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Identifying molecular characteristics that are associated with aggressive cancer phenotypes through gene expression profiling can help predict treatment responses and clinical outcomes. Claudins are deregulated in colorectal cancer (CRC). In CRC, increased claudin-1 expression results in epithelial-to-mesenchymal transition and metastasis, while claudin-7 functions as a tumor suppressor. In this study, we have developed a molecular signature based on claudin-1 and claudin-7 associated with poor patient survival and chemoresistance. This signature was validated using an integrated approach including publicly available datasets and CRC samples from patients who either responded or did not respond to standard-of-care treatment, CRC cell lines, and patient-derived rectal and colon tumoroids. Transcriptomic analysis from a patient dataset initially yielded 23 genes that were differentially expressed along with higher claudin-1 and decreased claudin-7. From this analysis, we selected a claudins-associated molecular signature including PIK3CA, SLC6A6, TMEM43, and ASAP-1 based on their importance in CRC. The upregulation of these genes and their protein products was validated using multiple CRC patient datasets, in vitro chemoresistant cell lines, and patient-derived tumoroid models. Additionally, blocking these genes improved 5-FU sensitivity in chemoresistant CRC cells. Our findings propose a new claudin-based molecular signature that associates with poor prognosis as well as characteristics of treatment-resistant CRC including chemoresistance, metastasis, and relapse.

Excessive sympathoexcitation characterizes the chronic heart failure (CHF) state. An exaggerated cardiac sympathetic afferent reflex (CSAR) contributes to this sympathoexcitation. Prior studies have demonstrated that the CSAR to capsaicin [transient receptor potential (TRP) vanilloid 1 agonist] is exaggerated in CHF animal models. We recently discovered that capsaicin application to the lung visceral pleura in anesthetized, vagotomized, open-chested rats increases mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). We named this response the pulmonary spinal afferent reflex (PSAR). Due to the similarities between TRP vanilloid 1 and TRP ankyrin 1 (TRPA1) channels as well as the excessive sympathoexcitation of CHF, we hypothesized that stimulation of the CSAR and PSAR with a specific TRPA1 agonist would result in an augmented response in CHF rats (coronary ligation model) compared with sham control rats. In response to a TRPA1 agonist, both CSAR and PSAR in sham rats resulted in biphasic changes in MAP and increases in HR and RSNA 10–12 wk postmyocardial infarction (post-MI). These effects were blunted in CHF rats. Assessment of TRPA1 expression levels in cardiopulmonary spinal afferents by immunofluorescence, quantitative RT-PCR, and Western blot analysis 10–12 wk post-MI all indicates reduced expression in CHF rats but no reduction at earlier time points. TRPA1 protein was reduced in a dorsal root ganglia cell culture model of inflammation and simulated tissue ischemia, raising the possibility that the in vivo reduction of TRPA1 expression was, in part, caused by CHF-related tissue ischemia and inflammation. These data provide evidence that reflex responses to cardiopulmonary spinal afferent TRPA1 stimulation may be attenuated in CHF rather than enhanced. NEW & NOTEWORTHY Excessive sympathoexcitation characterizes chronic heart failure (CHF). The contribution of transient receptor potential ankyrin 1 (TRPA1) channel-mediated reflexes to this sympathoexcitation is unknown. We found that application of TRPA1 agonist to the heart and lung surface resulted in increased heart rate and sympathetic output and a biphasic change in mean arterial pressure in control rats. These effects were attenuated in CHF rats, decreasing the likelihood that TRPA1 channels contribute to cardiopulmonary afferent sensitization in CHF.

Neuronal connectivity is dependent on size and shape of the dendritic arbor. However, mechanisms controlling dendritic arborization, especially in the peripheral nervous system, are not completely understood. Previous studies have shown that bone morphogenetic proteins (BMPs) are important initiators of dendritic growth in peripheral neurons. In this study, we examined the hypothesis that post-transcriptional regulation mediated by microRNAs (miRNAs) is necessary f or BMP-7 induced dendritic growth in these neurons. To examine the role of miRNAs in BMP-7-induced dendritic growth, microarray analyses was used to profile miRNA expression in cultured sympathetic neurons from the superior cervical ganglia of embryonic day 21 rat pups at 6 and 24 h after treatment with BMP-7 (50 ng/mL). Our data showed that BMP-7 significantly regulated the expression of 43 of the 762 miRNAs. Of the 43 miRNAs, 22 showed robust gene expression;14 were upregulated by BMP-7 and 8 were downregulated by BMP-7. The expression profile for miR-335, miR-664-1*, miR-21 and miR-23b was confirmed using qPCR analyses. Functional studies using morphometric analyses of d endritic growth in cultured sympathetic neurons transfected with miRNA mimics and inhibitors indicated that miR-664-1*, miR-23b and miR-21 regulated early stages of BMP-7 induced dendritic growth. In summary, our data provide evidence for miRNA-mediated post-transcriptional regulation as important downstream component of BMP-7 signaling during early stages of dendritic growth in sympathetic neurons.

Despite advances in the cytotoxic and targeted therapy, resistance to chemotherapy remains one of the greatest challenges in long-term management of metastatic colorectal cancer, which eventually contributes to patient death as tumors accumulate means of evading treatment. We have recently demonstrated that expression of the tight junction protein claudin-1 increases while claudin-7 expression decreases with human colon cancer (CRC) progression and metastasis. Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) are critically implicated in cancer metastasis and chemoresistance. Taking this to account, we performed a computational assessment of 250 patient datasets from two different cancer centers (Vanderbilt and Moffitt) and identified the gene sets whose expression increased proportionally with claudin-1 expression and decreased with claudin-7, and vice versa, and correlated these gene sets it with chemoresistance, EMT and CSC markers. We identified a resulting 23-gene set biomarker signature, out of these gene clusters. Some of the important candidates based on literature and their function in colorectal cancer which we focused on were SLC6A6, PIK3CA, ASAP1, TMEM and E2F2. This signature was then validated using the TCGA database. To further evaluate functional relevance of this biomarker signature, we developed oxaliplatin resistant DLD-1 and HT29 colon cancer cells. The chemoresistance of these cells were confirmed by determining the IC50 values DLD (Parental) (4.5μM) and HT29 (Parental) (10.08 μM), after exposing them to increasing conc. of clinically relevant dose of oxaliplatin DLD (Oxaliplatin-R) (16.35 μM) and HT29 (Oxaliplatin-R) (20.05 μM). To our interest, we observed a significant upregulation of SLC6A6, PIK3CA, ASAP1, TMEM and downregulation of E2F2 in DLDOxaR and HT29OxaR cells compared to parental cells. Moreover, DLDOxaR and HT29OxaR cells possessed significantly increased expression of EMT markers such as α-SMA, vimentin, Snail and Slug along with enrichment of the stem cell markers like CD133, CD44 and Aldh1, colony and sphere forming ability. A concomitant decrease in E-cadherin characterized these cells. A similar increase in the expressions of SLC6A6, PIK3CA, ASAP1, TMEM and decrease in E2F2 was observed in colon cancer mouse models as well as chemoresistant patient samples. Taken together, we propose a new predictive biomarker signature which may offer insights into identifying new therapies required to overcome the acquired resistance of colon cancer towards Oxaliplatin and uncover potential molecular pathways involved in treatment failure to help guide therapeutic alternative. Citation Format: Saiprasad Gowrikumar, Kristina Pravoverov, Caroline Selisteda, Kiran D. Bastola, Steven Chen, Joshua J. Smith, Mary K. Washington, Amar B. Singh, Punita Dhawan. A novel biomarker signature in predicting chemoresistance in colorectal cancer: Potential application in chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3126.