Loss of RAB25 Cooperates with Oncogenes in the Transformation of Human Mammary Epithelial Cells (HMECs) to Give Rise to Claudin-Low Tumors.

The loss of RAB25 expression-RAS superfamily of GTPase characteristic of numerous breast cancers-corresponds with H-RAS point mutations, particularly in triple-negative breast cancers (TNBC), a subtype associated with a poor prognosis. To address the poorly understood factors dictating the progression of TNBC tumors, we examine the cooperative effects that loss of RAB25 expression in human mammary epithelial cell (HMEC) lines with H-RAS mutations confers in tumorigenesis. HMECs were immortalized by transduction with LXSN CDK4 R24C, a mutant form of cyclin-dependent kinase, followed by transduction with hTERT, a catalytic subunit of the telomerase enzyme. We found that with the loss of RAB25 and overexpression of mutant H-RAS61L, immortal HMECs transformed toward anchorage-independent growth and acquired an increased ability to migrate. Furthermore, cells express low CD24, high CD44, and low claudin levels, indicating stem-like properties upon transformation. Besides, loss of RAB25 and overexpression of H-RAS61L resulted in increased expression of transcription factors Snail and Slug that drive these cells to lose E-cadherin and undergo epithelial-mesenchymal transition (EMT). This study confirms that loss of RAB25 and overexpression of mutant H-RAS can drive HMECs toward a mesenchymal stem-like state. Our findings reveal that RAB25 functions as a tumor suppressor gene, and loss of RAB25 could serve as a novel biomarker of the claudin-low type of TNBC.

Mechanism of inert inflammation in an immune checkpoint blockade-resistant tumor subtype bearing transcription elongation defects.

The clinical response to immune checkpoint blockade (ICB) correlates with tumor-infiltrating cytolytic T lymphocytes (CTLs) prior to treatment. However, many of these inflamed tumors resist ICB through unknown mechanisms. We show that tumors with transcription elongation deficiencies (TEdef+), which we previously reported as being resistant to ICB in mouse models and the clinic, have high baseline CTLs. We show that high baseline CTLs in TEdef+ tumors result from aberrant activation of the nucleic acid sensing-TBK1-CCL5/CXCL9 signaling cascade, which results in an immunosuppressive microenvironment with elevated regulatory T cells and exhausted CTLs. ICB therapy of TEdef+ tumors fail to increase CTL infiltration and suppress tumor growth in both experimental and clinical settings, suggesting that TEdef+, along with surrogate markers of tumor immunogenicity such as tumor mutational burden and CTLs, should be considered in the decision process for patient immunotherapy indication.

Targeting of Cdc42 GTPase in regulatory T cells unleashes antitumor T-cell immunity.

BACKGROUND: Cancer immunotherapy has taken center stage in cancer treatment. However, the current immunotherapies only benefit a small proportion of patients with cancer, necessitating better understanding of the mechanisms of tumor immune evasion and improved cancer immunotherapy strategies. Regulatory T (Treg) cells play an important role in maintaining immune tolerance through inhibiting effector T-cell function. In the tumor microenvironment, Treg cells are used by tumor cells to counteract effector T cell-mediated tumor suppression. Targeting Treg cells may thus unleash the antitumor activity of effector T cells. While systemic depletion of Treg cells can cause excessive effector T-cell responses and subsequent autoimmune diseases, controlled targeting of Treg cells may benefit patients with cancer. METHODS: Treg cells from Treg cell-specific heterozygous Cdc42 knockout mice, C57BL/6 mice treated with a Cdc42 inhibitor CASIN, and control mice were examined for their homeostasis and stability by flow cytometry. The autoimmune responses in Treg cell-specific heterozygous Cdc42 knockout mice, CASIN-treated C57BL/6 mice, and control mice were assessed by H&E staining and ELISA. Antitumor T-cell immunity in Treg cell-specific heterozygous Cdc42 knockout mice, CASIN-treated C57BL/6 mice, humanized NSGS mice, and control mice was assessed by challenging the mice with MC38 mouse colon cancer cells, KPC mouse pancreatic cancer cells, or HCT116 human colon cancer cells. RESULTS: Treg cell-specific heterozygous deletion or pharmacological targeting of Cdc42 with CASIN does not affect Treg cell numbers but induces Treg cell instability, leading to antitumor T-cell immunity without detectable autoimmune reactions. Cdc42 targeting causes an additive effect on immune checkpoint inhibitor anti-programmed cell death protein-1 antibody-induced T-cell response against mouse and human tumors. Mechanistically, Cdc42 targeting induces Treg cell instability and unleashes antitumor T-cell immunity through carbonic anhydrase I-mediated pH changes. CONCLUSIONS: Rational targeting of Cdc42 in Treg cells holds therapeutic promises in cancer immunotherapy.

Graded RhoA GTPase Expression in Treg Cells Distinguishes Tumor Immunity From Autoimmunity.

RhoA of the Rho GTPase family is prenylated at its C-terminus. Prenylation of RhoA has been shown to control T helper 17 (Th17) cell-mediated colitis. By characterizing T cell-specific RhoA conditional knockout mice, we have recently shown that RhoA is required for Th2 and Th17 cell differentiation and Th2/Th17 cell-mediated allergic airway inflammation. It remains unclear whether RhoA plays a cell-intrinsic role in regulatory T (Treg) cells that suppress effector T cells such as Th2/Th17 cells to maintain immune tolerance and to promote tumor immune evasion. Here we have generated Treg cell-specific RhoA-deficient mice. We found that homozygous RhoA deletion in Treg cells led to early, fatal systemic inflammatory disorders. The autoimmune responses came from an increase in activated CD4+ and CD8+ T cells and in effector T cells including Th17, Th1 and Th2 cells. The immune activation was due to impaired Treg cell homeostasis and increased Treg cell plasticity. Interestingly, heterozygous RhoA deletion in Treg cells did not affect Treg cell homeostasis nor cause systemic autoimmunity but induced Treg cell plasticity and an increase in effector T cells. Importantly, heterozygous RhoA deletion significantly inhibited tumor growth, which was associated with tumor-infiltrating Treg cell plasticity and increased tumor-infiltrating effector T cells. Collectively, our findings suggest that graded RhoA expression in Treg cells distinguishes tumor immunity from autoimmunity and that rational targeting of RhoA in Treg cells may trigger anti-tumor T cell immunity without causing autoimmune responses.

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers.

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)-we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-κB pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers-as it portends a significant roadblock against immunotherapy-the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

Suppression of tumor antigen presentation during aneuploid tumor evolution contributes to immune evasion.

Anti-tumor immune responses impede tumor formation, and cancers have evolved many mechanisms of immune evasion. Confirming earlier findings, we show that human tumors with high chromosomal instability (CIN+) are significantly less immunogenic, as judged by tumor lymphocyte infiltration, compared to those with more stable genomes (CIN-). This finding is paradoxical, as genomic instability is expected to evoke an innate immune response. Importantly, CIN+ tumors and cell lines exhibited suppressed expression of proteins involved in MHC class I antigen presentation at least partly due to DNA hypermethylation of the corresponding genes. Using a mouse model of the in vivo evolution of aneuploid tumors, we found that the induction of chromosomal instability in tumor cells is highly immunogenic due to the activation of the STING/TBK1 pathway and consequent increased interferon signaling and antigen presentation. However, tumors evolving under immune pressure suppress the STING/TBK1 and antigen presentation pathways and evade anti-tumor immune responses. In contrast, CIN+ tumors that develop under low immune pressure in both humans and mice retain efficient MHC class I antigen presentation and immunogenicity. Altogether, this study identifies an important mechanism of immune evasion in chromosomally unstable tumors.

Defective transcription elongation in a subset of cancers confers immunotherapy resistance.

The nature and role of global transcriptional deregulations in cancers are not fully understood. We report that a large proportion of cancers have widespread defects in mRNA transcription elongation (TE). Cancers with TE defects (TEdeff) display spurious transcription and defective mRNA processing of genes characterized by long genomic length, poised promoters and inducible expression. Signaling pathways regulated by such genes, such as pro-inflammatory response pathways, are consistently suppressed in TEdeff tumors. Remarkably, TEdeff correlates with the poor response and outcome in immunotherapy, but not chemo- or targeted therapy, -treated renal cell carcinoma and metastatic melanoma patients. Forced pharmacologic or genetic induction of TEdeff in tumor cells impairs pro-inflammatory response signaling, and imposes resistance to the innate and adaptive anti-tumor immune responses and checkpoint inhibitor therapy in vivo. Therefore, defective TE is a previously unknown mechanism of tumor immune resistance, and should be assessed in cancer patients undergoing immunotherapy.

Characterization of immortalized human mammary epithelial cell line HMEC 2.6.

Primary human mammary epithelial cells have a limited life span which makes it difficult to study them in vitro for most purposes. To overcome this problem, we have developed a cell line that was immortalized using defined genetic elements, and we have characterized this immortalized non-tumorigenic human mammary epithelial cell line to establish it as a potential model system. human mammary epithelial cells were obtained from a healthy individual undergoing reduction mammoplasty at SIU School of Medicine. The cells were transduced with CDK4R24C followed by transduction with human telomerase reverse transcriptase. Post all manipulation, the cells displayed a normal cell cycle phase distribution and were near diploid in nature, which was confirmed by flow cytometry and karyotyping. In vitro studies showed that the cells were anchorage dependent and were non-invasive in nature. The cell line expressed basal epithelial markers such as cytokeratin 7, CD10, and p63 and was negative for the expression of estrogen receptor and progesterone receptor. Upon G-band karyotyping, the cell line displayed the presence of a few cytogenic abnormalities, including trisomy 20 and trisomy 7, which are also commonly present in other immortalized mammary cell lines. Furthermore, the benign nature of these cells was confirmed by multiple in vitro and in vivo experiments. Therefore, we think that this cell line could serve as a good model to understand the molecular mechanisms involved in the development and progression of breast cancer and to also assess the effect of novel therapeutics on human mammary epithelial cells.

CD24 Expression May Play a Role as a Predictive Indicator and a Modulator of Cisplatin Treatment Response in Head and Neck Squamous Cellular Carcinoma.

Platinum-based therapy is most often used to treat advanced cases of head and neck cancers, but only a small fraction of the patient population responds to cisplatin, with a median survival time of less than a year. Although gene signatures and molecular etiology of head and neck cancers have been previously described, none of them are predictive indicators of cisplatin treatment response in particular. Therefore, currently, there is a lack of clinically employable predictive indicators of the disease beyond HPV status to specifically predict patients' response to platinum-based therapy. It beckons a substantial effort to look for predictive indicators of cisplatin treatment response. In this regard, CD24 expression level appears to be a significant molecular phenotype of cisplatin-resistant residual cells in laryngeal carcinoma lines. CD24 expression level directly affects cisplatin sensitivity and affects the expression of critical apoptotic, stem and drug resistance genes. A relatively small retrospective patient tumor analysis suggests that CD24 high tumors go on to show an unfavorable response to cisplatin treatment. Overall, based on the strength of further analysis, CD24 presents a strong rationale to be utilized as a predictive indicator to stratify head and neck cancer patients for platinum-based therapy. It also provides a rationale for using CD24 as a therapeutic adjuvant target along with standard cisplatin therapy.

HPV and CSC in HNSCC cisplatin resistance.

Cisplatin (DNA intercalator), a standard chemotherapy drug often used to treat head and neck squamous cellular carcinoma (HNSCC) has very low response rates in recurrent disease of HNSCC, this is a major clinical problem today. However, a valuable window to look in to the underlying molecular aspects of a favorable and unfavorable cisplatin response is offered by a distinct disease entity in HNSCC - HPV+ OPSCC. It responds far more favorably to cisplatin than non-HPV driven HNSCC. Another intriguing aspect of head and neck cancer biology is the emergence of the CD44+ cancer stem cell - the tumor initiating population that in all likelihood is the root cause of therapeutic resistance. The critical question is, are there any differences between the CD44+ CSC population of an HPV+ OPSCC and a non-HPV HNSCC? In this regard, the inverse relation between EGFR levels and HPV status in OPSCC may be a common thread that connects the better response rates of HPV+ OPSCC with the contribution of CD44+ stem cells of HNSCC to chemoresistance.

Hyaluronan synthase HAS2 promotes tumor progression in bone by stimulating the interaction of breast cancer stem-like cells with macrophages and stromal cells.

The molecular mechanisms that operate within the organ microenvironment to support metastatic progression remain unclear. Here, we report that upregulation of hyaluronan synthase 2 (HAS2) occurs in highly metastatic breast cancer stem-like cells (CSC) defined by CD44(+)/CD24(-)/ESA(+) phenotype, where it plays a critical role in the generation of a prometastatic microenvironment in breast cancer. HAS2 was critical for the interaction of CSCs with tumor-associated macrophages (TAM), leading to enhanced secretion of platelet-derived growth factor-BB from TAMs, which then activated stromal cells and enhanced CSC self-renewal. Loss of HAS2 in CSCs or treatment with 4-methylumbelliferone, an inhibitor of HAS, which blocks hyaluronan production, drastically reduced the incidence and growth of metastatic lesions in vitro or in vivo, respectively. Taken together, our findings show a critical role of HAS2 in the development of a prometastatic microenvironment and suggest that HAS2 inhibitors can act as antimetastatic agents that disrupt a paracrine growth factor loop within this microenvironment.

KAI1 gene is engaged in NDRG1 gene-mediated metastasis suppression through the ATF3-NFkappaB complex in human prostate cancer.

NDRG1 and KAI1 belong to metastasis suppressor genes, which impede the dissemination of tumor cells from primary tumors to distant organs. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of the KAI1 gene. In this report, we found that ectopic expression of NDRG1 was able to augment endogenous KAI1 gene expression in prostate cancer cell lines, whereas silencing NDRG1 was accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of the KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFκB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional downstream target of the NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through an intrinsic transcriptional cascade.