Creeping yeast: a simple, cheap and robust protocol for the identification of mating type in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an exceptional genetic system, with genetic crosses facilitated by its ability to be maintained in haploid and diploid forms. Such crosses are straightforward if the mating type/ploidy of the strains is known. Several techniques can determine mating type (or ploidy), but all have limitations. Here, we validate a simple, cheap and robust method to identify S. cerevisiae mating types. When cells of opposite mating type are mixed in liquid media, they 'creep' up the culture vessel sides, a phenotype that can be easily detected visually. In contrast, mixtures of the same mating type or with a diploid simply settle out. The phenotype is observable for several days under a range of routine growth conditions and with different media/strains. Microscopy suggests that cell aggregation during mating is responsible for the phenotype. Yeast knockout collection analysis identified 107 genes required for the creeping phenotype, with these being enriched for mating-specific genes. Surprisingly, the RIM101 signaling pathway was strongly represented. We propose that RIM101 signaling regulates aggregation as part of a wider, previously unrecognized role in mating. The simplicity and robustness of this method make it ideal for routine verification of S. cerevisiae mating type, with future studies required to verify its molecular basis.

Correction: Loss of tuberous sclerosis complex 2 sensitizes tumors to nelfinavir-bortezomib therapy to intensify endoplasmic reticulum stress-induced cell death.

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Energy Stress-Mediated Cytotoxicity in Tuberous Sclerosis Complex 2-Deficient Cells with Nelfinavir and Mefloquine Treatment.

To find new anti-cancer drug therapies, we wanted to exploit homeostatic vulnerabilities within Tuberous Sclerosis Complex 2 (TSC2)-deficient cells with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivity. We show that nelfinavir and mefloquine synergize to selectively evoke a cytotoxic response in TSC2-deficient cell lines with mTORC1 hyperactivity. We optimize the concentrations of nelfinavir and mefloquine to a clinically viable range that kill cells that lack TSC2, while wild-type cells tolerate treatment. This new clinically viable drug combination causes a significant level of cell death in TSC2-deficient tumor spheroids. Furthermore, no cell recovery was apparent after drug withdrawal, revealing potent cytotoxicity. Transcriptional profiling by RNA sequencing of drug treated TSC2-deficient cells compared to wild-type cells suggested the cytotoxic mechanism of action, involving initial ER stress and an imbalance in energy homeostatic pathways. Further characterization revealed that supplementation with methyl pyruvate alleviated energy stress and reduced the cytotoxic effect, implicating energy deprivation as the trigger of cell death. This work underpins a critical vulnerability with cancer cells with aberrant signaling through the TSC2-mTORC1 pathway that lack flexibility in homeostatic pathways, which could be exploited with combined nelfinavir and mefloquine treatment.

Loss of tuberous sclerosis complex 2 sensitizes tumors to nelfinavir-bortezomib therapy to intensify endoplasmic reticulum stress-induced cell death.

Cancer cells lose homeostatic flexibility because of mutations and dysregulated signaling pathways involved in maintaining homeostasis. Tuberous Sclerosis Complex 1 (TSC1) and TSC2 play a fundamental role in cell homeostasis, where signal transduction through TSC1/TSC2 is often compromised in cancer, leading to aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 hyperactivation increases the basal level of endoplasmic reticulum (ER) stress via an accumulation of unfolded protein, due to heightened de novo protein translation and repression of autophagy. We exploit this intrinsic vulnerability of tumor cells lacking TSC2, by treating with nelvinavir to further enhance ER stress while inhibiting the proteasome with bortezomib to prevent effective protein removal. We show that TSC2-deficient cells are highly dependent on the proteosomal degradation pathway for survival. Combined treatment with nelfinavir and bortezomib at clinically relevant drug concentrations show synergy in selectively killing TSC2-deficient cells with limited toxicity in control cells. This drug combination inhibited tumor formation in xenograft mouse models and patient-derived cell models of TSC and caused tumor spheroid death in 3D culture. Importantly, 3D culture assays differentiated between the cytostatic effects of the mTORC1 inhibitor, rapamycin, and the cytotoxic effects of the nelfinavir/bortezomib combination. Through RNA sequencing, we determined that nelfinavir and bortezomib tip the balance of ER protein homeostasis of the already ER-stressed TSC2-deficient cells in favor of cell death. These findings have clinical relevance in stratified medicine to treat tumors that have compromised signaling through TSC and are inflexible in their capacity to restore ER homeostasis.

Exploiting cancer vulnerabilities: mTOR, autophagy, and homeostatic imbalance.

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) at lysosomes plays a pivotal role in cell growth control where an array of large multiprotein complexes relay nutrient, energy, and growth signal inputs through mTORC1. In cancer cells, such regulation often becomes disconnected, leading to uncontrolled cell growth and an elevation in cellular stress. Consequently, cancer cells often lose homeostatic balance as they grow in unfavorable conditions, i.e. when nutrients and energy are limited yet mTORC1 is still aberrantly activated. Cancer cells lose signaling flexibility because of hyperactive mTORC1 that leads to heightened cellular stress and loss of nutrient and energy homeostasis, all of which are potential avenues for cancer therapy. Cancer cells often enhance mTORC1 to drive cell growth and proliferation, while also maintaining their survival. Autophagy regulation by mTORC1 is critically involved in nutrient and energy homeostasis, cell growth control, and survival. Studying mTORC1 and autophagy as a potential therapeutic target for cancer treatment has been the focus of a wide range of research over the past few decades. This review will explore the signaling pathways central to mTORC1 and autophagy regulation, and cancer vulnerabilities while considering anticancer therapies.

Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively induces death of mTORC1 hyperactive cells.

Uncontrolled cell growth in Tuberous Sclerosis Complex occurs due to inappropriate activation of mechanistic (mammalian) target of rapamycin complex 1 (mTORC1). The current therapy, rapamycin, produced promising clinical trial results, but patient tumours regrow if treatment is discontinued, revealing rapamycin has cytostatic properties rather than a cytotoxic effect. Taking advantage of the enhanced levels of endoplasmic reticulum (ER) stress present in TSC2-null cells, we investigated drug combinations producing a cytotoxic response. We found a nelfinavir and salinomycin combination specifically killed TSC2-deficient, mTORC1 hyperactive cells. Cytotoxicity was rescued by reducing protein synthesis, either through mTORC1 inhibition or cycloheximide treatment. This indicates that the drug combination targets the cells by tipping the protein homeostasis balance of the already metabolically stressed TSC2-deficient cells in favour of cell death. Furthermore, this drug combination also inhibited tumour formation in TSC2-deficient cell models and caused tumour spheroid death in 3D culture. Importantly, the 3D assay could differentiate the cytostatic agent, rapamycin, from the cytotoxic nelfinavir/salinomycin combination. Sporadic cancer cell lines with hyperactive mTORC1 signalling were also susceptible to this nelfinavir/salinomycin drug combination. This work indicates that the protein homeostasis pathway is an attractive therapeutic target in both Tuberous Sclerosis Complex and mTORC1-driven sporadic cancers.

Endoplasmic reticulum stress and cell death in mTORC1-overactive cells is induced by nelfinavir and enhanced by chloroquine.

Inappropriate activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is common in cancer and has many cellular consequences including elevated endoplasmic reticulum (ER) stress. Cells employ autophagy as a critical compensatory survival mechanism during ER stress. This study utilised drug-induced ER stress through nelfinavir in order to examine ER stress tolerance in cell lines with hyper-active mTORC1 signalling. Our initial findings in wild type cells showed nelfinavir inhibited mTORC1 signalling and upregulated autophagy, as determined by decreased rpS6 and S6K1 phosphorylation, and SQTSM1 protein expression, respectively. Contrastingly, cells with hyper-active mTORC1 displayed basally elevated levels of ER stress which was greatly exaggerated following nelfinavir treatment, seen through increased CHOP mRNA and XBP1 splicing. To further enhance the effects of nelfinavir, we introduced chloroquine as an autophagy inhibitor. Combination of nelfinavir and chloroquine significantly increased ER stress and caused selective cell death in multiple cell line models with hyper-active mTORC1, whilst control cells with normalised mTORC1 signalling tolerated treatment. By comparing chloroquine to other autophagy inhibitors, we uncovered that selective toxicity invoked by chloroquine was independent of autophagy inhibition yet entrapment of chloroquine to acidified lysosomal/endosomal compartments was necessary for cytotoxicity. Our research demonstrates that combination of nelfinavir and chloroquine has therapeutic potential for treatment of mTORC1-driven tumours.