Avijit Majumdar

Targeting TGF-β signaling in cancer.

Introduction: The transforming growth factor-β (TGF-β) signaling pathway has a pivotal role in tumor suppression and yet, paradoxically, in tumor promotion. Functional context dependent insights into the TGF-β pathway are crucial in developing TGF-β-based therapeutics for cancer. Areas covered: This review discusses the molecular mechanism of the TGF-β pathway and describes the different ways of tumor suppression by TGF-β. It is then explained how tumors can evade these effects and how TGF-β contributes to further growing and spreading of some of the tumors. In the last part of the review, the data on targeting TGF-β pathway for cancer treatment is assessed. This review focuses on anti-TGF-β based treatment and other options targeting activated pathways in tumors where the TGF-β tumor suppressor pathway is lost. Pre-clinical as well up to date results of the most recent clinical trials are given. Expert opinion: Targeting the TGF-β pathway can be a promising direction in cancer treatment. However, several challenges still exist, the most important are differentiating between the carcinogenic effects of TGF-β and its other physiological roles, and delineating the tumor suppressive versus the tumor promoting roles of TGF-β in each specific tumor. Future studies are needed in order to find safer and more effective TGF-β-based drugs.

Hepatic stem cells and transforming growth factor β in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. It arises from modulation of multiple genes by mutations, epigenetic regulation, noncoding RNAs and translational modifications of encoded proteins. Although >40% of HCCs are clonal and thought to arise from cancer stem cells (CSCs), the precise identification and mechanisms of CSC formation remain poorly understood. A functional role of transforming growth factor (TGF)-β signalling in liver and intestinal stem cell niches has been demonstrated through mouse genetics. These studies demonstrate that loss of TGF-β signalling yields a phenotype similar to a human CSC disorder, Beckwith–Wiedemann syndrome. Insights into this powerful pathway will be vital for developing new therapeutics in cancer. Current clinical approaches are aimed at establishing novel cancer drugs that target activated pathways when the TGF-β tumour suppressor pathway is lost, and TGF-β itself could potentially be targeted in metastases. Studies delineating key functional pathways in HCC and CSC formation could be important in preventing this disease and could lead to simple treatment strategies; for example, use of vitamin D might be effective when the TGF-β pathway is lost or when wnt signalling is activated.

Mutational Profiles Reveal an Aberrant TGF-β-CEA Regulated Pathway in Colon Adenomas

Mutational processes and signatures that drive early tumorigenesis are centrally important for early cancer prevention. Yet, to date, biomarkers and risk factors for polyps (adenomas) that inordinately and rapidly develop into colon cancer remain poorly defined. Here, we describe surprisingly high mutational profiles through whole-genome sequence (WGS) analysis in 2 of 4 pairs of benign colorectal adenoma tissue samples. Unsupervised hierarchical clustered transcriptomic analysis of a further 7 pairs of adenomas reveals distinct mutational signatures regardless of adenoma size. Transitional single nucleotide substitutions of C:G>T:A predominate in the adenoma mutational spectrum. Strikingly, we observe mutations in the TGF-β pathway and CEA-associated genes in 4 out of 11 adenomas, overlapping with the Wnt pathway. Immunohistochemical labeling reveals a nearly 5-fold increase in CEA levels in 23% of adenoma samples with a concomitant loss of TGF-β signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-β signaling. Our study uncovers diverse mutational processes underlying the transition from early adenoma to cancer. This has broad implications for biomarker-driven targeting of CEA/TGF-β in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression.

Abstract 67: Genomic and mutational profiling of human colon adenomas reveals early driver mutations and a TGF-β-CEA regulated profile

Introduction: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world with 143,700 newly diagnosed cases in 2012. The rapid development of cancers in small, sessile adenomas could be a contributing factor that may have been overlooked. Identifying high risk patients through genomic analysis of adenomas could potentially lead to an early intervention therapy. Methods: Whole Genome Sequence (WGS) and Whole Exome Sequence (WES) analyses were performed for 4 pairs of normal controls (two from proximal colon and two from the distal colon) and colorectal adenomas (2 sessile serrated (SSA) and 2 tubulovillous adenomas (TVA) less than 1 cm in size). Transcriptome sequence analysis was performed in seven pairs of control-test matched adenomas (6 TVA and 1 SSA). Expression level of CEA and TGF-β pathway members were carried out on 30 non dysplastic adenomas and normal colon tissues by immunohistochemistry. Results: 1. Hyper-mutator profiles were observed in two of the samples (1 SSA and 1 TVA) by WGS with 1709 mutations after normalization with normal paired samples. 2. The samples showed an average mutation frequency of 0.55 mutations per 10 6 bases. 3. Aberrant mutational profiles was detected in seven of the eleven adenomas, with distinct mutational signatures among the samples, two with high, two intermediate and three low mutational rates. 4. Six of the eleven adenomas (1 SSA and 4 TVAs) showed alteration in the Wnt and p53 pathway. 5. Transitional single nucleotide substitutions of C:T>G:A in the mutational spectrum were observed in 37% of the samples. 6. Subtle localized hyper mutation (kataegis) was observed among two of the samples. 7. Five out of eleven adenomas showed mutations in the TGF-β (transforming growth factor-β) and CEA pathways members, overlapping with Wnt/p53 mutations in four adenomas. 8. Analyses of expression level of CEA and TGF-β pathway members in 30 non dysplastic tissues revealed a marked increase (over 8 fold) in CEA expression in 25% of adenoma samples which was linked to concomitant loss of TGF-β signaling. 9. Further functional studies revealed that CEA associated with TGF-β Type I receptor and disruption of TGF-β tumor suppressor signaling with activation of STAT3. Conclusions: Our studies indicate that small adenomas both TVAs and SSAs can resemble CRCs in genomic profiling and may reflect a high risk population. Genetic and mechanistic analyses reveal that disruption of CEA/TGF-β pathway in early adenomas may reflect a new and early role for these pathways in CRC. This study further supports the biomarker driven targeting of CEA/TGF-β in high risk adenomas and can be used as a prognostic marker for early detection of aggressive adenoma-CRC progression. Citation Format: Vipin K. Menon, Raju S. Gottumukkala, Jian Chen, Xiaoping Su, Nipun Mistry, Avijit Majumdar, Ji-Hyun Shin, Shulin Li, Kirti Shetty, Xifeng Wu, Brian Weston, Ethan Miller, John R. Stroehlein, Marta L. Davila, Mehnaz A. Shafi, Asif Rashid, Bhaskar V. Kallakury, Selvi Thirumurthi, John S. McMurray, Sue-Hwa Lin, Wilma Jogunoori, Lopa Mishra. Genomic and mutational profiling of human colon adenomas reveals early driver mutations and a TGF-β-CEA regulated profile. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 67. doi:10.1158/1538-7445.AM2015-67

Abstract 4703: Genome & exome analysis of early colon cancers reveals new targets

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Advanced colorectal cancer (CRC) remains lethal and difficult to treat with over 140,000 new cases a year in the United States. One contributing factor that has emerged is the rapid development of cancers in small, sessile adenomas(polyps) (less than 1cm), which might be overlooked. Results: Using whole genome and whole exome sequence (WGS) analyses of four pairs of colorectal adenoma (early CRC) tissue samples along with their normal counterparts, we observe dramatic genomic instability in two of the samples, suggesting that these two adenomas could be on the brink of overt CRC. We proceeded to analyze a further 20 samples through RNA sequence and/or immunohistochemical analysis. The two polyps are associated with hyper mutated profile of multiple genes including Carcinoembryonic antigen (CEA) and CEACAM6 in one of the samples. Analyses of the expression levels of CEA and TGF-β signaling pathway members in 20 early adenomas and normal colon tissues demonstrate a marked increase in CEA expression in 25% of adenoma samples linked to a concomitant loss of TGF-β signaling. We proceeded to mechanistic studies examining CEA and it role in inactivating the tumor suppressive arm of transforming growth factor-β (TGF-β) signaling, providing crucial new information for CEA in promoting aggressive CRC. We found that CEA interacts directly with the transforming growth factor-β (TGF-β) receptor 1 (TBR1) with inhibition of downstream TGF-β tumor suppressor signaling through its B3 domain. Moreover, we previously found that deletion of Smad3/4 adaptor β2SP results in loss of TGF-β signaling, with dramatic and spontaneous formation of adenomas as well as CRC. Ectopic expression of CEA also induces CRC metastases, potentially indirectly through activation of STAT3 and stimulating production of IL-6, IL-10 and others, in addition to inactivating TGF-β tumor suppressor signaling. Conclusions: Our data suggest that enhanced CEA levels and oncogenic mutations in the colon adenomas can be used as prognostic markers for early detection of aggressive CRC and the B3 domain of CEA can be targeted by monoclonal antibody/peptide inhibitors/small molecule inhibitors targeting CEA and CRC cell proliferation. Citation Format: Avijit Majumdar, Jian Chen, Sue Hwa Lin, Lior Katz, Gottumukkala Subba Raju, Kirti Shetty, Jon White, Xifeng Wu, Asif Rashid, John S. McMurray, Kenna R. Shaw, Xiaoping Su, Brian Weston, Selvi Thirumurthi, Aiwu Ruth He, Lopa Mishra. Genome & exome analysis of early colon cancers reveals new targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4703. doi:10.1158/1538-7445.AM2014-4703

618 A Novel Somatic Mutation of β2sp Inactivates the TGF-β Pathway and Promotes Alcohol Induced HCC

A S L D A b st ra ct s vehicle:1337±552 vs crecont, FGF-19:196±50 and c-foshypo, vehicle: 1395±315 vs c-foshypo, FGF-19:125±8]. ASBT protein levels were not reduced in the ileum of the c-fosintmice treated with FGF-19 [c-fosint-, vehicle: 1413±83 vs c-fosint-, FGF19: 1453±79]. FGF-19 treatment led to a marked increase in phospho-ERK1/2, JNK1 and JNK2 in both wild type and c-fosintmice [1) pERK1/2: wt: 2199±771 vs FGF-19: 8517±1170, c-fosint-,vehicle: 2963±1200 vs c-fosint-,FGF19: 9215±2200; 2) JNK1: wt: 268±130 vs FGF-19: 1433±249, c-fosint-,vehicle: 251±75 vs c-fosint-,FGF19: 1526±202; 3) JNK2: wt: 261±46 vs FGF-19: 897±152, c-fosint-,vehicle: 201±55 vs c-fosint-,FGF19: 1440±257]. p-c-jun was markedly increased in ileal nuclear extracts from wild type and c-fosintmice treated with FGF-19 [wt: 71±7 vs FGF-19: 1119±218; c-fos-/-,vehicle: 80±11 vs c-fos-/-,FGF19: 1179±245]. Nuclear pc-fos was increased in wild type mice but not in the c-fosintmice where c-fos was not expressed [wt: 1564±352 vs FGF-19: 7862±2635]. ASBT expression was repressed in the gallbladder in response to FGF-19 treatment of all mice including the c-fosintmice. Conclusion: Activation and phosphorylation of c-fos is critical to FGF-19 mediated repression of ASBT.

Abstract 1775: High resolution characterization of human hepatocellular cancer (HCC) reveals a novel inactivating mutation in the TGF-β pathway that promotes alcohol induced HCC.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: A crucial factor in protection from liver injury, fibrosis and cancer by agents such as alcohol, aflatoxins and viral hepatitis is the enforcement of genomic stability. However, sensors for genotoxicity leading to aberrant DNA repair remain elusive. Because inactivation of the TGF-β pathway results in DNA damage, alcohol toxicity and HCC, TGF-β has been implicated as a critical promoter of genomic stability and tumor suppression; however, the framework by which this occurs is not known. We performed Whole-genome (WGS) and Exome sequencing (WES) on 6 pairs human HCC samples and identified inactivation of TGF-beta pathway members as a prominent characteristic of alcohol induced HCC, potentially through inactivation of a TGF-b adaptor, β2SP. We therefore hypothesized that the TGF-β/β2SP/Smad3 pathway is crucial for protection against aldehyde genotoxicity through enforcing genomic stability. Materials & Methods: We performed Whole-genome (WGS) and Exome sequencing (WES) on 6 pairs human HCC samples. β2SP mutant mice were treated with alcohol to determine their susceptibility to aldehyde-induced developmental abnormalities. Primary MEFs from β2SP+/+, β2SP+/− and β2SP−/− mice were treated with colcemid for cytogenetic analysis, and they were also treated with multiple DNA damaging agents and γ-irradiation to evaluate Mdc1, NBS1 and Rad51 foci formation. ChIP assays were performed to determine the recruitment of β2SP/Smad3/Smad4 at FancD2 promoter. Results: (1) Through our WGS and WES analyses, we discovered a novel inactivating mutation of TGF-β/β2SP pathway in alcohol-associated HCC. This β2SP mutation results in functional disruption of TGF-β signaling. (2) Alcohol treatment in the absence of β2SP mice results in teratogenicity and spontaneous fetal alcohol-like phenotype (FAS). (3) Loss of β2SP results in premature replicative senescence and marked hypersensitivity to DNA interstrand crosslinking agents, such as mitomycin C (MMC). (4) DNA damage response induces nuclear localization of β2SP in a TGF-β-dependent manner. (5) Suppression of β2SP impairs the recruitment of DNA repair proteins in the nucleus and consequently the repair of DNA double-strand breaks. (6) Loss of β2SP correlates with loss of FANCD2 expression, but not of any of the other 12 Fanconi anemia complementation factors. Moreover, our results indicate that β2SP/Smad3/Smad4 regulate expression of FANCD2 at the transcriptional level. (7) The expression of β2SP and FANCD2 is correlated in alcoholic hepatitis and cirrhotic livers. Conclusions: Through whole genome and Exome sequencing we found a novel inactivating mutation in β2SP which results in loss of TGF-β signaling pathway. Importantly, these results could potentially lead to new therapeutics targeting toxin-induced DNA damage and tumorigenesis. Citation Format: Vivek Shukla, Jian Chen, Jiun-Sheng Chen, Ying Li, Lior Katz, Avijit Majumdar, Lei Li, Walter Hittleman, Xiaoping Su, Junjie Chen, Xifeng Wu, Patrizia Farci, Lopa Mishra. High resolution characterization of human hepatocellular cancer (HCC) reveals a novel inactivating mutation in the TGF-β pathway that promotes alcohol induced HCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1775. doi:10.1158/1538-7445.AM2013-1775

Abstract 4335: CEA: A therapeutic target for the treatment of advanced colorectal cancer.

Introduction: Advanced colorectal cancer (CRC) remains the second most common cause of cancer mortality worldwide. Carcinoembryonic antigen (CEA) represents one of the few biomarkers approved by the U.S. Food and Drug Administration (FDA). Ectopic expression of CEA induces CRC metastases, potentially indirectly through activation of STAT3, stimulating production of IL-6, IL-10 and others. Yet until recently a clear mechanism of CEA in tumorigenesis has remained elusive. Our study demonstrated that CEA inactivates the tumor suppressive arm of transforming growth factor-β (TGF-β) signaling, providing crucial new mechanistic information for CEA in promoting aggressive CRC. Moreover, we previously found that deletion of Smad3/4 adaptor β2SP results in loss of TGF-β signaling, with dramatic and spontaneous formation of CRC, presenting a strong mouse model of CRC. We hypothesized that CEA interacts through a specific site targeting the TGF-β pathway potentially TGF-βRI leading to aggressive colorectal cancers. Impeding this interaction by using specific antibodies or peptide inhibitors may be used as a therapeutic agent to treat advanced CRC. Methods & Results: We observed that CEA interacts directly with the transforming growth factor-β (TGF-β) receptor 1 (TGF-βR1) and causes inhibition of downstream TGF-β tumor suppressor signaling. Targeting CEA with either an anti-CEA specific antibody or siRNA-mediated CEA silencing restores the tumor suppressive properties of TGF-β signaling. Detailed analysis of this interaction revealed the D7 fragment of CEA responsible for binding with TGF-β receptor I and capable of inhibiting tumor suppressor function of TGF-β signaling as evident by the ability of D7 fragment to reduce phospho -Smad3 level upon TGF-β stimulation. In human CRC, increased CEA levels are associated with loss of TGF-β signaling. Immunohistochemical analysis of the CRC revealed induction of phospho STAT3 level compared to normal colon tissue. To impede the CEA mediated suppression of growth inhibitory TGF-β signaling we are currently using antibodies which block this interaction and reinforce tumor suppressor TGF-β signaling. Conclusions: Our results demonstrate that CEA induces CRC metastasis through interacting with TGF-βR1 and suppressing growth inhibitory TGF-β signaling pathway which is mediated by the D7 fragment of CEA. Inhibiting the interaction of CEA and TGF-βR1 using peptide inhibitors/antibodies /small molecule inhibitors will be very useful in preventing CRC cell growth and proliferation. Thus the study holds promise to generate novel therapeutic agents for the treatment of lethal metastatic CRC. Citation Format: Avijit Majumdar, Ying Li, Sue-Hwa Lin, Lopa Mishra. CEA: A therapeutic target for the treatment of advanced colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4335. doi:10.1158/1538-7445.AM2013-4335

Abstract 897: CEA induces colorectal cancer metastases by disrupting TGF-β signaling

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Advanced colorectal cancer (CRC) remains the second most common cause of cancer mortality worldwide. Carcinoembryonic antigen (CEA) represents one of the few biomarkers approved by the U.S. Food and Drug Administration (FDA) as signifying metastatic CRC, potentially through activation of STAT3, stimulating production of IL-6, IL-10 and others. Yet until recently a clear functional role of CEA in tumorigenesis has remained elusive. Our study demonstrated that CEA inactivates the tumor suppressive arm of transforming growth factor-β (TGF-β) signaling, providing crucial new mechanistic information for CEA in promoting aggressive CRC. Moreover, we previously found that deletion of Smad3/4 adaptor β2SP results in loss of TGF-β signaling, with dramatic and spontaneous formation of CRC, presenting a strong mouse model of CRC. We hypothesized that the disruption of the TGF-β tumor suppressor pathway by CEA (through TGF-β receptor TαR1, β2SP, Smad3 and Smad4) leads to aggressive colorectal cancers. Methods & Results: We observed that CEA plays a role in CRC metastasis by interacting directly with the transforming growth factor-β (TGF-β) receptor 1 (TαR1) and inhibiting downstream TGF-β tumor suppressor signaling. Targeting CEA with either an anti-CEA specific antibody or siRNA- mediated CEA silencing restores the tumor suppressive properties of TGF-β signaling. Ectopic expression of CEA induces CRC metastases. In human CRC, increased CEA levels are associated with loss of TGF-β signaling. Detailed analysis of the interaction between CEA and TαR1 by generating deletion fragments of CEA further revealed that the N terminal domain of CEA containing Ig like domain 1 is responsible for binding with TαR1. Our data also demonstrate that this fragment can impede the growth inhibitory TGF-β signaling by inhibiting the phospho SMAD3 level upon TGF-β treatment. Immunohistochemical analysis of the CRC revealed induction of phospho STAT3 level compared to normal colon tissue and STAT3 inhibitor inhibited the proliferation of cells with impaired TGF-β signaling. A novel triterpenoid molecule RTA402 inhibits CRC cell proliferation both through inhibition of STAT3 and restoration of TGF-β pathway in cell lines with activated CEA. Conclusions: Our results demonstrate that CEA induces CRC metastasis through interacting with TαR1 and suppressing growth inhibitory TGF-β signaling pathway. Inhibiting the interaction of CEA and TαR1 using peptide inhibitors will be very useful in preventing CRC cell growth and proliferation. It will be interesting to see the tumor suppressive effect of these peptide inhibitors and/or RTA402 in the consistently aggressive mouse model of CRC with high CEA levels. Thus the study holds promise to generate novel therapeutic agents for the treatment of lethal metastatic CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 897. doi:1538-7445.AM2012-897