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Because survival of patients with metastatic colorectal cancer remain poor, there is an urgent need to identify potential novel druggable targets that are associated with colorectal cancer progression. One such target, basic leucine zipper and W2 domains 2 (BZW2), is involved in regulation of protein translation, and its overexpression is associated with human malignancy. Thus, we investigated the expression and regulation of BZW2, assessed its role in activation of WNT/β-catenin signaling, identified its downstream molecules, and demonstrated its involvement in metastasis of colorectal cancer. In human colorectal cancers, high mRNA and protein expression levels of BZW2 were associated with tumor progression. BZW2-knockdown reduced malignant phenotypes, including cell proliferation, invasion, and spheroid and colony formation. BZW2-knockdown also reduced tumor growth and metastasis; conversely, transfection of BZW2 into BZW2 low-expressing colorectal cancer cells promoted malignant features, including tumor growth and metastasis. BZW2 expression was coordinately regulated by microRNA-98, c-Myc, and histone methyltransferase enhancer of zeste homolog 2 (EZH2). RNA sequencing analyses of colorectal cancer cells modulated for BZW2 identified P4HA1 and the long noncoding RNAs, MALAT1 and NEAT1, as its downstream targets. Further, BZW2 activated the Wnt/β-catenin signaling pathway in colorectal cancers expressing wild-type β-catenin. In sum, our study suggests the possibility of targeting BZW2 expression by inhibiting EZH2 and/or c-Myc. Implications: FDA-approved small-molecule inhibitors of EZH2 can indirectly target BZW2 and because BZW2 functions as an oncogene, these inhibitors could serve as therapeutic agents for colorectal cancer.

S5. MALAT1 and NEAT1 are downstream target genes of BZW2 in CRC.

S2. BZW2 expression in CRC.

S1. BZW2 mRNA overexpression in CRC.

Supplementary figure legends 1-8, and supplementary tables 1-5

S4. BZW2 is involved in CRC tumor growth.

S7. miR-98-5p regulates BZW2 in CRC cells.

S6. MYC regulates BZW2 in CRC cells.

Because survival of patients with metastatic colorectal cancer remain poor, there is an urgent need to identify potential novel druggable targets that are associated with colorectal cancer progression. One such target, basic leucine zipper and W2 domains 2 (BZW2), is involved in regulation of protein translation, and its overexpression is associated with human malignancy. Thus, we investigated the expression and regulation of BZW2, assessed its role in activation of WNT/β-catenin signaling, identified its downstream molecules, and demonstrated its involvement in metastasis of colorectal cancer. In human colorectal cancers, high mRNA and protein expression levels of BZW2 were associated with tumor progression. BZW2-knockdown reduced malignant phenotypes, including cell proliferation, invasion, and spheroid and colony formation. BZW2-knockdown also reduced tumor growth and metastasis; conversely, transfection of BZW2 into BZW2 low-expressing colorectal cancer cells promoted malignant features, including tumor growth and metastasis. BZW2 expression was coordinately regulated by microRNA-98, c-Myc, and histone methyltransferase enhancer of zeste homolog 2 (EZH2). RNA sequencing analyses of colorectal cancer cells modulated for BZW2 identified P4HA1 and the long noncoding RNAs, MALAT1 and NEAT1, as its downstream targets. Further, BZW2 activated the Wnt/β-catenin signaling pathway in colorectal cancers expressing wild-type β-catenin. In sum, our study suggests the possibility of targeting BZW2 expression by inhibiting EZH2 and/or c-Myc.Implications:FDA-approved small-molecule inhibitors of EZH2 can indirectly target BZW2 and because BZW2 functions as an oncogene, these inhibitors could serve as therapeutic agents for colorectal cancer.

S8. EZH2 and BZW2 correlation in CRC cells.

S3. BZW2-knockdown reduces cell invasion.

Supplementary Figure 6 from Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human malignancies, with an overall 5-year survival rate of KRAS are almost uniformly present in early-stage disease, with subsequent inactivating mutations in p16INK4A, p53, and SMAD4 occurring in more advanced lesions. In this study, we have tested whether the loss of Smad4 would cooperate with an activating KrasG12D mutation to promote progression to PDAC using the Pdx1-Cre transgenic system to activate KrasG12D and delete Smad4 in all pancreatic lineages including the ductal epithelium. Analysis of double-mutant mice showed that loss of Smad4 significantly accelerated the progression of pancreatic intraepithelial neoplasias (mPanIN) and promoted a high incidence of intraductal papillary mucinous neoplasia and active fibrosis compared with Pdx1-Cre;KrasG12D or Pdx1-Cre;Smad4lox/lox mice. Occasionally, double-mutant mice progressed to locally invasive PDAC with little evidence of metastases by 6 months of age and without the detectable loss of p53 or p16Ink4A expression or function. The loss of Smad4 only seemed to promote disease progression in the presence of the activated KrasG12D allele because we observed no abnormal pathology within the pancreata of 23 Pdx1-Cre;Smad4lox/lox animals that were analyzed up to 8 months of age. This indicates that Smad4 is dispensable for normal pancreatic development but is critical for at least partial suppression of multiple KrasG12D-dependent disease-associated phenotypes. [Cancer Res 2007;67(17):8121–30]

Supplementary Figure 1 from Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

Supplementary Figure 3 from Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

Supplementary Figure 4 from Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

Supplementary Figure 2 from Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

Supplementary Figure 5 from Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

Reliable preclinical models are needed for screening new cancer drugs. Thus, we developed an improved 3D tumor organoid model termed “organoid raft cultures” (ORCs). Development of ORCs involved culturing tumors ex vivo on collagen beds (boats) with grid supports to maintain their morphological structure. The ORCs were developed from patient-derived xenografts (PDXs) of colon cancers excised from immune-deficient mice (NOD/SCID/IL2Rgammanull). We utilized these new models to evaluate the efficacy of an investigational drug, Navitoclax (ABT-263). We tested the efficacy of ABT-263, an inhibitor of BCL-2 family proteins, in these ORCs derived from a PDX that showed high expression of antiapoptotic BCL2 family proteins (BCL-2, BCL-XL, and BCL-W). Hematoxylin and eosin staining evaluation of PDXs and corresponding ORCs indicated the retention of morphological and other histological integrity of ORCs. ORCs treated with ABT-263 showed decreased expression of antiapoptotic proteins (BCL2, BCL-XL and BCL-W) and increased proapoptotic proteins (BAX and PUMA), with concomitant activation of caspase 3. These studies support the usefulness of the ORCs, developed from PDXs, as an alternative to PDXs and as faster screening models.

Background In silico deconvolution of invasive immune cell infiltration in bulk breast tumors helps characterize immunophenotype, expands treatment options, and influences survival endpoints. In this study, we identify the differential expression (DE) of the LM22 signature to classify immune‐rich and ‐poor breast tumors and evaluate immune infiltration by receptor subtype and lymph node metastasis. Methods Using publicly available data, we applied the CIBERSORT algorithm to estimate immune cells infiltrating the tumor into immune‐rich and immune‐poor groups. We then tested the association of receptor subtype and nodal status with immune‐rich/poor phenotype. We used DE to test individual signature genes and over‐representation analysis for related pathways. Results CCL19 and CXCL9 expression differed between rich/poor signature groups regardless of subtype. Overexpression of CHI3L2 and FES was observed in triple negative breast cancers (TNBCs) relative to other subtypes in immune‐rich tumors. Non‐signature genes, LYZ, C1QB, CORO1A, EVI2B, GBP1, PSMB9, and CD52 were consistently overexpressed in immune‐rich tumors, and SCUBE2 and GRIA2 were associated with immune‐poor tumors. Immune‐rich tumors had significant upregulation of genes/pathways while none were identified in immune‐poor tumors. Conclusions Overall, the proportion of immune‐rich/poor tumors differed by subtype; however, a subset of 10 LM22 genes that marked immune‐rich status remained the same across subtype. Non‐LM22 genes differentially expressed between the phenotypes suggest that the biologic processes responsible for immune‐poor phenotype are not yet well characterized., Our approach classified 2369 patients into two groups (immune‐rich, and immune‐poor) using an immune cell expression signature. The overall frequency of immune‐rich versus immune‐poor tumors differed by receptor subtype, however the specific signature genes remained the same, suggesting that signature expression is a stronger determinant of immune infiltration than receptor subtype.

Introduction: Colorectal cancer (CRC) is the third leading cause of cancer-associated deaths in United States. Despite advances in therapeutic modalities, the overall survival rate for metastatic CRC remains poor. Therefore, there is an urgent need to identify potential drug targets, which are associated with CRC progression and metastasis. One such target is Basic leucine zipper and W2 domains 2 (BZW2), also known as translation initiation regulator eIF5-mimic protein 1 (5MP1), a member of the basic-region leucine zipper (bZIP) superfamily of transcription factors. BZW2 is upregulated in some human cancers; however, its function in CRC malignancy is unknown. Thus, the role of BZW2 in CRC is studied. Methods: To address BZW2 role in CRC progression and metastasis, (1) we compared the RNA expression of BZW2 in human CRCs with their matched normal tissues (controls), (2) assessed the protein expression of BZW2 in primary CRCs and in CRCs which metastasized to liver, (3) as CRC is a molecularly heterogeneous disease, we conducted in vitro experiments, in CRC cell lines with various molecular alterations [P53, KRAS, BRAF, EGFR, or microsatellite stable/microsatellite instable (MSS/MSI) status], such as colony and spheroid formation to evaluate role of BZW2 on cell proliferation, (4) RNA-seq experiments with BZW2 knockdown (shRNA) to identify its downstream targets, and (5) to evaluate BZW2 role in CRC progression and metastasis, using BZW2 knockdown cells in in vivo models of NOD/SCID/IL2γ-receptor null (NSG) mice to assess tumor growth and the extent of metastasis. Results: Our results demonstrated that BZW2 is upregulated in CRC tissues, at RNA and protein levels, as compared to their matched controls. A higher expression was also noted in CRC liver metastatic lesions compared to matched controls. Immunohistochemical results, on invasive CRC tissues further confirmed the findings of high expression of BZW2 in human CRCs. Our in vitro results with BZW2 knockdown in CRC cells (HCT116, SW480, and HT29 cells) showed reduced colony and spheroid formation. RNA-seq results comparing the expression in BZW2 knockdown cells with their controls, demonstrated key genes were modulated upon BZW2 knockdown, including those for P4HA1 and the long non-coding RNAs (lncRNAs), MALAT1 and NEAT1. Our in vivo experiments with SW480 xenografts showed smaller tumors in BZW2 knockdown as compared to those of controls. Knockdown of BZW2 in HT-29 luciferase-tagged cells, reduced metastasis in NSG mice, potentiating the fact that decreasing BZW2 would affect metastasis. Conclusions: The results provide an evidence that BZW2 functions as an oncogene in CRC, regardless of their status of P53, KRAS, BRAF, EGFR, and MSS/MSI. Therefore, BZW2 may be an effective target for blocking CRC progression, and development of small molecule inhibitors of BZW2 could serve as potential therapeutic agents for CRC. Citation Format: Prachi Bajpai, Sumit Agarwal, Farrukh Afaq, Michael Behring, Hyung-Gyoon Kim, Darshan Shimoga Chandrashekar, Shajan Peter, Sameer Al Diffalha, Moh’d Khushman, Andreas Seeber, Sooryanarayana Varambally, Upender Manne. BZW2 a potential target to inhibit colorectal cancer growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1297.

This study demonstrates overexpression of TRIP13 in colorectal cancers is independent of patient's gender, age, race/ethnicity, pathologic stage (primary and liver metastatic lesions), and p53 and microsatellite instability status. Furthermore, it establishes role of TRIP13 in colorectal cancer metastasis and identifies COL6A3, TREM2, SHC3, and KLK7 as its downstream targets. Additionally, TRIP13 activates EGFR‐AKT pathway via its interaction with FGFR4., Overexpression of TRIP13, a member of the AAA‐ATPase family, is linked with various cancers, but its role in metastasis is unknown in colorectal cancer (CRC). In the current study, we investigated the role TRIP13 in experimental metastasis and its involvement in regulation of WNT/β‐catenin and EGFR signaling pathways. Evaluation of formalin‐fixed paraffin‐embedded (FFPE) and frozen tissues of adenomas and CRCs, along with their corresponding normal samples, showed that TRIP13 was gradually increased in its phenotypic expression from adenoma to carcinoma and that its overexpression in CRCs was independent of patient's gender, age, race/ethnicity, pathologic stage, and p53 and microsatellite instability (MSI) status. Moreover, liver metastases of CRCs showed TRIP13 overexpression as compared to matched adjacent liver tissues, indicating the biological relevance of TRIP13 in CRC progression and metastasis. TRIP13 knockdown impeded colony formation, invasion, motility, and spheroid‐forming capacity of CRC cells irrespective of their p53 and MSI status. Furthermore, xenograft studies demonstrated high expression of TRIP13 contributed to tumor growth and metastasis. Depletion of TRIP13 in CRC cells decreased metastasis and it was independent of the p53 and MSI status. Furthermore, TRIP13 interacted with a tyrosine kinase, FGFR4; this interaction could be essential for activation of the EGFR‐AKT pathway. In addition, we demonstrated the involvement of TRIP13 in the Wnt signaling pathway and in the epithelial–mesenchymal transition. Cell‐based assays revealed that miR‐192 and PNPT1 regulate TRIP13 expression in CRC. Additionally, RNA sequencing of CRC cells with TRIP13 knockdown identified COL6A3, TREM2, SHC3, and KLK7 as downstream targets that may have functional relevance in TRIP13‐mediated tumor growth and metastasis. In summary, our results demonstrated that TRIP13 promotes tumor growth and metastasis regardless of p53 and MSI status, and indicated that it is a target for therapy of CRC.

Colorectal cancer (CRC) is the fifth most diagnosed cancer in Sub-Saharan Africa. In Kenya, CRC incidence rates tripled from 1997 to 2017. In the Moi Teaching and Referral Hospital, Moi University, there has been an increase in CRC cases, notably for younger patients. A suggested pathobiology for this increase is gut microbiome dysbiosis. Since, for the Kenyan CRC patient population, microbiome studies are rare, there is a need for a better understanding of how microbiome dysbiosis influences CRC epidemiology in Kenya. In this single-center study, the focus was on profiling the gut microbiome of Kenyan CRC patients and healthy volunteers and evaluating associations between microbiome profiles and the age of CRC patients.The gut mucosa-associated microbiome of 18 CRC patients and 18 healthy controls were determined by 16S rRNA sequencing and analyzed for alpha and beta diversity, differential abundance, and microbial metabolic profiling.Alpha diversity metrics showed no significant differences, but beta diversity metrics showed dissimilarities in the microbial communities between CRC patients and healthy controls. The most underrepresented species in the CRC group wereMicrobiome and microbial metabolic profiles of CRC patients are different from those of healthy individuals. CRC microbiome dysbiosis, particularly

BackgroundFor several cancers, including those of the breast, young age at diagnosis is associated with an adverse prognosis. Although this effect is often attributed to heritable mutations such as BRCA1/2, the relationship between pathologic features, young age of onset, and prognosis for breast cancer remains unclear. In the present study, we highlight links between age of onset and lymph node metastasis (NM) in US women with breast cancer.MethodsCase listings from Surveillance, Epidemiology, and End Result (SEER) 18 registry data for women with breast cancer, which include information on race, were used. NM and its associated outcomes were evaluated for a subset of women with receptor subtype information and then compared against a larger, pre-subtype validation set of data from the same registry. Age of diagnosis was a 5-category variable; under 40 years, 40-49 years, 50-59 years, 60-69 years and 70+ years. Univariate and adjusted multivariate survival models were applied to both sets of data.ResultsAs determined with adjusted logistic regression models, women under 40 years old at diagnosis had 1.55 times the odds of NM as women 60-69 years of age. The odds of NM for (HR = hormone receptor) HR+/HER2+, HR-/HER2+, and triple-negative breast cancer subtypes were significantly lower than those for HR+/HER2-. In subtype-stratified adjusted models, age of diagnosis had a consistent trend of decreasing odds of NM by age category, most noticeable for HR+ subtypes of luminal A and B. Univariate 5-year survival by age was worst for women under 40 years, with NM attributable for 49% of the hazard of death from cancer in adjusted multivariate models.ConclusionsLymph node metastasis is age-dependent, yet not all molecular subtypes are clearly affected by this relationship. For

e15597 Background: In the US, colorectal cancer (CRC) is the third most common cancer. Patients receiving regorafenib, a multiple-kinase inhibitor, recommended to manage metastatic CRCs (mCRCs), has a modest improvement in median overall survival but it is associated with several toxicities. Our present study addresses regorafenib-induced toxicity concerns by combining regorafenib with a novel dual JAK-HDAC inhibitor (JAK-HDACi). The rationale for the dual inhibitor drug selection is due to the facts that the JAK/STAT/SOCS pathway is modulated in CRCs, and concurrent inhibition of JAK sensitizes solid tumors to HDACi. This study focused on evaluating the efficacy and reducing regorafenib-induced toxicity with this novel therapeutic combination in CRC preclinical models. Methods: We evaluated the toxicity of the JAK-HDACi, regorafenib, and their combination in normal colonic cells (CRL-1807) and their efficacy in CRC cell lines (HCT116, RKO, HT29, and SW480) exhibiting various statuses of p53, KRAS, BRAF, EGFR, and microsatellite instability, by conducting colony formation, cell proliferation, and cell cycle arrest assays. Kinome profiling and whole transcriptomic analysis were performed. Their efficacy was assessed in vivo in a CRC patient-derived xenograft (PDX) model, and experimental metastasis was evaluated in NSG mice using luciferase-tagged HT29 cells. Non-invasive, whole-body bioluminescence imaging was performed. Tumor tissues were harvested and stored at −80°C or prepared formalin-fixed paraffin-embedded blocks for Hematoxylin and Eosin (H&E) and immunostaining. Serum analysis was performed to evaluate liver and kidney functions to assess the toxicity. Results: At 500 nM concentrations, there was no pronounced death of CRL-1807 cells, but reduced number of colonies in CRC cells. Drug treatments decreased phosphorylation of STAT3 and ERK1/2 and cell viability, wherein the reduction was robust in the combination. The combination reduced activity of various kinases, as evident through kinome profiling. In SW480 cells, the combination caused G0-G1 cell arrest and decreased the S phase. RNA-seq results revealed modulation of key pathways: apoptosis, ECM-receptor interaction, and focal adhesion. The PDX model showed that the combination treatment reduced tumor growth, as evidenced in H&E staining with higher necrosis and reduced Ki67 staining. Experimental metastasis, bioluminescence imaging, and histological examination showed pronounced reduction in metastasis in mice treated with the combination. Serum chemistry profiles showed that the treatments did not cause systemic toxicity to mice used in either model. Conclusions: The combination therapy with the JAK-HDACi and regorafenib was more effective than the single agents with no evident toxicity. These findings lend credence to a clinical trial to assess this combination for treatment of patients with advanced CRC.

Triple negative breast cancers (TNBCs) are molecularly heterogeneous, and the link between their aggressiveness with African ancestry is not established. We investigated primary TNBCs for gene expression among self-reported race (SRR) groups of African American (AA, n = 42) and European American (EA, n = 33) women. RNA sequencing data were analyzed to measure changes in genome-wide expression, and we utilized logistic regressions to identify ancestry-associated gene expression signatures. Using SNVs identified from our RNA sequencing data, global ancestry was estimated. We identified 156 African ancestry-associated genes and found that, compared to SRR, quantitative genetic analysis was a more robust method to identify racial/ethnic-specific genes that were differentially expressed. A subset of African ancestry-specific genes that were upregulated in TNBCs of our AA patients were validated in TCGA data. In AA patients, there was a higher incidence of basal-like two tumors and altered TP53, NFB1, and AKT pathways. The distinct distribution of TNBC subtypes and altered oncologic pathways show that the ethnic variations in TNBCs are driven by shared genetic ancestry. Thus, to appreciate the molecular diversity of TNBCs, tumors from patients of various ancestral origins should be evaluated.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with an extremely poor prognosis. There is an urgent need to identify new therapeutic targets and also understand the mechanism of PDAC progression that leads to aggressiveness of the disease. To find therapeutic targets, we analyzed data related to PDAC transcriptome sequencing and found overexpression of the de novo purine metabolic enzyme phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS). Immunohistochemical analysis of PDAC tissues showed high expression of the PAICS protein. To assess the biological roles of PAICS, we used RNA interference and knock down of its expression in PDAC cell lines that caused a reduction in PDAC cell proliferation and invasion. Furthermore, results of chorioallantoic membrane assays and pancreatic cancer xenografts demonstrated that PAICS regulated pancreatic tumor growth. Our data also showed that, in PDAC cells, microRNA-128 regulates and targets PAICS. PAICS depletion in PDAC cells caused upregulation in E-cadherin, a marker of the epithelial-mesenchymal transition. In PDAC cells, a BET inhibitor, JQ1, reduced PAICS expression. Thus, our investigations show that PAICS is a therapeutic target for PDAC and, as an enzyme, is amenable to targeting by small molecules.

Triple negative breast cancers (TNBCs) are molecularly heterogeneous, and the link between their aggressiveness with African ancestry is not established. We investigated primary TNBCs for gene expression among self-reported race (SRR) groups of African American (AA, n=42) and European American (EA, n=33) women. Using The Cancer Genome Atlas (TCGA) approaches, we analyzed RNA sequencing data to measure changes in genome-wide expression and used logistic regressions to identify ancestry-associated gene expression signatures. To determine global ancestry, GATK best practices were followed for variant calling, and used the 1000 Genomes Project as reference data. We identified >150 African ancestry-associated genes and found that, compared to SRR, quantitative genetic analysis was a more robust method to identify racial/ethnic-specific genes that were differentially expressed. A subset of African ancestry-specific genes that were upregulated in TNBCs of our AA patients were validated in TCGA data. In AA patients, there was a higher incidence of basal-like 2 tumors and altered TP53, NFB1, and AKT pathways. The distinct distribution of TNBC subtypes and altered oncologic pathways show that the ethnic variations in TNBCs are driven by shared genetic ancestry. Thus, to appreciate the molecular diversity of TNBCs, tumors from patients of various ancestral origins should be evaluated.

Deposition, remodeling, and signaling of the extracellular matrix facilitate tumor growth and metastasis. Here, we demonstrated that an enzyme, collagen prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), which is involved in collagen synthesis and deposition, had elevated expression in colorectal cancers (CRCs) as compared to normal colonic tissues. The expression of P4HA1 in CRCs was independent of patient's age, race/ethnicity, gender, pathologic stage and grade, tumor location, and microsatellite instability (MSI) and p53 status. By modulating P4HA1 with shRNA, there was a reduction in malignant phenotypes of CRCs, including cell proliferation, colony formation, invasion, migration, and tumor growth, in mice regardless of their p53 and MSI status. Immunoblot analysis of excised xenograft tumors developed from cells with silenced PH4HA1 showed low levels of proliferating cell nuclear antigen. Further, in CRC mouse models, silencing of P4HA1 in HT29 cells resulted in less metastasis to liver and bone. P4HA1 expression was regulated by miR-124, and inhibition of cell growth was noted for CRC cells treated with miR-124. Furthermore, low levels of the transcriptional repressor EZH2 reduced P4HA1 expression in CRC cells. Inhibition of P4HA1 with the small molecule inhibitor diethyl-pythiDC decreased AGO2 and MMP1, which are P4HA1 target molecules, and reduced the malignant phenotypes of CRC cells. Treatment of CRC patient-derived xenografts that exhibit high expression of P4HA1 with diethyl-pythiDC resulted in tumor regression. Thus, the present study shows that P4HA1 contributes to CRC progression and metastasis and that targeting of P4HA1 with diethyl-pythiDC could be an effective therapeutic strategy for aggressive CRCs.

Background: Cancer is among the leading causes of death worldwide. Hence, new drugs and faster screening models are needed. However, costly experiments for determining the therapeutic efficacy of drugs in animals before they reach clinical trials make the process difficult. To address this issue, we developed an alternative strategy to screen drugs against human tumors. 3D tumor organoids are termed Organoid Raft Cultures (ORCs) due to the strategy involved in culturing tumors ex vivo on collagen beds with grid supports to maintain their morphological structure and molecular makeup. Methodology: After receiving informed consent of patients and IRB approval at the University of Alabama at Birmingham, patients with histologically confirmed colon cancers were recruited. Portions of collected colon tumors were then implanted into the flanks of mice to generate Patient-Derived Xenografts (PDXs). Once their volumes reached 2000 mm2, tumors were excised, and ORCs were generated. This approach addresses two concerns: 1) how well are the morphological features retained when the tumors are grown ex vivo, and 2): can we accomplish faster screening of the efficacy of drugs. In the present study, we used a tumor in which we identified, by DNA-sequencing, a TP53 missense mutation at amino acid R248Q (a change from arginine to glutamine). We tested PRIMA-1Met (APR-246), an agent that reactivates mutant p53 by causing a conformational change and restores its function as a tumor suppressor. Excised xenograft tumor tissue was cut into small pieces and seeded on mucosa-stromal equivalent consisting of buffered collagen and fibroblasts in 24-well plates and allowed to grow for 2-3 days. Next, the tissue assembly was detached and placed on raft shaped stainless steel grids in 6-well plates containing ORC media. These cultures, termed ORCs, were then divided into groups of APR-246 treated and untreated. After 7 days, tissues were formalin fixed, and slides of 5-micron thickness were cut for Hematoxylin and eosin (H&E), p53 and TUNEL staining. Results: H&E staining of PDXs and ORCs showed the normal glandular morphology of colon cancers. This conventional characteristic of adenocarcinoma was retained along with other morphologic features, indicating the usefulness of ORCs. Additionally, comparisons of p53 and TUNEL staining of untreated vs APR-246-treated ORCs showed that lack of nuclear accumulation of mutant p53 in this p53 mutant tissue and induced apoptosis by APR-246 treatment, indicating the efficacy of the drug and the usefulness of the ORCs as alternative screening model. Conclusion: ORCs are economical and will be useful for screening new drugs/therapies ex vivo, allowing faster drug screening and thus moving new agents to clinical trials. This work was supported in part by the ELKUS foundation. (*Equal contributing first coauthors) Citation Format: Sanjib Nilam Banerjee, Prachi Bajpai, Amr Elkholy, Dianne W. Moore, Hyung Gyoon Kim, Sumit Agarwal, Michael Behring, Sameer Al Diffalha, Upender Manne. Development of 3D organoid raft cultures of colon cancer as a model to screen the therapeutic efficacy of PRIMA-1Met (APR-246) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB249.

In the USA, colorectal cancer (CRC) is a leading cause of cancer deaths in men and women. Two primary molecular subtypes of CRCs are those with microsatellite instability (MSI) and those that are microsatellite stable (MSS). MSI-CRCs have a dysfunction of the mismatch repair (MMR) genes and are less aggressive; MSS-CRCs have active mismatch repair genes, account for 85% of sporadic CRCs, and are more aggressive. CRCs with MSI have a favorable prognosis and are candidates for immunotherapy; those with MSS have a poor prognosis and are resistant to various therapies. Our previous gene expression profiling studies of CRCs showed an association of high expression of tripartite motif-containing 29 (TRIM29) with MSS and p53 mutations. TRIM29, also known as ataxia telangiectasia group D-complementing (ATDC) protein, binds to p53 and inhibits its function. Since point mutations of p53 occur in 50% of CRCs and are associated with the MSS phenotype and with aggressiveness of CRCs, we assessed TRIM29 expression in CRC cells exhibiting various microsatellite and p53 status. With RNA sequencing and qPCR validation, we found higher expression of TRIM29 in MSS and p53-mutated types of CRC cells (HT29 and SW480) as compared to cells exhibiting MSI and the p53 wild-type (HCT116, LOVO, RKO and LS174T) as well as cells with MSS and the p53 wild-type (T84). TRIM29 knockdown studies with HT29 and SW480 CRC cells showed that TRIM29 contributes to the aggressive progression of cells with MSS and p53 mutations. Preclinical studies with experimental animals demonstrated that MSS and p53-mutated CRC cells (HT29) with high expression of TRIM29 metastasized more extensively to liver and bone relative to cells with stably inhibited TRIM29. Further, gene expression profiling of stably inhibited TRIM29 cells showed downregulation of P4HA1, ALDOC, and hexokinase2, enzymes that are involved in the hypoxia pathway. In summary, the present studies show that overexpression of TRIM29 contributes to CRC proliferation, invasion, migration, tumor growth, and experimental metastasis. Thus, targeting of TRIM29 with small molecule inhibitors could be an effective strategy for treatment of CRCs with MSS and p53 mutations. This work was supported in part by an NIH grant (3U54CA118948) and by a grant from the ELKUS foundation. Citation Format: Sumit Agarwal, Michael Behring, Prachi Bajpai, Amr Elkohly, Hyung-Gyoon Kim, Darshan S. Chandrashekar, Nirzari Gupta, Sameer Al Diffalha, Sooryanarayana Varambally, Upender Manne. TRIM29 mediates metastasis though hypoxia-regulated proteins in microsatellite-stable and p53-mutated colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB245.

Triple negative breast cancers (TNBCs) are molecularly heterogeneous, and the link between their aggressiveness with African ancestry is not established. We investigated primary TNBCs for gene expression among self-reported race (SRR) groups of African American and European American women. RNA sequencing data was analyzed to measure changes in genome-wide expression, and we utilized logistic regressions to identify ancestry-associated gene expression signatures. Using SNVs identified from our RNA sequencing data, global ancestry was estimated. We identified 156 African ancestry-associated genes and found that, compared to SRR, quantitative genetic analysis was a more robust method to identify racial/ethnic-specific genes that were differentially expressed. A subset of African ancestry-specific genes that were upregulated in TNBCs of our AA patients were validated in TCGA data. In AA patients, there was a higher incidence of basal-like 2 tumors and altered TP53, NFB1, and AKT pathways. The distinct distribution of TNBC subtypes and altered oncologic pathways show that the ethnic variations in TNBCs are driven by shared genetic ancestry. Thus, to appreciate the molecular diversity of TNBCs, tumors from patients of various ancestral origins should be evaluated. We created a novel tool to determine the heterogenic status of TNBC, which accounts for proportional differences multiple cell 'subtypes'. Citation Format: Melissa B. Davis, Rachel Martini, Lisa A. Newman, Olivier Elemento, Jason White, Akanksha Verma, Indrani Datta, Indra Adrianto, Yalei Chen, Kevin Gardner, Hyung-Gyoon Kim, Windy D. Coleman, Isam-Eldin Eltoum, Andra Frost, William Grizzle, Andrea Sboner, Upender Manne, Clayton Yates. Distinct heterogeneous subtypes of Triple-Negative Breast Cancer, associated with African Ancestry [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr IA34.

Colorectal cancer (CRC) is the third most common cancer in the United States. Most CRCs in patients receiving therapeutic agents are resistant to apoptotic stimuli. Two protein families, caspase enzymes and B-cell lymphoma-2 (Bcl-2) family proteins are involved in apoptosis. Caspase enzymes promote apoptosis, functioning in a cascade, of which caspase 3 is the pivotal protein. Members of the Bcl-2 family, Bcl-2, Bcl-xL, and Bax, are functionally opposed: Bcl-2 and Bcl-xL inhibit apoptosis, whereas Bax counteracts this effect. Only Bcl-2 family members that have the BH3-domain trigger apoptosis by binding to the pro-survival proteins, thereby neutralizing their functional activity. The approach of “BH-3 mimetics” was utilized for discovery and development of navitoclax (ABT-263), a small-molecule that mimics BH3-only proteins and thus induces apoptosis. Despite advances made in targeted therapy, the mechanism of action of ABT-263 is less studied, particularly for CRCs, which have over-expression of anti-apoptotic proteins, supporting avoidance of cell death, a hallmark of cancer. To identify the cellular mechanism of ABT-263 on apoptosis, we quantified the expression of anti-apoptotic Bcl-2-family members in CRC tumor samples relative to their corresponding controls and found several-fold higher expression of Bcl2, Bcl-xL, and Mcl-1 in the CRC tumor samples. Since ABT-263 is a classical drug for Bcl-2 inhibition, we studied its effect (2.5 µM for 24 hr) on RKO CRC cells, which have high endogenous expression of Bcl-2, by measuring gene expression profiles of Bcl-2 family members by qPCR, along with other proteins involved in apoptosis. ABT-263 treatment resulted in down-regulation of anti-apoptotic modulators, Bcl-2, Bcl-xL, and Bcl-w, by 27%, 29%, and 13%, respectively; these effects were concomitant with two-fold higher expression of caspase 3. Flow cytometry results with Annexin V/PI staining showed that ABT-263 induced apoptosis in RKO cells with 1.59-, 2.59-, and 2.76-fold elevations of early apoptotic, late apoptotic, and necrotic cells. Further, ABT-263 (2.5 µM) lowered cell proliferation by 15% after 24 hr. Similar lower gene expressions of Bcl-2 family members were evident for LS174T CRC cells, which also exhibit high endogenous Bcl-2 expression, when treated with ABT-263. These results highlight a previously unexplored effect of ABT-263-induced apoptosis in CRC cells, by upregulating caspase 3, through inhibition of Bcl-2 family members. These studies were supported by the Richard Elkus, MD, Eminent Scholars Program in Gastro-Intestinal Cancer Research at University of Alabama at Birmingham. Citation Format: Prachi Bajpai, Amr Elkholy, Sumit Agarwal, Hyung Gyoon Kim, Michael Behring, Upender Manne. Navitoclax (ABT-263) induces apoptosis in colon cancer cells by upregulating caspase 3 through inhibition of Bcl-2 family members [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 63.

Inter-ethnic differential gene expression in stage II recurrent colorectal cancers Prachi Bajpai1, Amr Elholy1, Michael Behring1, Dongquan Chen2, 3, Kevin Hale1, Sumit Agrawal1, Hyung-Gyoon Kim1, Trafina Jadhav1, Temegsen Samuel4, Upender Manne1, 3 1Department of Pathology, 2Division of Preventive Medicine, Department of Medicine, University of Alabama at Birmingham (UAB), Birmingham, Alabama, USA, 3O’Neal Comprehensive Cancer Center of UAB, Birmingham; 4Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, Alabama, USA Background and Objective: For colorectal cancer (CRC), the second most common cause of cancer-related death in the US, there are racial/ethnic disparities in incidence and mortality. Of these patients, 25-35% with pathologic Stage II CRCs exhibit recurrence after surgery with curative intent. Relative to Caucasian (CA) patients, African American (AA) patients with CRC have a 20% higher stage-specific mortality. The present study delineates a distinct gene expression profile, specific for Stage II CRCs, based on disease recurrence and patient race/ethnicity. Methods: We obtained gene expression profiles for Stage II CRCs from 16 AA and 30 CA patients by the use of Affymetrix GeneChip microarrays. Samples from patients with recurrent disease, within 5 years post-surgery, were compared to those with non-recurrent CRCs within 5 years post-surgery. For comparison between AA with CA patients, gene expression was assessed. Results: For recurrent CRCs, there were nine genes common to AA and CA patients, suggesting a common gene signature specific for Stage II disease. For tumors, relative to their corresponding normal tissues, there was higher expression of five of these genes; the direction of association with disease recurrence was distinct with race/ethnicity. GFM2, DCAF17, and GEN1 had a positive association with recurrence in CAs and a negative association with recurrence in AAs. Additionally, for recurrent patients, TAPBP and FEZ were upregulated in AAs and downregulated in CAs (False Discovery Rate Citation Format: Prachi Bajpai, Amr Elholy, Michael Behring, Dongquan Chen, Kevin Hale, Sumit Agrawal, Hyung-Gyoon Kim, Trafina Jadhav, Temegsen Samuel, Upender Manne. Inter-ethnic differential gene expression in stage II recurrent colorectal cancers [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C044.