rna-situ-hybridization-with-sequential-protein-immunofluorescence

RNA <em>In situ</em> Hybridization with Sequential Protein Immunofluorescence in Tandem Assay

The functionality of cells within a host does not depend on isolated signals. Instead, all components, from the smallest RNA molecules to the largest ...

The University of Texas MD Anderson Cancer Center

Lung cancer remains the leading cause of cancer death. Genome sequencing of lung tumors from patients with squamous cell carcinoma has identified SMAD4 to be frequently mutated. Here, we use a mouse model to determine the molecular mechanisms by which Smad4 loss leads to lung cancer progression. Mice with ablation of Pten and Smad4 in airway epithelium develop metastatic adenosquamous tumors. Comparative transcriptomic and in vivo cistromic analyses determine that loss of PTEN and SMAD4 results in ELF3 and ErbB2 pathway activation due to decreased expression of ERRFI1, a negative regulator of ERBB2 in mouse and human cells. The combinatorial inhibition of ErbB2 and Akt signaling attenuate tumor progression and cell invasion, respectively. Expression profile analysis of human lung tumors substantiated the importance of the ErbB2/Akt/ELF3 signaling pathway as both a prognostic biomarker and a therapeutic drug target for treating lung cancer.

ErbB2 Pathway Activation upon Smad4 Loss Promotes Lung Tumor Growth and Metastasis.

Krüppel-like factor 12 (KLF12) is a transcription factor that plays a role in normal kidney development and repression of decidualization. KLF12 is frequently elevated in esophageal adenocarcinoma and has been reported to promote gastric cancer progression. Here, we examined the role of KLF12 in colorectal cancer (CRC). Indeed, KLF12 promotes tumor growth by directly activating early growth response protein 1 (EGR1). The levels of KLF12 and EGR1 correlate synergistically with a poor prognosis. These results indicate that KLF12 likely plays an important role in CRC and could serve as a potential prognostic marker and therapeutic target.

Krüppel-Like Factor 12 Promotes Colorectal Cancer Growth through Early Growth Response Protein 1.

Somatic mutations can promote malignant transformation of airway epithelial cells and induce inflammatory responses directed against resultant tumors. Tumor-infiltrating T lymphocytes (TIL) in early-stage non-small cell lung cancer (NSCLC) secrete distinct proinflammatory cytokines, but the contribution of these TILs to tumor development and metastasis remains unknown. We show here that TILs in early-stage NSCLC are biased toward IL17A expression (Th17) when compared with adjacent tumor-free tissue, whereas Th17 cells are decreased in tumor infiltrating locoregional lymph nodes in advanced NSCLC. Mice in which  and  ( ) are deleted from airway epithelial cells develop spontaneous tumors, that share genetic signatures with squamous- (SQ.2b), and adeno- (AD.1) subtypes of human NSCLC.  mice globally lacking in  ( ) showed decreased tumor latency and increased metastasis. Th17 cells were required for recruitment of CD103 dendritic cells, and adoptive transfer of -sufficient CD4 T cells reversed early tumor development and metastasis in  mice. Together, these findings support a key role for Th17 cells in TILs associated with the  model of NSCLC and suggest therapeutic and biomarker strategies for human SQ2b and AD1 lung cancer. .

IL17A Regulates Tumor Latency and Metastasis in Lung Adeno and Squamous SQ.2b and AD.1 Cancer.

Microsomal prostaglandin E2 synthase 1 (mPGES1) was evaluated as an important downstream effector of the COX2 pathway responsible for tumor-mediated immunosuppression in melanoma.

The COX2 Effector Microsomal PGE2 Synthase 1 is a Regulator of Immunosuppression in Cutaneous Melanoma.

Mechanisms of lung squamous cell carcinoma (LSCC) development are poorly understood. Here, we report that JNK1/2 activities attenuate Lkb1-deficiency-driven LSCC initiation and progression through repressing ΔNp63 signaling. In vivo Lkb1 ablation alone is sufficient to induce LSCC development by reducing MKK7 levels and JNK1/2 activities, independent of the AMPKα and mTOR pathways. JNK1/2 activities is positively regulated by MKK7 during LSCC development. Pharmaceutically elevated JNK1/2 activities abates Lkb1 dependent LSCC formation while compound mutations of Jnk1/2 and Lkb1 further accelerate LSCC progression. JNK1/2 is inactivated in a substantial proportion of human LSCC and JNK1/2 activities positively correlates with survival rates of lung, cervical and head and neck squamous cell carcinoma patients. These findings not only determine a suppressive role of the stress response regulators JNK1/2 on LSCC development by acting downstream of the key LSCC suppresser Lkb1, but also demonstrate activating JNK1/2 activities as a therapeutic approach against LSCC.

JNK represses Lkb-deficiency-induced lung squamous cell carcinoma progression.

Soluble forms of receptors play distinctive roles in modulating signal-transduction pathways. Soluble CD74 (sCD74) has been identified in sera of inflammatory diseases and implicated in their pathophysiology; however, few relevant data are available in the context of cancer. Here we assessed the composition and production mechanisms, as well as the clinical significance and biological properties, of sCD74 in melanoma. Serum sCD74 levels were significantly elevated in advanced melanoma patients compared with normal healthy donors, and the high ratio of sCD74 to macrophage-migration inhibitory factor (MIF) conferred significant predictive value for prolonged survival in these patients (p = 0.0035). Secretion of sCD74 was observed primarily in melanoma cell lines as well as a THP-1 line of macrophages from monocytes and primary macrophages, especially in response to interferon-γ (IFN-γ). A predominant form that showed clinical relevance was the 25-KDa sCD74, which originated from the 33-KDa isoform of CD74. The release of this sCD74 was regulated by either a disintegrin and metalloproteinase-mediated cell-surface cleavage or cysteine-protease-mediated lysosomal cleavage, depending on cell types. Both recombinant and THP-1 macrophage-released endogenous sCD74 suppressed melanoma cell growth and induced apoptosis under IFN-γ stimulatory conditions via inhibiting the MIF/CD74/AKT-survival pathway. Our findings demonstrate that the interplay between sCD74 and MIF regulates tumor progression and determines patient outcomes in advanced melanoma.

Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma.

Correction: Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma.

The arachidonic acid pathway participates in immunosuppression in various types of cancer. Our previous observation detailed that microsomal prostaglandin E2 synthase 1 (mPGES-1), an enzyme downstream of cyclooxygenase 2 (COX-2), limited antitumor immunity in melanoma; in addition, genetic depletion of mPGES-1 specifically enhanced immune checkpoint blockade therapy. The current study set out to distinguish the roles of mPGES-1 from those of COX-2 in tumor immunity and determine the potential of mPGES-1 inhibitors for reinforcing immunotherapy in melanoma. Genetic deletion of mPGES-1 showed different profiles of prostaglandin metabolites from that of COX-2 deletion. In our syngeneic mouse model, mPGES-1-deficient cells exhibited similar tumorigenicity to that of COX-2-deficient cells, despite a lower ability to suppress PGE2 synthesis by mPGES-1 depletion, indicating the presence of factors other than PGE2 that are likely to regulate tumor immunity. RNA-sequencing analysis revealed that mPGES-1 depletion reduced the expressions of collagen-related genes, which have been found to be associated with immunosuppressive signatures. In our mouse model, collagen was reduced in mPGES-1-deficient tumors, and phenotypic analysis of tumor-infiltrating lymphocytes indicated that mPGES-1-deficient tumors had fewer TIM3 exhausted CD8 T cells compared with COX-2-deficient tumors. CAY10678, an mPGES-1 inhibitor, was equivalent to celecoxib, a selective COX-2 inhibitor, in reinforcing anti-PD-1 treatment. Our study indicates that mPGES-1 inhibitors represent a promising adjuvant for immunotherapies in melanoma by reducing collagen deposition and T-cell exhaustion.

Inhibition of Microsomal Prostaglandin E2 Synthase Reduces Collagen Deposition in Melanoma Tumors and May Improve Immunotherapy Efficacy by Reducing T-cell Exhaustion.

K-ras mutant lung adenocarcinoma (KM-LUAD) is a difficult-to-treat cancer subtype in which chronic inflammation pervades the tumor immune microenvironment (TIME). Pro-inflammatory pathways dampen the response to treatments, including immune checkpoint inhibitors, necessitating therapies that target this inflammatory signaling network in the TIME. One of the lynchpins of chronic inflammation in KM-LUAD is signal transducer and activator of transcription 3 (STAT3).

Selective inhibition of canonical STAT3 signaling suppresses K-ras mutant lung tumorigenesis and reinvigorates anti-tumor immunity.

KRAS mutations frequently co-occur with alterations in STK11/LKB1 and/or KEAP1, defining an aggressive subset of lung cancers resistant to immuno- and chemotherapy. While LKB1 loss is associated with vulnerability to DNA damage response-based therapies, the impact of KEAP1 alterations remains unknown. We demonstrate that KEAP1-NRF2 pathway drives a compensatory modulation of ATR-CHK1 signaling, enhancing vulnerability to ATR inhibitors (ATRi), particularly in the setting of increased replication stress associated with LKB1 loss. ATRi shows enhanced anti-tumor activity in LKB1 and/or KEAP1-deficient non-small cell lung cancer (NSCLC) models and synergizes with gemcitabine. ATRi also enhances antitumor immunity and mitigates the immunosuppressed phenotype of LKB1/KEAP1-deficient tumors. In the HUDSON trial, LKB1/KEAP1-deficient NSCLC patients demonstrate enhanced benefits to the ATRi ceralasertib plus durvalumab. These findings suggest that alterations in the KEAP1-NRF2 pathway and/or LKB1 are associated with enhanced sensitivity to ATRi and could serve as biomarkers for predicting response to ATRi combination regimens.

Article	Year
ErbB2 Pathway Activation upon Smad4 Loss Promotes Lung Tumor Growth and Metastasis. Cell reports\| PubMed ID: 25753424	2015
Krüppel-Like Factor 12 Promotes Colorectal Cancer Growth through Early Growth Response Protein 1. PloS one\| PubMed ID: 27442508	2016
IL17A Regulates Tumor Latency and Metastasis in Lung Adeno and Squamous SQ.2b and AD.1 Cancer. Cancer immunology research\| PubMed ID: 29653981	2018
The COX2 Effector Microsomal PGE2 Synthase 1 is a Regulator of Immunosuppression in Cutaneous Melanoma. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 30538110	2019
JNK represses Lkb-deficiency-induced lung squamous cell carcinoma progression. Nature communications\| PubMed ID: 31089135	2019
Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma. Cell death & disease\| PubMed ID: 35121729	2022
Correction: Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma. Cell death & disease\| PubMed ID: 35501311	2022
Inhibition of Microsomal Prostaglandin E2 Synthase Reduces Collagen Deposition in Melanoma Tumors and May Improve Immunotherapy Efficacy by Reducing T-cell Exhaustion. Cancer research communications\| PubMed ID: 37529399	2023
Selective inhibition of canonical STAT3 signaling suppresses K-ras mutant lung tumorigenesis and reinvigorates anti-tumor immunity. Frontiers in immunology\| PubMed ID: 40356899	2025
KEAP1 and STK11/LKB1 alterations enhance vulnerability to ATR inhibition in KRAS mutant non-small cell lung cancer. Cancer cell\| PubMed ID: 40645185	2025

Sungnam Cho

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