Multiple Endocrine Neoplasia Type 1 Regulates TGFß-Mediated Suppression of Tumor Formation and Metastasis in Melanoma.

Over the past few decades, the worldwide incidence of cutaneous melanoma, a malignant neoplasm arising from melanocytes, has been increasing markedly, leading to the highest rate of skin cancer-related deaths. While localized tumors are easily removed by excision surgery, late-stage metastatic melanomas are refractory to treatment and exhibit a poor prognosis. Consequently, unraveling the molecular mechanisms underlying melanoma tumorigenesis and metastasis is crucial for developing novel targeted therapies. We found that the multiple endocrine neoplasia type 1 (MEN1) gene product Menin is required for the transforming growth factor beta (TGFß) signaling pathway to induce cell growth arrest and apoptosis in vitro and prevent tumorigenesis in vivo in preclinical xenograft models of melanoma. We further identified point mutations in two MEN1 family members affected by melanoma that led to proteasomal degradation of the MEN1 gene product and to a loss of TGFß signaling. Interestingly, blocking the proteasome degradation pathway using an FDA-approved drug and RNAi targeting could efficiently restore MEN1 expression and TGFß transcriptional responses. Together, these results provide new potential therapeutic strategies and patient stratification for the treatment of cutaneous melanoma.

Genome-wide in vivo CRISPR screen identifies TGFß3 as actionable biomarker of palbociclib resistance in triple negative breast cancer.

Triple negative breast cancer (TNBC) remains exceptionally challenging to treat. While CDK4/6 inhibitors have revolutionized HR + breast cancer therapy, there is limited understanding of their efficacy in TNBC and meaningful predictors of response and resistance to these drugs remain scarce. We conducted an in vivo genome-wide CRISPR screen using palbociclib as a selection pressure in TNBC. Hits were prioritized using microarray data from a large panel of breast cancer cell lines to identify top palbociclib sensitizers. Our study defines TGFß3 as an actionable determinant of palbociclib sensitivity that potentiates its anti-tumor effects. Mechanistically, we show that chronic palbociclib exposure depletes p21 levels, contributing to acquired resistance, and that TGFß3 treatment can overcome this. This study defines TGFß3 as an actionable biomarker that can be used to improve patient stratification for palbociclib treatment and exploits the synergistic interaction between CDK4/6 and TGFß3 to propose a new combinatorial treatment for TNBC.

Targeting the DYRK1A kinase prevents cancer progression and metastasis and promotes cancer cells response to G1/S targeting chemotherapy drugs.

Metastatic cancer remains incurable as patients eventually loose sensitivity to targeted therapies and chemotherapies, further leading to poor clinical outcome. Thus, there is a clear medical gap and urgent need to develop efficient and improved targeted therapies for cancer patients. In this study, we investigated the role of DYRK1A kinase in regulating cancer progression and evaluated the therapeutic potential of DYRK1A inhibition in invasive solid tumors, including colon and triple-negative breast cancers. We uncovered new roles played by the DYRK1A kinase. We found that blocking DYRK1A gene expression or pharmacological inhibition of its kinase activity via harmine efficiently blocked primary tumor formation and the metastatic tumor spread in preclinical models of breast and colon cancers. Further assessing the underlying molecular mechanisms, we found that DYRK1A inhibition resulted in increased expression of the G1/S cell cycle regulators while decreasing expression of the G2/M regulators. Combined, these effects release cancer cells from quiescence, leading to their accumulation in G1/S and further delaying/preventing their progression toward G2/M, ultimately leading to growth arrest and tumor growth inhibition. Furthermore, we show that accumulation of cancer cells in G1/S upon DYRK1A inhibition led to significant potentiation of G1/S targeting chemotherapy drug responses in vitro and in vivo. This study underscores the potential for developing novel DYRK1A-targeting therapies in colon and breast cancers and, at the same time, further defines DYRK1A pharmacological inhibition as a viable and powerful combinatorial treatment approach for improving G1/S targeting chemotherapy drugs treatments in solid tumors.

Transforming Growth Factor-ß/Smad Signaling Inhibits Melanoma Cancer Stem Cell Self-Renewal, Tumor Formation and Metastasis.

The secreted protein transforming growth factor-beta (TGFß) plays essential roles, ranging from cell growth regulation and cell differentiation in both normal and cancer cells. In melanoma, TGFß acts as a potent tumor suppressor in melanoma by blocking cell cycle progression and inducing apoptosis. In the present study, we found TGFß to regulate cancer stemness in melanoma through the Smad signaling pathway. We discovered that TGFß/Smad signaling inhibits melanosphere formation in multiple melanoma cell lines and reduces expression of the CD133+ cancer stem cell subpopulation in a Smad3-dependent manner. Using preclinical models of melanoma, we further showed that preventing Smad3/4 signaling, by means of CRISPR knockouts, promoted both tumorigenesis and lung metastasis in vivo. Collectively, our results define new functions for the TGFß/Smad signaling axis in melanoma stem-cell maintenance and open avenues for new therapeutic approaches to this disease.

Combined in vitro/in vivo genome-wide CRISPR screens in triple negative breast cancer identify cancer stemness regulators in paclitaxel resistance.

Triple negative breast cancer (TNBC) is defined as lacking the expressions of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC patients exhibit relatively poor clinical outcomes due to lack of molecular markers for targeted therapies. As such chemotherapy often remains the only systemic treatment option for these patients. While chemotherapy can initially help shrink TNBC tumor size, patients eventually develop resistance to drug, leading to tumor recurrence. We report a combined in vitro/in vivo genome-wide CRISPR synthetic lethality screening approach in a relevant TNBC cell line model to identify several targets responsible for the chemotherapy drug, paclitaxel resistance. Computational analysis integrating in vitro and in vivo data identified a set of genes, for which specific loss-of-function deletion enhanced paclitaxel resistance in TNBC. We found that several of these genes (ATP8B3, FOXR2, FRG2, HIST1H4A) act as cancer stemness negative regulators. Finally, using in vivo orthotopic transplantation TNBC models we showed that FRG2 gene deletion reduced paclitaxel efficacy and promoted tumor metastasis, while increasing FRG2 expression by means of CRISPR activation efficiently sensitized TNBC tumors to paclitaxel treatment and inhibited their metastatic abilities. In summary, the combined in vitro/in vivo genome-wide CRISPR screening approach proved effective as a tool to identify novel regulators of paclitaxel resistance/sensitivity and highlight the FRG2 gene as a potential therapeutical target overcoming paclitaxel resistance in TNBC.

In vivo genome-wide CRISPR screen reveals breast cancer vulnerabilities and synergistic mTOR/Hippo targeted combination therapy.

Triple negative breast cancer (TNBC) patients exhibit poor survival outcomes and lack effective targeted therapies. Using unbiased in vivo genome-wide CRISPR screening, we interrogated cancer vulnerabilities in TNBC and identified an interplay between oncogenic and tumor suppressor pathways. This study reveals tumor regulatory functions for essential components of the mTOR and Hippo pathways in TNBC. Using in vitro drug matrix synergy models and in vivo patient-derived xenografts, we further establish the therapeutic relevance of our findings and show that pharmacological inhibition of mTORC1/2 and oncoprotein YAP efficiently reduces tumorigenesis in TNBC. At the molecular level, we find that while verteporfin-induced YAP inhibition leads to apoptosis, torin1-mediated mTORC1/2 inhibition promotes macropinocytosis. Torin1-induced macropinocytosis further facilitates verteporfin uptake, thereby greatly enhancing its pro-apoptotic effects in cancer cells. Overall, our study underscores the power and robustness of in vivo CRISPR genome-wide screens in identifying clinically relevant and innovative therapeutic modalities in cancer.

TGFß/cyclin D1/Smad-mediated inhibition of BMP4 promotes breast cancer stem cell self-renewal activity.

Basal-like triple-negative breast cancers (TNBCs) display poor prognosis, have a high risk of tumor recurrence, and exhibit high resistance to drug treatments. The TNBC aggressive features are largely due to the high proportion of cancer stem cells present within these tumors. In this study, we investigated the interplay and networking pathways occurring between TGFß family ligands in regulating stemness in TNBCs. We found that TGFß stimulation of TNBCs resulted in enhanced tumorsphere formation efficiency and an increased proportion of the highly tumorigenic CD44high/CD24low cancer stem cell population. Analysis of the TGFß transcriptome in TNBC cells revealed bone morphogenetic protein4 (BMP4) as a main TGFß-repressed target in these tumor cells. We further found that BMP4 opposed TGFß effects on stemness and potently decreased cancer stem cell numbers, thereby acting as a differentiation factor in TNBC. At the molecular level, we found that TGFß inhibition of BMP4 gene expression is mediated through the Smad pathway and cyclin D1. In addition, we also found BMP4 to act as a pro-differentiation factor in normal mammary epithelial cells and promote mammary acinar formation in 3D cell culture assays. Finally, and consistent with our in vitro results, in silico patient data analysis defined BMP4 as a potential valuable prognosis marker for TNBC patients.

Identification of MFGE8 and KLK5/7 as mediators of breast tumorigenesis and resistance to COX-2 inhibition.

BACKGROUND: Cyclooxygenase 2 (COX-2) promotes stemness in triple negative breast cancer (TNBC), highlighting COX-2 as a promising therapeutic target in these tumors. However, to date, clinical trials using COX-2 inhibitors in breast cancer only showed variable patient responses with no clear significant clinical benefits, suggesting underlying molecular mechanisms contributing to resistance to COX-2 inhibitors. METHODS: By combining in silico analysis of human breast cancer RNA-seq data with interrogation of public patient databases and their associated transcriptomic, genomic, and clinical profiles, we identified COX-2 associated genes whose expression correlate with aggressive TNBC features and resistance to COX-2 inhibitors. We then assessed their individual contributions to TNBC metastasis and resistance to COX-2 inhibitors, using CRISPR gene knockout approaches in both in vitro and in vivo preclinical models of TNBC. RESULTS: We identified multiple COX-2 associated genes (TPM4, RGS2, LAMC2, SERPINB5, KLK7, MFGE8, KLK5, ID4, RBP1, SLC2A1) that regulate tumor lung colonization in TNBC. Furthermore, we found that silencing MFGE8 and KLK5/7 gene expression in TNBC cells markedly restored sensitivity to COX-2 selective inhibitor both in vitro and in vivo. CONCLUSIONS: Together, our study supports the establishment and use of novel COX-2 inhibitor-based combination therapies as future strategies for TNBC treatment.

Prolactin receptor-driven combined luminal and epithelial differentiation in breast cancer restricts plasticity, stemness, tumorigenesis and metastasis.

Dedifferentiation increased cellular plasticity and stemness are established derivers of tumor heterogeneity, metastasis and therapeutic failure resulting in incurable cancers. Therefore, it is essential to decipher pro/forward-differentiation mechanisms in cancer that may serve as therapeutic targets. We found that interfering with expression of the receptor for the lactogenic hormone prolactin (PRLR) in breast cancer cells representative of the luminal and epithelial breast cancer subtypes (hormone receptor positive (HR+) and HER2-enriched (HER2-E) resulted in loss of their differentiation state, enriched for stem-like cell subpopulations, and increased their tumorigenic capacity in a subtype-specific manner. Loss of PRLR expression in HR+ breast cancer cells caused their dedifferentiation generating a mesenchymal-basal-like phenotype enriched in CD44+ breast cancer stem-like cells (BCSCs) showing high tumorigenic and metastatic capacities and resistance to anti-hormonal therapy. Whereas loss of PRLR expression in HER2-E breast cancer cells resulted in loss of their luminal differentiation yet enriched for epithelial ALDH+ BCSC population showing elevated HER2-driven tumorigenic, multi-organ metastatic spread, and resistance to anti-HER2 therapy. Collectively, this study defines PRLR as a driver of precise luminal and epithelial differentiation limiting cellular plasticity, stemness, and tumorigenesis and emphasizing the function of pro/forward-differentiation pathways as a foundation for the discovery of anti-cancer therapeutic targets.

Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways.

Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo. CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors. SIGNIFICANCE: In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects.

Unprecedented tunability of riboswitch structure and regulatory function by sub-millimolar variations in physiological Mg2.

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer-ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Transforming Growth Factor-beta Regulation of Ephrin Type-A Receptor 4 Signaling in Breast Cancer Cellular Migration.

Breast cancer consists of a range of tumor subtypes with different clinical characteristics, disease prognosis, and treatment-response. Luminal breast cancer has the best prognosis while basal-like breast cancer (BLBC) represents the worst subtype. Transforming growth factor-beta (TGFß) plays a prominent role in stimulating the migration and invasion of malignant breast cancer cells contributing to tumor progression. In this study, we identified the Ephrin type-A receptor 4 (EPHA4) as a novel target of TGFß in breast cancer. Moreover, we show that TGFß induction of EPHA4 gene expression is specific to basal-like tumors and is required for TGFß-mediated cell migration. We further addressed the mechanism and found EPHA4 to be required for TGFß-mediated cell migration in breast cancer through TGFß-induced short term and long term activation of RhoGTPases. Finally, our data revealed a strong association between high EPHA4 expression and advanced tumor stage, aggressive BLBC molecular subtype and poor prognosis. Importantly, we found significant co-expression of EPHA4 and the TGFß receptor type-2 (TGFßR2) in breast cancer subtypes associated with increased tumor relapse and drug resistance. Together, this study highlight the important role of the TGFß/EPHA4 signaling axis in mediating tumor aggressiveness and poor patient survival in human breast cancer.

Single-Molecule Approaches for the Characterization of Riboswitch Folding Mechanisms.

Riboswitches are highly structured RNA molecules that control genetic expression by altering their structure as a function of metabolite binding. Accumulating evidence suggests that riboswitch structures are highly dynamic and perform conformational exchange between structural states that are important for the outcome of genetic regulation. To understand how ligand binding influences the folding of riboswitches, it is important to monitor in real time the riboswitch folding pathway as a function of experimental conditions. Single-molecule FRET (sm-FRET) is unique among biophysical techniques to study riboswitch conformational changes as it allows to both monitor steady-state populations of riboswitch conformers and associated interconversion dynamics. Since FRET fluorophores can be attached to virtually any nucleotide position, FRET assays can be adapted to monitor specific conformational changes, thus enabling to deduce complex riboswitch folding pathways. Herein, we show how to employ sm-FRET to study the folding pathway of the S-adenosylmethionine (SAM) and how this can be used to understand very specific conformational changes that are at the heart of riboswitch regulation mechanism.