BIRMINGHAM, Ala. – Metastatic bladder cancer is generally incurable, so new therapies are an urgent need. Researchers at the University of Alabama at Birmingham now report a potential treatment for a quarter of bladder cancers.

Their discovery, published in the journal JCI Insight, comes from mechanistic insights of gene changes and altered signaling pathways that revealed therapeutically actionable targets and mechanisms of resistance.

The UAB research focused on mutations in the gene ARID1A, a key part of a chromatin-modifying complex in the cell nucleus. Chromatin is a mix of DNA and proteins that tightly packages the 5-foot-long chromosomal DNA inside each cell, and chromatin modifiers act to help turn on or turn off gene expression. ARID1A-inactivating mutations are seen in about 20 percent of bladder tumors

The foundations for this research were various previous studies in cancer. First, a histone methyltransferase called EZH2 is considered to be an oncogene that causes normal cells to become cancer cells because it is overexpressed in many aggressive cancers and is thought to drive growth of the cells as shown by the lead authors and others. EZH2 can silence genes, including those that act to suppress growth of a tumor, called tumor suppressors. Second, previous studies have shown that mutations in ARID1A sensitize cells to pharmacologic inhibition with the EZH2-specific small molecule GSK-126.

So the researchers, led by James “Jed” Ferguson III, M.D., Ph.D., and Sooryanarayana Varambally, Ph.D., hypothesized that bladder cancer cells with ARID1A mutations would show sensitivity to EZH2 inhibition, which could be utilized as a therapeutic target for patients with ARID1A-deficient bladder cancer. Ferguson is an assistant professor in the UAB Department of Urology, and Varambally is a professor in the UAB Department of Pathology Division of Molecular and Cellular Pathology.

Analysis of existing datasets by the researchers showed that up to 29 percent of bladder cancers have nonsense or truncating mutations in ARID1A, and these tumors also have high expression of EZH2. Furthermore, several existing bladder cancer cell lines have mutations in ARID1A, which the researchers call ARID1Amut, while other bladder cancer cell lines did not have ARID1A mutations.

Using these cell lines, the researchers showed that the ARID1Amut cancer cells — but not ARID1A wild-type cancer cells — were sensitive to EZH2 inhibition by GSK-126 in cell culture, as measured by viability and proliferation. In mouse xenografts, using both the bladder cancer cell lines and patient-derived bladder cancer cells, GSK-126 halted growth of cancer cells containing ARID1Amut but not those with wild-type ARID1A.

Ferguson and Varambally next hypothesized that ARID1A-deficient cells become sensitive to GSK-126 due to transcriptional upregulation of specific tumor suppressors that remain transcriptionally repressed in ARID1A wild-type cells. They compared gene expression of GSK-126-treated ARID1A-wild-type bladder cancer cell lines with GSK-126-treated ARID1A knockdown cell lines. The ARID1A knockdown cells have less ARID1A and show a sensitivity to GSK-126 inhibition similar to that of ARID1Amut cells.

Out of about 70 differentially expressed genes, one was notable — the tumor suppressor PIKIP1 that acts by attenuating the cell-signaling pathway PI3K/AKT/mTOR. This pathway is important in regulating cell proliferation and growth in healthy cells; but in many cancers, it is overactive and causes unrestrained cell proliferation.

Healthy cells with wild-type ARID1A use the MAPK signaling pathway, but the researchers found that cells with ARID1Amut or ARID1A knockdowns use a different pathway. These cancer cells are dependent on PI3K signaling to activate the PI3K/AKT/mTOR pathway for survival, a signaling that is due to an upregulation of a relatively uncharacterized regulatory subunit of PI3K called PIK3R3.

Researchers confirmed this in clinical lysates of human bladder cancers, showing that tumors deficient in ARID1A protein had elevated levels of PIK3R3 and phosphoAKT, where phosphoAKT is the active form of AKT in the PI3K/AKT/mTOR pathway.

Of potential clinical importance, the researchers showed that ARID1A-deficient bladder cancer was sensitive to combination therapies with the EZH2 inhibitor GSK-126 and several inhibitors of PI3K, acting together in a synergistic manner.

“Thus, our studies suggest that bladder cancers with ARID1A mutations can be treated with inhibitors of EZH2 and/or PI3K,” Ferguson said. Ferguson and Varambally note that ARID1A is frequently mutated across a wide variety of human cancers, including bladder, gastric, pancreatic and ovarian cancers. With FDA approved EZH2 inhibitors available, the lead authors discussed the possibility of targeting EZH2 in cancer in a previous review that appeared in the journal Cancer Research.

Further mechanistic studies by the researchers showed that the EZH2-inhibitor sensitivity in ARID1A-deficient bladder cancer cells is due to upregulation of PIK3IP1, a protein inhibitor of PI3K signaling. The researchers also showed, for the first time, that PIK3IP1 inhibits PI3K signaling by inducing proteasomal degradation of PIK3R3. In support of these findings, they showed that PIK3R3 upregulation is necessary and sufficient for PI3K/AKT pathway activation and increased bladder cancer cell proliferation. Similarly, they showed that PIK3IP1 upregulation was necessary and sufficient for GSK-126-mediated cell death in ARID1A-deficient bladder cancer cells.

Co-first authors of the study, “ARID1A-deficient bladder cancer is dependent on PI3K signaling and sensitive to EZH2 and PI3K inhibitors,” are Hasibur Rehman, UAB Department of Pathology, and Darshan S. Chandrashekar, UAB Department of Pathology.

Other co-authors besides Ferguson, Varambally, Rehman and Chandrashekar, are Chakravarthi Balabhadrapatruni, Saroj Nepal, Sai Akshaya Hodigere Balasubramanya, Abigail K. Shelton, Kasey R. Skinner, Sumit Agarwal, Alyncia D. Robinson, George J. Netto, Upender Manne and C. Ryan Miller, UAB Department of Pathology; AiHong Ma and Ting Rao, Renmin Hospital of Wuhan University, Wuhan, China; Marie-Lisa Eich, University Hospital Cologne, Cologne, Germany; Gurudatta Naik, O’Neal Comprehensive Cancer Center at UAB; and Chong-xian Pan and Guru Sonpavde, Harvard Medical School, Boston, Massachusetts.

Support came from a Research Development Award from the U.S. Department of Veterans Affairs BLRD service, Veterans Affairs Merit Review grant 1I01BX003840, U.S. Department of Defense grant W81XWH1910588, Research Development Award from Veterans Affairs VISN7 and National Institutes of Health grant U54 CA233306.

At UAB, Urology and Pathology are departments in the Marnix E. Heersink School of Medicine.

Varambally is a senior scientist and Ferguson an associate scientist in the O’Neal Comprehensive Cancer Center at UAB.

Read the release on EurekAlert.

BIRMINGHAM, Ala. – A new study that analyzed protein levels in 2,002 primary tumors from 14 tissue-based cancer types identified 11 distinct molecular subtypes, providing systematic knowledge that greatly expands a searchable online database that has become a go-to platform for cancer data analysis by users worldwide.

The University of Alabama at Birmingham Cancer Data analysis portal, or UALCAN, was developed and released to public use in 2017 as a user-friendly portal for pan-cancer omics data analysis, including transcriptomics, epigenetics and proteomics. UALCAN has had nearly 920,000 site visits from researchers in more than 100 countries, and it has been cited more than 2,750 times.

“UALCAN is an effort to distribute comprehensive cancer data to researchers and clinicians in a user-friendly format to make discoveries and find needles in the haystack,” said Sooryanarayana Varambally, Ph.D., professor in the UAB Department of Pathology Division of Molecular and Cellular Pathology and director of UAB’s Translational Oncologic Pathology Research program. “Cancer detection, diagnosis, treatment, cure and research need a global team effort, and making sense of the huge amount of data involved needs a way to analyze and interpret these data.”

Cancer is a complex disease, and its initiation, progression and metastasis, the spread to distant organs, involves dynamic molecular changes in each type of cancer. Individual cancer patients show variations apart from some of the common genomic events.

In the new study, Varambally worked with longtime collaborator Chad Creighton, Ph.D., Baylor College of Medicine, Houston, Texas. Creighton led the proteomic study, published in Nature Communications, “Proteogenomic characterization of 2002 human cancers reveals pan-cancer molecular subtypes and associated pathways.” This extends two early proteomics studies published in 2019 and 2021.

Previously the team performed RNA transcripts analysis, providing the data to researchers through UALCAN, to determine which pathways the myriad forms of cancer use to aid growth, spread and aggressiveness. With this recent study, the team performed and incorporated large-scale proteomics analysis. The data and results provide new ideas for further research and possible therapeutic interventions.

A proteome is the complement of proteins expressed in a cell or tissue, and these can be measured quantitatively through recent technological advances in mass-spectrometry. In cells, DNA makes mRNA, and mRNA makes protein, processes known as the central dogma of molecular biology. Proteins are major functional moieties of cells, crucial in cell metabolism, structure, growth, signaling and movement.

The cancer types represented in the UALCAN proteomic dataset include breast, colorectal, gastric, glioblastoma, head and neck, liver, lung adenocarcinoma, lung squamous, ovarian, pancreatic, pediatric brain, prostate, renal, and uterine cancers. The number of tumors in each cancer type in the study ranged from 76 to 230, with an average of 143. Intriguingly, the pan-cancer, proteome-based subtypes the current study found cut across tumor lineages.

The compendium proteomic dataset came from 17 individual studies. Corresponding multi-omics data were available for most of these tumors, including mRNA levels, DNA somatic small mutations and insertions/deletions, and DNA somatic copy number alterations.

In general, the researchers found the protein expression of genes across tumors broadly correlated with corresponding mRNA levels or copy number alterations. However, there were some notable exceptions.

They identified 11 distinct proteome-based pan-cancer subtypes — named s1 through s11 — that can provide insights into the deregulated pathways and processes in tumors that make them cancerous. Each subtype spanned multiple tissue-based cancer types, though subtype s11 was specific to brain tumors, spanning glioblastomas and pediatric brain tumors.

Each subtype expressed specific gene categories, some seen before in a previous, less comprehensive proteomic study. Three subtypes showed new gene categories: subtype s7 with “axon guidance” and “frizzled binding” genes, subtype s10 with “DNA repair” and “chromatin organization” genes, and subtype s11 with “synapse,” “dendrite” and “axon” genes.

At the DNA level, the study detailed differences among the proteome-based subtypes in overall copy number alterations of genes, and somatic mutations in subtypes associated with higher pathway activity, as inferred by proteome or transcriptome data.

“Our study results provide a framework for understanding the molecular landscape of cancers at the proteome level to integrate and compare the data with other molecular correlates of cancers,” Varambally said. “The associated datasets and gene-level associations represent a resource for the research community, including helping to identify gene candidates for functional studies and further develop candidates as diagnostic markers or therapeutic targets for specific subset of cancers.

“Furthermore, this study reinforces the notion that cancers should be comprehensively surveyed at the protein level, though expression profiling on tumors has historically been mostly limited to the RNA transcript level. Many of the analyses in this ever-evolving cancer data analysis platform are based on user or expert requests, and the team is indebted to the support and encouragement from the researchers who use this platform to make discoveries that make a difference in cancer research.”

Some of the large datasets for the UAB site are generated by consortiums like The Cancer Genome Atlas, or TCGA, and the Clinical Proteomic Tumor Analysis Consortium, or CPTAC, of the National Cancer Institute.

Precision targeting of cancer requires the identification of individual or subclass-specific genomic and molecular alterations. To help cancer researchers perform various data analyses for better understanding of these large datasets, Darshan Shimoga Chandrashekar, Ph.D., led the development of the UALCAN portal under the mentorship of Varambally. Updates to this continuously evolving portal were recently published in Neoplasia.

The UALCAN initiative and its continuous development involve contributions from a team of experts including bioinformaticians, computer scientists, statisticians, cancer biologists, pathologists and oncologists. “It is a team science approach to enable the global cancer research team to tackle cancer,” Varambally said.

Support came from National Institutes of Health grants CA125123 and CA118948 and United States Department of Defense grant W81XWH-19-1-0588.

Co-first authors of this study are Yiqun Zhang and Fengju Chen, Baylor College of Medicine, and Chandrashekar, UAB Department of Pathology Division of Molecular and Cellular Pathology.

Pathology is a department in the Marnix E. Heersink School of Medicine at UAB. Varambally is a senior scientist in the O’Neal Comprehensive Cancer Center and the Informatics Institute at UAB and is co-director of Cancer Biology Theme of Graduate Biomedical Sciences at UAB. He holds an adjunct position at the Michigan Center for Translational Pathology, the University of Michigan, Ann Arbor.

Read this release on EurekAlert.

by Jeff Hansen

Metastatic bladder cancer is generally incurable, so new therapies are an urgent need. Researchers at the University of Alabama at Birmingham now report a potential treatment for a quarter of bladder cancers.

Their discovery, published in the journal JCI Insight, comes from mechanistic insights of gene changes and altered signaling pathways that revealed therapeutically actionable targets and mechanisms of resistance.

The UAB research focused on mutations in the gene ARID1A, a key part of a chromatin-modifying complex in the cell nucleus. Chromatin is a mix of DNA and proteins that tightly packages the 5-foot-long chromosomal DNA inside each cell, and chromatin modifiers act to help turn on or turn off gene expression. ARID1A-inactivating mutations are seen in about 20 percent of bladder tumors.

The foundations for this research were various previous studies in cancer. First, a histone methyltransferase called EZH2 is considered to be an oncogene that causes normal cells to become cancer cells because it is overexpressed in many aggressive cancers and is thought to drive growth of the cells as shown by the lead authors and others. EZH2 can silence genes, including those that act to suppress growth of a tumor, called tumor suppressors. Second, previous studies have shown that mutations in ARID1A sensitize cells to pharmacologic inhibition with the EZH2-specific small molecule GSK-126.

So the researchers, led by James “Jed” Ferguson III, M.D., Ph.D., and Sooryanarayana Varambally, Ph.D., hypothesized that bladder cancer cells with ARID1A mutations would show sensitivity to EZH2 inhibition, which could be utilized as a therapeutic target for patients with ARID1A-deficient bladder cancer. Ferguson is an assistant professor in the UAB Department of Urology, and Varambally is a professor in the UAB Department of Pathology Division of Molecular and Cellular Pathology.

Analysis of existing datasets by the researchers showed that up to 29 percent of bladder cancers have nonsense or truncating mutations in ARID1A, and these tumors also have high expression of EZH2. Furthermore, several existing bladder cancer cell lines have mutations in ARID1A, which the researchers call ARID1Amut, while other bladder cancer cell lines did not have ARID1A mutations.

Using these cell lines, the researchers showed that the ARID1Amut cancer cells — but not ARID1A wild-type cancer cells — were sensitive to EZH2 inhibition by GSK-126 in cell culture, as measured by viability and proliferation. In mouse xenografts, using both the bladder cancer cell lines and patient-derived bladder cancer cells, GSK-126 halted growth of cancer cells containing ARID1Amut but not those with wild-type ARID1A.

Ferguson and Varambally next hypothesized that ARID1A-deficient cells become sensitive to GSK-126 due to transcriptional upregulation of specific tumor suppressors that remain transcriptionally repressed in ARID1A wild-type cells. They compared gene expression of GSK-126-treated ARID1A-wild-type bladder cancer cell lines with GSK-126-treated ARID1A knockdown cell lines. The ARID1A knockdown cells have less ARID1A and show a sensitivity to GSK-126 inhibition similar to that of ARID1Amut cells.

Out of about 70 differentially expressed genes, one was notable — the tumor suppressor PIKIP1 that acts by attenuating the cell-signaling pathway PI3K/AKT/mTOR. This pathway is important in regulating cell proliferation and growth in healthy cells; but in many cancers, it is overactive and causes unrestrained cell proliferation.

Healthy cells with wild-type ARID1A use the MAPK signaling pathway, but the researchers found that cells with ARID1Amut or ARID1A knockdowns use a different pathway. These cancer cells are dependent on PI3K signaling to activate the PI3K/AKT/mTOR pathway for survival, a signaling that is due to an upregulation of a relatively uncharacterized regulatory subunit of PI3K called PIK3R3.

Researchers confirmed this in clinical lysates of human bladder cancers, showing that tumors deficient in ARID1A protein had elevated levels of PIK3R3 and phosphoAKT, where phosphoAKT is the active form of AKT in the PI3K/AKT/mTOR pathway.

Of potential clinical importance, the researchers showed that ARID1A-deficient bladder cancer was sensitive to combination therapies with the EZH2 inhibitor GSK-126 and several inhibitors of PI3K, acting together in a synergistic manner.

“Thus, our studies suggest that bladder cancers with ARID1A mutations can be treated with inhibitors of EZH2 and/or PI3K,” Ferguson said. Ferguson and Varambally note that ARID1A is frequently mutated across a wide variety of human cancers, including bladder, gastric, pancreatic and ovarian cancers. With FDA approved EZH2 inhibitors available, the lead authors discussed the possibility of targeting EZH2 in cancer in a previous review that appeared in the journal Cancer Research.

Further mechanistic studies by the researchers showed that the EZH2-inhibitor sensitivity in ARID1A-deficient bladder cancer cells is due to upregulation of PIK3IP1, a protein inhibitor of PI3K signaling. The researchers also showed, for the first time, that PIK3IP1 inhibits PI3K signaling by inducing proteasomal degradation of PIK3R3. In support of these findings, they showed that PIK3R3 upregulation is necessary and sufficient for PI3K/AKT pathway activation and increased bladder cancer cell proliferation. Similarly, they showed that PIK3IP1 upregulation was necessary and sufficient for GSK-126-mediated cell death in ARID1A-deficient bladder cancer cells. 

Co-first authors of the study, “ARID1A-deficient bladder cancer is dependent on PI3K signaling and sensitive to EZH2 and PI3K inhibitors,” are Hasibur Rehman, UAB Department of Pathology, and Darshan S. Chandrashekar, UAB Department of Pathology. 

Other co-authors besides Ferguson, Varambally, Rehman and Chandrashekar, are Chakravarthi Balabhadrapatruni, Saroj Nepal, Sai Akshaya Hodigere Balasubramanya, Abigail K. Shelton, Kasey R. Skinner, Sumit Agarwal, Alyncia D. Robinson, George J. Netto, Upender Manne and C. Ryan Miller, UAB Department of Pathology; AiHong Ma and Ting Rao, Renmin Hospital of Wuhan University, Wuhan, China; Marie-Lisa Eich, University Hospital Cologne, Cologne, Germany; Gurudatta Naik, O’Neal Comprehensive Cancer Center at UAB; and Chong-xian Pan and Guru Sonpavde, Harvard Medical School, Boston, Massachusetts.

Support came from a Research Development Award from the U.S. Department of Veterans Affairs BLRD service, Veterans Affairs Merit Review grant 1I01BX003840, U.S. Department of Defense grant W81XWH1910588, Research Development Award from Veterans Affairs VISN7 and National Institutes of Health grant U54 CA233306.

At UAB, Urology and Pathology are departments in the Marnix E. Heersink School of Medicine.

Varambally is a senior scientist and Ferguson an associate scientist in the O’Neal Comprehensive Cancer Center at UAB.

Read this story on UAB News.