodrin depletion, as validated by real-time PCR studies.

Fodrin and its erythroid-specific isoform spectrin are actin-associated fibrous proteins that play crucial roles in the maintenance of structural integrity in mammalian cells, which is necessary for proper cell function. Normal cell morphology is altered in diseases, like various cancers and certain neuronal disorders. Fodrin and spectrin are two-chain (αβ) molecules that are encoded by paralogous genes and share many common features, but also demonstrate certain differences. Fodrin (in humans, typically, a heterodimer of the products of the SPTAN1 and SPTBN1 genes) is expressed in nearly all cell types, and is especially abundant in neuronal tissues, whereas spectrin (in humans, a heterodimer of the products of the SPTA1 and SPTB1 genes) is expressed almost exclusively in erythrocytes. To fulfill a role in such a variety of different cell types, it was anticipated that fodrin would need to be a more versatile scaffold than spectrin. Indeed, as summarized here, domains unique to fodrin and its regulation by Ca²⁺, calmoduln, and a variety of post-translational modifications (PTMs) endow fodrin with additional specific functions. However, how fodrin structural variations and misregulated PTMs may contribute to the etiology of various cancers and neurodegenerative diseases needs to be further investigated.

The cytoskeleton protein α-fodrin plays a major role in maintaining structural stability of membranes. It was also identified as part of the brain γ-tubulin ring complex, the major microtubule nucleator. Here, we investigated the requirement of α-fodrin for microtubule spindle assembly during mitotic progression. We found that α-fodrin depletion results in abnormal mitosis with uncongressed chromosomes, leading to prolonged activation of the spindle assembly checkpoint and a severe mitotic delay. Further, α-fodrin repression led to the formation of shortened spindles with unstable kinetochore-microtubule attachments. We also found that the mitotic kinesin CENP-E had reduced levels at kinetochores to likely account for the chromosome misalignment defects in α-fodrin-depleted cells. Importantly, we showed these cells to exhibit reduced levels of detyrosinated α-tubulin, which primarily drives CENP-E localization. Since proper microtubule dynamics and chromosome alignment are required for completion of normal mitosis, this study reveals an unforeseen role of α-fodrin in regulating mitotic progression. Future studies on these lines of observations should reveal important mechanistic insight for fodrin’s involvement in cancer. Graphical abstract

Non‐erythroid spectrin or fodrin is present as part of the γ‐tubulin ring complex (γ‐TuRC) in brain tissue and brain derived cells. Here, we show that fodrin, which is otherwise known for providing structural support to the cell membrane, interacts directly with γ‐tubulin within the γ‐TuRC through a GRIP2‐like motif. Turbidometric analysis of microtubule polymerization with nucleation‐potent γ‐TuRC isolated from HEK‐293 cells that lack fodrin and the γ‐TuRC from goat brain that contains fodrin shows inefficiency of the latter to promote nucleation. The involvement of fodrin was confirmed by the reduction in the microtubule polymerization efficiency of HEK‐293 derived γ‐TuRCs upon addition of purified brain fodrin. Thus, the interaction of fodrin with gamma‐tubulin is responsible for its inhibitory effect on γ‐tubulin mediated microtubule nucleation.

P53 is a tumour suppressor protein that plays a key role in many steps of apoptosis, and malfunctioning of this transcription factor leads to tumorigenesis. Prognosis of many tumours also depends upon the p53 status. Most of the clinically used anticancer compounds activate p53 dependent pathway of apoptosis and hence require p53 for their mechanism of action. Further, Ras/Raf/MEK/ERK axis is an important signaling pathway activated in many cancers. Dependence of diaminothiazoles, compounds that have gained importance recently due to their anticancer and anti angiogenic activities, were tested in cancer models with varying p53 or Ras/Raf mutational status. In this study we have used p53 mutated and knock out colon cancer cells and xenograft tumours to study the role of p53 in apoptosis mediated by diaminothiazoles. Colon cancer cell lines with varying mutational status for Ras or Raf were also used. We have also examined the toxicity and in vivo efficacy of a lead diaminothiazole 4-Amino-5-benzoyl-2-(4-methoxy phenylamino)thiazole (DAT1) in colon cancer xenografts. We have found that DAT1 is active in both in vitro and in vivo models with nonfunctional p53. Earlier studies have shown that extrinsic pathway plays major role in DAT1 mediated apoptosis. In this study, we have found that DAT1 is causing p53 independent upregulation of the death receptor 5 by activating the Ras/Raf/MEK/ERK signaling pathway both in wild type and p53 suppressed colon cancer cells. These findings are also confirmed by the in vivo results. Further, DAT1 is more efficient to induce apoptosis in colon cancer cells with mutated Ras or Raf. Minimal toxicity in both acute and subacute studies along with the in vitro and in vivo efficacy of DAT1 in cancers with both wild type and nonfunctional p53 place it as a highly beneficial candidate for cancer chemotherapy. Besides, efficiency in cancer cells with mutations in the Ras oncoprotein or its downstream kinase Raf raise interest in diaminothiazole class of compounds for further follow-up.