Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

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.

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.

Concomitant Expression of Prolactin Receptor and TGFß Receptors in Breast Cancer: Association with Less Aggressive Phenotype and Favorable Patient Outcome.

The epithelial-mesenchymal transition (EMT) process is known to play an essential role in tumor progression, metastasis and resistance to therapy. This report evaluated the prognostic value of co-expression of the receptor for prolactin (PRLR), a suppressor of EMT, and the receptors for transforming growth factor ß (TGFßRI and TGFßRII), an inducer of EMT, in association with different clinicopathological parameters using TMA of 102 breast cancer patients and publicly available data on breast cancer patients. Interestingly, the results revealed that malignant tissues had significantly lower levels of concomitant protein expression of these receptors in comparison to normal/benign breast tissue. In addition, a higher level of concomitant expression was also observed in less aggressive breast cancer phenotypes, including low grade tumors, luminal breast cancer subtype, and less advanced stages of the disease (lymph node negative and early stages). Moreover, the results also showed that the expression of a gene signature composed of PRLR/TGFßRI/TGFßRII correlates more with differentiated grade I tumors, and identified a subset of patients showing better survival outcomes evident in luminal B and HER-2 enriched molecular subtypes. Together, these results indicate that loss of the co-expression of PRLR, TGFßRI and TGFßRII is indicative of aggressiveness and poor patient survival outcomes in breast cancer.

Dasatinib sensitises triple negative breast cancer cells to chemotherapy by targeting breast cancer stem cells.

BACKGROUND: Patients with triple negative breast cancer (TNBC) exhibit poor prognosis and are at high risk of tumour relapse, due to the resistance to chemotherapy. These aggressive phenotypes are in part attributed to the presence of breast cancer stem cells (BCSCs). Therefore, targeting BCSCs is a priority to overcoming chemotherapy failure in TNBCs. METHODS: We generated paclitaxel (pac)-resistant TNBC cells which displayed higher sphere forming potential and percentage of BCSC subpopulations compared to the parental cells. A screen with various kinase inhibitors revealed dasatinib, a Src kinase family inhibitor, as a potent suppressor of BCSC expansion/sphere formation in pac-resistant TNBC cells. RESULTS: We found dasatinib to block pac-induced BCSC enrichment and Src activation in both parental and pac-resistant TNBC cells. Interestingly, dasatinib induced an epithelial differentiation of the pac-resistant mesenchymal cells, resulting in their enhanced sensitivity to paclitaxel. The combination treatment of dasatinib and paclitaxel not only decreased the BCSCs numbers and their sphere forming capacity but also synergistically reduced cell viability of pac-resistant cells. Preclinical models of breast cancer further demonstrated the efficiency of the dasatinib/paclitaxel combination treatment in inhibiting tumour growth. CONCLUSIONS: Dasatinib is a promising anti-BCSC drug that could be used in combination with paclitaxel to overcome chemoresistance in TNBC.

Differential expression of TGFß isoforms in breast cancer highlights different roles during breast cancer progression.

While TGFß plays a critical role in tumor formation and progression, the role and contribution of its three different isoforms remain unclear. In this study, we aimed at elucidating the prognostic value of the TGFß isoforms and assessed their expression levels in breast cancer patients at different stages of the disease. We found higher levels of TGFß1 and TGFß3 in cancer patients compared to normal tissues, with no significant changes in TGFß2 expression. Similarly, TGFß1 and TGFß3, but not TGFß2, showed higher expression levels in advanced lymph node-positive and metastatic tumors, suggesting different roles for the different isoforms in tumor progression and the metastatic process, while in the least aggressive molecular subtype (luminal A), expression of the three TGFß isoforms significantly correlated with expression of both TGFß receptors, such correlation only occurred between TGFß1 and TGFß3 and the TGFß type II receptor (TßRII) in the highly aggressive basal-like subtype. Interestingly, a distinct and somehow opposite pattern was observed in HER-2 tumors, only showing significant association pattern between TGFß2 and the TGFß type I receptor (TßRI). Finally, the three TGFß isoforms showed distinct association patterns with patient outcome depending on the different molecular subtype, highlighting context-dependent, differential prognostic values.

The Retinoblastoma Tumor Suppressor Protein (pRb)/E2 Promoter Binding Factor 1 (E2F1) Pathway as a Novel Mediator of TGFß-induced Autophagy.

TGFß is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Although autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFß signaling and autophagy is context-dependent and remains unclear. TGFß-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFß in hepatocarcinoma cells. In the present study, we define a novel process of TGFß-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFß is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFß tumor suppressive effects. We further determined that TGFß induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFß induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes and further highlight the central relevance of the pRb/E2F1 pathway downstream of TGFß signaling in tumor suppression.

The leukemia inhibitory factor (LIF) and p21 mediate the TGFß tumor suppressive effects in human cutaneous melanoma.

BACKGROUND: Cutaneous melanoma is the most lethal skin cancer and its incidence in developed countries has dramatically increased over the past decades. Localized tumors are easily treated by surgery, but advanced melanomas lack efficient treatment and are associated with very poor outcomes. Thus, understanding the processes underlying melanoma development and progression is critical. The Transforming Growth Factor beta (TGFß) acts as a potent tumor suppressor in human melanoma, by inhibiting cell growth and preventing cellular migration and invasion. METHODS: In this study, we aimed at elucidating the molecular mechanisms underlying TGFß-mediated tumor suppression. Human cutaneous melanoma cell lines, derived from different patients, were used to assess for cell cycle analysis, apoptosis/caspase activity and cell migration. Techniques involved immunoblotting, immunohistochemistry, real time PCR and luciferase reporter assays. RESULTS: We found the leukemia inhibitory factor (LIF) to be strongly up-regulated by TGFß in melanoma cells, defining LIF as a novel TGFß downstream target gene in cutaneous melanoma. Interestingly, we also showed that TGFß-mediated LIF expression is required for TGFß-induced cell cycle arrest and caspase-mediated apoptosis, as well as for TGFß-mediated inhibition of cell migration. Moreover, we found that TGFß-mediated LIF expression leads to activation of transcription of the cell cycle inhibitor p21 in a STAT3-dependent manner, and further showed that p21 is required for TGFß/LIF-mediated cell cycle arrest and TGFß-induced gene activation of several pro-apoptotic genes. CONCLUSIONS: Together, our results define the LIF/p21 signaling cascade as a novel tumor suppressive-like pathway in melanoma, acting downstream of TGFß to regulate cell cycle arrest and cell death, further highlight new potential therapeutic strategies for the treatment of cutaneous melanoma.

MicroRNA-584 and the protein phosphatase and actin regulator 1 (PHACTR1), a new signaling route through which transforming growth factor-ß Mediates the migration and actin dynamics of breast cancer cells.

TGF-ß plays an important role in breast cancer progression as a prometastatic factor, notably through enhancement of cell migration. It is becoming clear that microRNAs, a new class of small regulatory molecules, also play crucial roles in mediating tumor formation and progression. We found TGF-ß to down-regulate the expression of the microRNA miR-584 in breast cancer cells. Furthermore, we identified PHACTR1, an actin-binding protein, to be positively regulated by TGF-ß in a miR-584-dependent manner. Moreover, we found TGF-ß-mediated down-regulation of miR-584 and increased expression of PHACTR1 to be required for TGF-ß-induced cell migration of breast cancer cells. Indeed, both overexpression of miR-584 and knockdown of PHACTR1 resulted in a drastic reorganization of the actin cytoskeleton and reduced TGF-ß-induced cell migration. Our data highlight a novel signaling route whereby TGF-ß silences the expression of miR-584, resulting in enhanced PHACTR1 expression, and further leading to actin rearrangement and breast cancer cell migration.

Breast cancer anti-estrogen resistance 3 inhibits transforming growth factor ß/Smad signaling and associates with favorable breast cancer disease outcomes.

INTRODUCTION: This study helps to define the implications of breast cancer anti-estrogen resistance 3 (BCAR3) in breast cancer and extends the current understanding of its molecular mechanism of action. BCAR3 has been shown to promote cell proliferation, migration and attachment to extracellular matrix components. However, in a cohort of metastatic breast cancer patients who received tamoxifen treatment, high BCAR3 mRNA levels were associated with favorable progression-free survival outcome. These results suggest that, besides its established roles, BCAR3 may have additional mechanisms of action that regulate breast cancer aggressive phenotype. In this study, we investigated whether BCAR3 is a novel antagonist of the canonical transforming growth factor ß (TGFß) pathway, which induces potent migration and invasion responses in breast cancer cells. METHODS: We surveyed functional genomics databases for correlations between BCAR3 expression and disease outcomes of breast cancer patients. We also studied how BCAR3 could regulate the TGFß/Smad signaling axis using Western blot analysis, coimmunoprecipitation and luciferase assays. In addition, we examined whether BCAR3 could modulate TGFß-induced cell migration and invasion by using an automated imaging system and a confocal microscopy imaging-based matrix degradation assay, respectively. RESULTS: Relatively low levels of BCAR3 expression in primary breast tumors correlate with poor distant metastasis-free survival and relapse-free survival outcomes. We also found a strong correlation between the loss of heterozygosity at BCAR3 gene alleles and lymph node invasion in human breast cancer, further suggesting a role for BCAR3 in preventing disease progression. In addition, we found BCAR3 to inhibit Smad activation, Smad-mediated gene transcription, Smad-dependent cell migration and matrix digestion in breast cancer cells. Furthermore, we found BCAR3 to be downregulated by TGFß through proteasome degradation, thus defining a novel positive feedback loop mechanism downstream of the TGFß/Smad signaling pathway. CONCLUSION: BCAR3 is considered to be associated with aggressive breast cancer phenotypes. However, our results indicate that BCAR3 acts as a putative suppressor of breast cancer progression by inhibiting the prometastatic TGFß/Smad signaling pathway in invasive breast tumors. These data provide new insights into BCAR3's molecular mechanism of action and highlight BCAR3 as a novel TGFß/Smad antagonist in breast cancer.

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.

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.

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.

Engulfment protein GULP is regulator of transforming growth factor-ß response in ovarian cells.

Transforming growth factor ß (TGF-ß) is a key regulatory molecule with pleiotropic effects on cell growth, migration, and invasion. As a result, impairment of proper TGF-ß signaling is central to tumorigenesis and metastasis. The TGF-ß receptor V (TGFBRV or LRP1) has been shown to be responsible for TGF-ß-mediated cell growth inhibition in Chinese hamster ovary (CHO) cells. The LRP1 adapter protein GULP mediates internalization of the various LRP1-specific ligands, and we hypothesize that GULP acts as a novel regulator of TGF-ß signaling in ovarian cells. CHO cells that overexpress exogenous GULP (FL) demonstrate enhancement in growth inhibition, migration, and invasion from TGF-ß treatment, whereas cells that lack GULP (AS) show impairment of growth inhibition and decreased migration and invasion. The enhanced TGF-ß response in FL cells was confirmed by a prolonged TGF-ß-induced SMAD3 phosphorylation, whereas a shortening of the phosphorylation event is observed in AS cells. Mechanistically, the presence of GULP retains the TGF-ß in a signaling-competent early endosome for enhanced signaling. To address this mechanism in a physiological setting, TGF-ß insensitive ovarian adenocarcinoma cells (HEY) have a very low GULP expression level, similar to the observation made in a wide selection of human ovarian adenocarcinomas. Transfection of GULP into the HEY cells restored the TGF-ß responsiveness, as measured by SMAD3 phosphorylation and impairment of cell growth. Because GULP expression positively regulates TGF-ß signaling leading to growth inhibition, this may represent an attractive target to achieve TGF-ß responsiveness in ovarian cells.

Development of buffers for fast semidry transfer of proteins.

Western blot is an extensively used method for protein detection in cell biology. To optimize this procedure, here we examined a panel of buffers for their ability to efficiently transfer proteins from SDS-polyacrylamide gels onto nitrocellulose membranes in a short 12-min period, designated here as fast semidry transfer. Our results show for the first time that HEPES- and HEPPS/EPPS-based buffers represent the most efficient buffers for fast semidry transfer.

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.

A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFß-mediated breast cancer cell migration and invasion.

INTRODUCTION: Tumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFß)-mediated breast cancer cell migration and invasion. METHODS: A mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFß and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFß transcriptional activity was measured by a TGFß/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFß downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFß/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive. RESULTS: We found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFß. p21 and p/CAF regulates TGFß-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFß/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients. CONCLUSIONS: Together these results highlight an important role for p21 and p/CAF in promoting breast cancer cell migration and invasion at the transcriptional level and may open new avenues for breast cancer therapy.