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Supplementary Figure 8 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 2. Knockdown of Mfng has no effect on proliferation of MDA-MB231 or C0321 cells

Supplementary Information. Including Supplementary Figure Legends, Supplementary Methods, and Supplementary References.

Supplementary Tables 1-3 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 4 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Table 1. A list of top 200 differentially expressed genes in xenografts from injection of shMFNG MDA-MB231 cells vs. control MDA-MB231 cells

Supplementary Figure 7 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 9 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 1. Generation of the Mfng mutant mice and expression analysis of Mfng in the mouse mammary gland

Supplementary Figure Legends from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 2 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 4. Pharmacological inhibition of PI3Kγ has no effect on proliferation of MDA-MB231 cells, but shows an inhibitory effect on C0321 cells.

Supplementary Figure 3 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 6 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 3. A reactome functional interaction network for genes that are differentially expressed in xenografts of shMFNG and control MDA-MB231 cells

Supplementary Figure 5 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 1 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Table 2. Correlation studies between MFNG, NOTCH4 and PIK3CG gene expressions using 6 independent data sets

The transcription factor Prdm16 functions as a potent suppressor of transforming growth factor-beta (TGF-β) signaling, whose inactivation is deemed essential to the progression of pancreatic ductal adenocarcinoma (PDAC). Using the KrasG12D-based mouse model of human PDAC, we surprisingly found that ablating Prdm16 did not block but instead accelerated PDAC formation and progression, suggesting that Prdm16 might function as a tumor suppressor in this malignancy. Subsequent genetic experiments showed that ablating Prdm16 along with Smad4 resulted in a shift from a well-differentiated and confined neoplasm to a highly aggressive and metastatic disease, which was associated with a striking deviation in the trajectory of the premalignant lesions. Mechanistically, we found that Smad4 interacted with and recruited Prdm16 to repress its own expression, therefore pinpointing a model in which Prdm16 functions downstream of Smad4 to constrain the PDAC malignant phenotype. Collectively, these findings unveil an unprecedented antagonistic interaction between the tumor suppressors Smad4 and Prdm16 that functions to restrict PDAC progression and metastasis.

Notch3 promotes mammary luminal cell specification and forced Notch3 activation can induce mammary tumor formation. However, recent studies suggest a tumor-suppressive role for Notch3. Here, we report on Notch3 expression and functional analysis in the mouse mammary gland. Notch3 is expressed in the luminal compartment throughout mammary gland development, but switches to basal cells with initiation of post-lactational involution. Deletion of Notch3 caused a decrease of Notch activation in luminal cells and diminished luminal progenitors at puberty, as well as reduced alveolar progenitors during pregnancy. Parous Notch3−/− mammary glands developed hyperplasia with accumulation of CD24hiCD49flo cells, some of which progressed to invasive tumors with luminal features. Notch3 deletion abolished Notch activation in basal cells during involution, accompanied by altered apoptosis and reduced brown adipocytes, leading to expansion of parity-identified mammary epithelial cells (PI-MECs). Interestingly, the postpartum microenvironment is required for the stem cell activity of Notch3−/− PI-MECs. Finally, high expression of NOTCH3 is associated with prolonged survival in patients with luminal breast cancer. These results highlight an unexpected tumor-suppressive function for Notch3 in the parous mammary gland through restriction of PI-MEC expansion.

The prostate epithelium consists of predominantly luminal cells that express androgen receptor and require androgens for growth. As a consequence, the depletion of testicular androgens in patients with prostate cancer results in tumor regression. However, it eventually leads to a castration-resistant disease that is highly metastatic. In this report, a mouse model of metastatic prostate cancer was generated through the deletion of the tumor-suppressor gene Trp53 in conjunction with oncogenic activation of the proto-oncogene Kras. These mice developed early-onset metastatic prostate cancer with complete penetrance. Tumors from these mice were poorly differentiated adenocarcinoma, characterized by extensive epithelial-mesenchymal transition. With no or a very low level of androgen receptor expression, the tumor cells were resistant to androgen receptor inhibition. Pik3cg, encoding phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit γ (Pi3kγ), was highly expressed in these tumors, and pharmacologic inhibition of Pi3kγ blocked tumor cell growth in vitro, reversed epithelial-mesenchymal transition, and abated tumor metastasis in vivo. Immunohistochemistry analysis in human prostate cancer specimens showed that the expression of PIK3CG was significantly associated with advanced clinical stages. Taken together, these results suggest that PIK3CG plays an important role in the progression and metastasis of prostate cancer, and may represent a new therapeutic target in the metastatic castration-resistant prostate cancer.

A critical feature of cancer is the ability to induce immunosuppression and evade immune responses. Tumor-induced immunosuppression diminishes the efficacy of endogenous immune responses and decreases the efficacy of cancer immunotherapy. In this study, we describe a new immunosuppressive pathway in which adenosine promotes Cbl-b-mediated Notch1 degradation, causing suppression of CD8+ T-cells effector functions. Genetic KO and pharmacological inhibition of Cbl-b prevents Notch1 degradation in response to adenosine and reactivates its signaling. Reactivation of Notch1 results in enhanced CD8+ T-cell effector functions, anti-cancer response and resistance to immunosuppression. Our work demonstrates that targeting Cbl-b-Notch1 axis is a novel promising strategy for cancer immunotherapy.

Purpose Molecular targeting is a powerful approach for aggressive claudin-low breast cancer (CLBC). Overexpression of PI3K catalytic subunit gamma (PIK3CG) in human CLBC is offering a promising opportunity for targeted therapies. We utilized a specific inhibitor of PIK3CG combined with paclitaxel (PTX) to treat CLBC cells in vitro and in vivo. Patients and methods The tumor cells growth and apoptosis in vitro were analyzed by CCK8, plate clone formation assay, tumorsphere assay, Hoechst staining and flow cytometry. The invasion and metastasis ability of tumor cells in vitro were investigated by wound healing and transwell experiments. Critical gene expression levels were checked by qRT-PCR and Western blot. Xenograft models with CLBC cell lines in SCID mice were established to investigate the effect of combined drugs in vivo. Results We identified that PIK3CG was a potential therapeutic target for CLBC patients. Targeting PIK3CG potentiated CLBC cells growth inhibition in 2D and 3D cultures by PTX. Inhibition of PIK3CG activation could enhance CLBC cells apoptosis and migration suppression induced by PTX. Manipulating autophagy was a validated approach for the use of PIK3CG inhibitor. Using CLBC xenograft mice model, we found that CLBC tumors in vivo could be well treated by combined drugs of PIK3CG inhibitor and PTX. Conclusion We demonstrated that PIK3CG was a potential target for the therapy of CLBC and inhibition of PIK3CG activation could reinforce the therapeutic effect of this aggressive disease by PTX. The combined use of PIK3CG inhibitor and PTX might be a potential regimen for treating this subtype of breast cancer.

A long-standing question in the pancreatic ductal adenocarcinoma (PDAC) field has been whether alternative genetic alterations could substitute for oncogenic KRAS mutations in initiating malignancy. Here, we report that Neurofibromin1 (NF1) inactivation can bypass the requirement of mutant KRAS for PDAC pathogenesis. An in-depth analysis of PDAC databases reveals various genetic alterations in the NF1 locus, including nonsense mutations, which occur predominantly in tumors with wild-type KRAS. Genetic experiments demonstrate that NF1 ablation culminates in acinar-to-ductal metaplasia, an early step in PDAC. Furthermore, NF1 haploinsufficiency results in a dramatic acceleration of Kras

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive breast cancers. TNBC patients have a high risk of recurrence and metastasis, and current treatment options remain limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Emerging evidence suggests that cancer stem-like cells (CSCs) that escape chemo or radiation therapy in TNBC are often Notch-dependent. At the same time, there is evidence that active tumor immunity predicts good response to neo-adjuvant chemotherapy in TNBC. Notch inhibitors, including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC, where they eliminate CSCs resistant to chemotherapy. However, the success of GSIs in clinical trials is limited by their intestinal toxicity and adverse immunological effects. CD4 and CD8 T-cells, necessary to adaptive tumor immunity, require Notch1 for activation. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSI. SS has Gamma Secretase Modifier (GSM) activity. We confirmed that SS inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In contrast, SS did not inhibit Notch expression or cleavage in murine T cells. In C0321 tumors, which recapitulate human mesenchymal TNBC, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors. SS caused an increase in intra-tumoral CD11c+ dendritic cells, but decreased CD4 cells, which in this model are largely PD-1 positive (exhausted). CD8 cells were modestly increased. SS did not affect the number of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. We are currently investigating the mechanisms of this anti-tumor activity. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy agents. Repurposing an FDA-approved, safe agent for the treatment of TNBC would be significantly easier and more cost-effective than developing unproven investigational agents. Citation Format: Hossain F, Ucar D, Majumder S, Xu K, Ran Y, Minter L, Xi Y, Burow M, Golde T, Osborne B, Miele L. Repurposing sulindac sulfide as a notch inhibitor to target cancer stem-like cells in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-22-01.

Pancreatic ductal adenocarcinoma formation culminates in hyperactivation of transforming growth factor β signaling, which in turn induces selective depletion of β-cell mass and thereby leading to new-onset diabetes., Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease that remains incurable because of late diagnosis, which renders any therapeutic intervention challenging. Most PDAC patients develop de novo diabetes, which exacerbates their morbidity and mortality. How PDAC triggers diabetes is still unfolding. Using a mouse model of KrasG12D-driven PDAC, which faithfully recapitulates the progression of the human disease, we observed a massive and selective depletion of β-cells, occurring very early at the stages of preneoplastic lesions. Mechanistically, we found that increased TGF beta (TGF-β) signaling during PDAC progression caused erosion of β-cell mass through apoptosis. Suppressing TGF-β signaling, either pharmacologically through TGF-β immunoneutralization or genetically through deletion of Smad4 or TGF-β type II receptor (TβRII), afforded substantial protection against PDAC-driven β-cell depletion. From a translational perspective, both activation of TGF-β signaling and depletion of β-cells frequently occur in human PDAC, providing a mechanistic explanation for the pathogenesis of diabetes in PDAC patients, and further implicating new-onset diabetes as a potential early prognostic marker for PDAC.

Molecular targeting is a powerful approach for aggressive claudin-low breast cancer (CLBC). Overexpression of PI3K catalytic subunit gamma (PIK3CG) in human CLBC is offering a promising opportunity for targeted therapies. We utilized a specific inhibitor of PIK3CG combined with paclitaxel (PTX) to treat CLBC cells in vitro and in vivo.The tumor cells growth and apoptosis in vitro were analyzed by CCK8, plate clone formation assay, tumorsphere assay, Hoechst staining and flow cytometry. The invasion and metastasis ability of tumor cells in vitro were investigated by wound healing and transwell experiments. Critical gene expression levels were checked by qRT-PCR and Western blot. Xenograft models with CLBC cell lines in SCID mice were established to investigate the effect of combined drugs in vivo.We identified that PIK3CG was a potential therapeutic target for CLBC patients. Targeting PIK3CG potentiated CLBC cells growth inhibition in 2D and 3D cultures by PTX. Inhibition of PIK3CG activation could enhance CLBC cells apoptosis and migration suppression induced by PTX. Manipulating autophagy was a validated approach for the use of PIK3CG inhibitor. Using CLBC xenograft mice model, we found that CLBC tumors in vivo could be well treated by combined drugs of PIK3CG inhibitor and PTX.We demonstrated that PIK3CG was a potential target for the therapy of CLBC and inhibition of PIK3CG activation could reinforce the therapeutic effect of this aggressive disease by PTX. The combined use of PIK3CG inhibitor and PTX might be a potential regimen for treating this subtype of breast cancer.

Pancreatic cancer is one of the leading causes of cancer-associated mortality. The understanding of the expression pattern of key protein factors and their function in pancreatic cancer cells is therefore vital for the diagnosis and treatment of this malignancy. The results of the present study reveal that the levels of neurogenic locus notch homolog protein 2 (Notch2) and phosphorylated (p)-SMAD family member 2 decreased, whereas the expression of Notch3 and phosphoinositide-3 kinase catalytic subunit-γ protein increased in human pancreatic cancer tissues compared with tumor-adjacent tissues. Using the human pancreatic cancer MIA PaCa-2 cell line, it was observed that retinoblastoma-associated protein (RB) and p-RB expression were inhibited and p-AKT was upregulated when Notch signaling was activated in MIA PaCa-2 cells. Furthermore, inhibition of phosphoinositide-3 kinase catalytic subunit-γ (PIK3CG) activity by AS-605240 was able to block the growth and migration of MIA PaCa-2 cells. In conclusion, the results of the present study demonstrate that the Notch signal pathway may be involved in pancreatic carcinogenesis by modulating RB and p-AKT. PIK3CG may therefore be a potential target gene for the treatment of pancreatic cancer.

Background Kras mutations and increased Notch activation occur frequently in gallbladder cancer. However, their roles in gallbladder carcinogenesis have not been defined. This study was aimed at determining whether expression of mutant Kras was sufficient to induce gallbladder carcinoma and whether Notch deregulation played a role in this context. Methods We determined Cre recombination activity of Pdx1-Cre in the gallbladder using a reporter strain and examined gallbladder tumor development in the KrasLSL- G12D/+;Pdx1-Cre mice. We analyzed expression of Notch pathway genes in the mouse gallbladder by immunohistochemistry, quantitative RT-PCR, and Western blot analysis. We also determined the effect of Jag1 deletion on Kras-induced gallbladder tumor development. Results Pdx1-Cre exhibits robust recombination activity in the gallbladder epithelium. KrasLSL-G12D/+;Pdx1-Cre mice form early onset adenoma in the gallbladder and adjacent biliary tract with complete penetrance, albeit short of invasive adenocarcinoma. KrasG12D upregulates expressions of Notch2, Notch3, Notch4, Jag1 and downstream target genes Hes1, Hey1 and Hey2, and deletion of Jag1 partially suppresses KrasG12D-induced adenoma development. Conclusions KrasG12D induces gallbladder adenoma and Notch plays a key role in Kras-initiated gallbladder tumorigenesis.

The Notch signaling pathway has been implicated in the development as well as tumorigenesis of the mammary gland. Notch3 promotes luminal cell specification and marks a highly clonogenic luminal progenitor population. Aberrant Notch3 activation has been shown to promote the growth of basal breast cancer as well as resistance to hormonal therapy in metastatic luminal breast cancer. Interestingly, recent studies also suggest a tumor-suppressive function of Notch3 in mammary epithelium through regulation of estrogen receptor α (ER). Here we report the expression and functional analysis of Notch3 in the mouse mammary gland. X-gal staining of the Notch3β-Geo/+ mammary gland showed that Notch3 was highly expressed in luminal cells throughout mammary development with exception at the initiation of post-lactational involution, when Notch3 expression was restricted to the basal cells. Deletion of Notch3 caused decreased Notch activation in the CD24HiCD49fLo subpopulation of the pubescent mammary gland, accompanied by a significant decrease in the number of CD24HiCD49fLoCD61+ luminal progenitor cells. Whole-mount preparation of the Notch3 knockout mammary glands showed normal development during puberty, pregnancy, lactation and involution. Surprisingly, parous Notch3 knockout mice developed mammary ductal hyperplasia by 10 months of age, some of which progressed to ductal carcinoma in situ, and ultimately to invasive and metastatic cancer. Parous Notch3 knockout mice exhibited an expansion of the CD24HiCD49fLo subpopulation, and mammary tumors from these mice were composed predominantly of CD24HiCD49fLo cells. The vast majority of these tumors were ER-positive and relatively well differentiated, with a papillary and/or glandular pattern. They expressed the luminal marker cytokeratin 8, and some of them co-expressed cytokeratin 14, a basal marker. All Notch3 knockout mammary tumors showed high level expression of Cyclin D1. These results suggest that Notch3 may prevent ER-positive breast cancer through suppression of CD24HiCD49fLo progenitor cell self-renewal in the postpartum mammary gland. In addition to its impact on mammary epithelium, deletion of Notch3 altered the mammary microenvironment, in particular, brown adipocyte differentiation in the mammary fat pad. Further experiments will be performed to define a potential role for Notch3-regulated brown adipose tissue in breast cancer initiation and progression. Finally, analysis of TCGA and METABRIC human breast cancer data sets revealed a negative correlation between expressions of NOTCH3 and ESR1 (encoding estrogen receptor α), and association of high NOTCH3 expression with enhanced survival of patients with luminal A and luminal B subtype breast tumors. Taken together, Notch3 can act as a tumor suppressor in the mammary epithelium, and possibly in the mammary microenvironment as well, to prevent development and metastasis of ER-positive breast cancer. Citation Format: Wen-Cheng Chung, Sean E. Egan, Keli Xu. Notch3 functions as a tumor suppressor in ER-positive breast cancer [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 3111.

Jag1 functions as a tumor suppressor in delaying pancreatic cancer precursor, while at the same time, promotes a phenotypic switch from benign cystic lesions to invasive carcinoma of the pancreas., Notch signaling exerts both oncogenic and tumor-suppressive functions in the pancreas. In this study, deletion of Jag1 in conjunction with oncogenic KrasG12D expression in the mouse pancreas induced rapid development of acinar-to-ductal metaplasia and early stage pancreatic intraepithelial neoplasm; however, culminating in cystic neoplasms rather than ductal adenocarcinoma. Most cystic lesions in these mice were reminiscent of serous cystic neoplasm, and the rest resembled intraductal papillary mucinous neoplasm. Jag1 expression was lost or decreased in cystic lesions but retained in adenocarcinoma in these mice, so was the expression of Sox9. In pancreatic cancer patients, JAG1 expression is higher in cancerous tissue, and high JAG1 is associated with poor overall survival. Expression of SOX9 is correlated with JAG1, and high SOX9 is also associated with poor survival. Mechanistically, Jag1 regulates expression of Lkb1, a tumor suppressor involved in the development of pancreatic cystic neoplasm. Collectively, Jag1 can act as a tumor suppressor in the pancreas by delaying precursor lesions, whereas loss of Jag1 promoted a phenotypic switch from malignant carcinoma to benign cystic lesions.

A prominent function of TGIF1 is suppression of transforming growth factor beta (TGF‐β) signaling, whose inactivation is deemed instrumental to the progression of pancreatic ductal adenocarcinoma (PDAC), as exemplified by the frequent loss of the tumor suppressor gene SMAD4 in this malignancy. Surprisingly, we found that genetic inactivation of Tgif1 in the context of oncogenic Kras, Kras(G12D), culminated in the development of highly aggressive and metastatic PDAC despite de‐repressing TGF‐β signaling. Mechanistic experiments show that TGIF1 associates with Twist1 and inhibits Twist1 expression and activity, and this function is suppressed in the vast majority of human PDACs by Kras(G12D)/MAPK‐mediated TGIF1 phosphorylation. Ablating Twist1 in Kras (G12D) ;Tgif1 (KO) mice completely blunted PDAC formation, providing the proof‐of‐principle that TGIF1 restrains Kras(G12D)‐driven PDAC through its ability to antagonize Twist1. Collectively, these findings pinpoint TGIF1 as a potential tumor suppressor in PDAC and further suggest that sustained activation of TGF‐β signaling might act to accelerate PDAC progression rather than to suppress its initiation.