Jh Carlson

Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers.

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronection-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP.

Abstract P5-03-14: MLN0128 regulates survival signaling by AKT and its downstream effectors in HER2+ breast cancer model

Evading apoptosis is considered to be a hallmark of cancers including breast cancer, since mutations in apoptotic regulators invariably accompany tumorigenesis. Chemotherapeutic agents induce apoptosis, and hence disruption of apoptosis during tumor progression may promote drug resistance. AKT is an apoptotic regulator that is activated in HER2+ breast tumor cells and promotes anti-HER2 therapy resistance in vitro . Nevertheless, how mTORC1/C2-AKT signaling disables apoptosis and its contribution to clinical drug resistance are not clear yet. Using HER2 amplified breast cancer cells [BT474 ( HER2+/ Trastuzumab-sensitive), BT474HerR ( HER2+/ Trastuzumab-resistant), HCC1954 and MDA-MB453 (both are HER2 +/ PIK3CA kinase domain mutated)], we show that mTORC1/C2 inhibitor; MLN0128 abrogates AKT (Ser473), Survivin and controls its downstream effectors of apoptotic signaling molecules (e.g. cleaved Caspase 3/9, cleaved PARP, MCL and BIM). MLN0128 also induces annexinV positive cells and regulates cellular proliferation (ON-TOP 3D colony formation and real-time proliferation assay). Additionally, increased cleaved Caspase 3 and decreased MCL1 expression were also observed following MLN0128 treatment in HER2+ xenograft model along with tumor growth inhibition. Our studies provide strong experimental evidence that high apoptotic signaling –specifically reduced MCL1 and increased cleaved-CASPASE3 expression expedite the response of targeted therapy that directly inhibits mTORC1/C2-AKT signaling. Citation Format: De PK, Carlson JH, Sun Y, Lin X, Williams C, Dey N, Leyland-Jones BR. MLN0128 regulates survival signaling by AKT and its downstream effectors in HER2+ breast cancer model. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-14.

Abstract P3-14-08: Preclinical efficacy of targeting c-MET by ARQ197 in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer cell lines

Background: Triple-negative breast cancer (TNBC), accounts for 15% of all invasive breast cancers (BCs) and has the poorest survival outcome of all BC subtypes. Due to its heterogeneity, TNBC lacks validated therapeutic targets compared with other BC subtypes (Sohn et al., 2014; Foulkes et al., 2010). Therefore, improved approaches to treatment of these cancers are unmet needed. Several molecular targets including: epidermal growth factor receptor (EGFR), poly ADP ribose polymerase (PARP), and hepatocyte growth factor receptor (c-MET) are under clinical investigation for the treatment of this disease (De et al., 2014; Cleator et al., 2007). The MET oncogene encodes a membrane-bound tyrosine kinase implicated in the formation and/or progression of several cancer types including TNBC, and several studies have shown c-Met overexpression to be an independent predictor of poor outcome in BC (Ho-Yen et al., 2014), c-MET may play a critical role in the development of the most aggressive BCs and may be a rational therapeutic target (Graveel et al., 2009). Currently inhibitors targeting c-MET (including ARQ197) are undergoing clinical trials in a variety of cancers including TNBC (Gaule et al., 2014; ClinicalTrials.gov). Recently, PARP inhibitors in combination with chemotherapy, has shown promising results in TNBC in clinical and preclinical studies (Tutt et al., 2010; De et al., 2014). We argue that, blocking the PARP-mediated nuclear machinery for repairing DNA-damage in presence of cytotoxic DNA damaging agents in conjunction with co-targeting c-Met pathway dependent downstream effectors may have a robust anti-tumor activity in TNBC cell lines. Methodology: BT-20 (PIK3CA mutated, H1047R), HCC70 (PTEN null), HCC1937 (PTEN null), MDA-MB-231 (KRAS/BRAF mutated), MDA-MB-468 (PTEN null) and SUM149PT (BRCA1 mutated) cells were used for this study. Growth inhibition, survival/proliferation, colony formation and apoptosis were examined using MTT assay, 2D proliferative /growth assay, 3D-ON-TOP assays, and annexinV staining respectively. Results: 1) For all TNBC cell lines, the IC50 of single agent ARQ197 was from 0.5 µM to 1.5 µM (following 96 hours treatment) 2) ARQ197 as a single agent or in combination with ABT888 or in triple combination dose dependently decreased cell growth/proliferation 3) annexin V positive cells were increased following treatment with single agent ARQ197 or in combination with ABT888 or in triple combination 4) 70-99% anti-proliferative activities were observed on 3D-ON TOP colony formation assay with ARQ197 alone or in combination in all tested cell lines. Conclusion: Our preclinical in vitro drug sensitivity data suggest that administration of c-MET inhibitor may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC models. Mechanism studies are ongoing, the results of which will be presented in the meeting. Citation Format: Sun Y, Carlson JH, Lin X, De PK, Williams C, Hausman S, Dey N, Leyland-Jones BR. Preclinical efficacy of targeting c-MET by ARQ197 in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer cell lines. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-08.

Abstract P4-08-04: Navigating genomic landscape to find a PI3K-signaling algorithm for a rational combinatin in precision medicine

Background: Treatment of BC is conventionally based on the presence/absence of ER/PR or HER2 status of the primary tumor. We have enriched this approach by including major genetic and proteomic changes in tumors of individual patients in order to develop a better treatment-rationale based on an alteration driven signaling algorithm. Methods: Genomic and proteomic data from 75 BC patients seen in our center were retrospectively analyzed. Patients were re-biopsied after consultation and samples were characterized (IHC for ER, PR, and HER2; FFPE samples for genomic [Foundation Medicine] and proteomic analyses [Theranostics]). In vivo studies were conducted using xenograft models. Results: Although alterations of PIK3CA, PIK3R1, AKT, PTEN, MDM2, MDM4, TSC1, mTOR and RICTOR are most frequently observed in our patients, there is a distinct pattern of alteration(s) of the PI3K pathway genes in different subtypes of BC. A total of 76 genes were altered in 48 ER+BC patients. In 79% of ER+BC patients the above mentioned PI3K pathway genes were altered. Analyzing the set of alterations of genes in individual patients, we observed that within these 48 patients 25% exhibited alterations in more than one node of the pathway; the most common combination (alterations) being the amplification/mutation of PIK3CA with the amplification of MDM2/4 genes. The percentage of patients belonging to HER2+ & TNBC exhibiting similar alterations in the PI3K pathway genes were significantly lower (∼40%). Our previous in vivo studies demonstrated that GDC-0980 and BEZ235 enhanced the antitumor activity of ABT888 plus carboplatin in TNBC or trastuzumab in HER2+ BC respectively and blocked the growth of established xenograft tumors by 80% to 90% with a concomitant decrease in tumor Ki67, pS6RP and CD31. Mechanistically the action of the PI3K-mTOR pathway targeted drug(s) was tested using cell line based models of BC subtypes pertaining to their respective genomic alterations. A combination of a pan-PI3K pathway inhibitor, GDC-0941 or isoform-specific inhibitors along with AI, trastuzumab, or HRD inhibitors (PARP) blocked proliferative signals and enhanced apoptosis (cleaved caspase3) in ER+/PIK3CA mutated, HER2+/PIK3CA mutated or PTEN-null TNBC cells respectively as demonstrated by WB, flow cytometry, cell proliferation, viability and cytotoxicity assays. A recent study demonstrated that exposure to chemotherapy induced a phenotypic shift or cell state transition towards a transient CD44Hi/CD24Hi chemotherapy-tolerant state, leading to the activation of downstream non-receptor tyrosine kinase signaling towards an emerging adaptive resistance (Goldman et al., Nature Comm. 2015). Hence drug combination(s) are being tested for their effect on CD44/CD24 expression levels, results of which will be presented in the meeting. Conclusion: Plotting the genetic alterations from the patient on the signaling landscape will be useful in cracking the code leading to improved treatment options. Patient specific in-depth plotting of genetic alterations of the PI3K-mTOR pathway and the relevance of these alterations in the context of (1) mechanisms of PI3K-mTOR pathway targeted drugs and (2) cell signaling are critical in determining choice of drugs in BC subtypes. Citation Format: Carlson JH, Krie A, Williams C, Sun Y, Lin X, Williams K, Klein J, Friedman L, De P, Dey N, Leyland-Jones B. Navigating genomic landscape to find a PI3K-signaling algorithm for a rational combinatin in precision medicine. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-04.

Erratum to “Doubling Down on the PI3K-AKTmTOR Pathway Enhances the Antitumor Efficacy of PARP Inhibitor in Triple Negative Breast Cancer Model beyond BRCA-ness” [Neoplasia 16 (2014) 43–72]

[This corrects the article DOI: 10.1593/neo.131694.].

HER2 amplification and overexpression between primary and liver metastatic breast cancer: Significance and implication.

150 Background: The systemic management of metastatic breast cancer (MBC) is mostly based on the ER or HER2 status of the primary tumor. However, the hormonal status or the amplificaction/overexpression HER2 may change in every metastatic site because of the effects of the long-term treatment of metastatic cancer with endocrine therapy, chemotherapy, or targeted agents. The purpose of this study was to investigate the frequency of change in HER2 expression in primary and distant metastatic tumors (especially in liver) in HER2+ breast cancer patients. METHODS We retrospectively analyzed the results of 31 consecutive metastatic breast cancer patients that were seen in our center over 4 months from February 2014 through May 2014. All patients were rebiopsied after consultation and samples were sent for standard immunohistochemistry (IHC) for ER, PR, and HER2 and formalin-fixed, paraffin-embedded (FFPE) samples were sent for genomic (Foundation Medicine) and proteomic analysis (Theranostics). All results from the metastatic samples were compared to the baseline IHC and/or FISH results for HER2. RESULTS A change in HER2 status was observed in 26% of the cases. 16% of cases underwent a negative to positive conversion in HER2 status while 10% of cases underwent a positive to negative conversion. It is notable that all 5 patients that underwent a negative to positive conversion in HER2 status had biopsies taken from metastatic disease in the liver. Overall, 45% of patients with metastatic disease in the liver had a negative to positive conversion in HER2 status. CONCLUSIONS The results of this study emphasize the significance of confirming HER2 expression in a recurrence lesion. This discordance may be due to the increasing level of genetic instability occurring throughout disease progression that can significantly influence the alterations of the HER2 gene. If feasible, HER2 reassessment in metastatic lesions should be carefully taken into account, especially for metastases coming from non-HER2 amplified breast cancer. Although HER2 status is usually appraised in primary tumor, knowledge of the HER2 status in metastases may be of potential value for therapeutic decision making.

Abstract 544: Dual PI3K/mTOR inhibitor, GDC-0980 sensitizes BRCA-competent triple negative breast cancer cells to ABT888 in the presence of carboplatin by impairing DNA-damage repair: A proof of concept combination of PI3K/mTOR inhibitor with PARP inhibitor .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: PARP is a promising target in the Triple Negative subset of Breast Cancer, the most aggressive subset characterized by poor prognosis and limited option for targeted therapy. The PI3K pathway, in addition to its pro-proliferative effects on tumor cells, also contributes to the repair of DSB in both a kinase-dependent and independent manner (Kumar, 2010) directly by controlling relevant enzymes (Friedman, 2009) or indirectly by regulating the expression of repair-related proteins. PI3K inhibition impairs BRCA1/2 expression in BRCA-proficient cells leading to the DNA damage, and HR impairment (Juvekar, 2012; Ibrahim, 2012). Purpose: We hypothesize that an additional inhibition of HR and NHEJ by GDC-0980 in conjunction with the inhibition of classical PI3K-mTOR survival signals, will induce more effective anti-proliferative and pro-apoptotic signals in BRCA-competent TNBC cells when combined with PARP inhibitor (impaired DNA-SSB-repair), and carboplatin (increased DNA-DSB). Methods: We tested effects of ABT888, and GDC-0980 (alone, and combination), on cell survival/ proliferation in a panel of 5-7 BRCA-wt and mutant TNBC cell lines using MTT assay, CelltiterGLO, and cell cycle analyses (PI & EDU). Clonogenic growth was tested by 3D ON-TOP assay and soft-agar assay. Apoptosis (Annexin V, Apoptosis Array), and cell signaling marker(s) (WB) were investigated. Results: The results show that, (1) the range of EC50s for GDC-0980 in cells (HCC70, HCC1143, HCC1937, MDA-MB231, MDA468, BT20) varied from 0.2 μM to 5 μM; PIK3CA mutated (H1047R) BT20 cells exhibited the lowest EC50, while RAS/RAF mutated MDA-MB231 cells exhibited the highest EC50, (2) PTEN-null and ATM kinase mutated MDA-MB468 cells were the most sensitive to ABT888 in the dose-response study by clonogenic assay (ABT888 failed to show a response by MTT assay), (3) out of three doses of GDC-0980 (200 nM, 100 nM, and 50 nM) tested in combination with ABT888 (10μM) plus carboplatin (10 μM), BRCA-competent MDA-MB468 cells were found to be more sensitive to the combination at 50 nM GDC-0980 than BRCA-competent MDA-MB231 cells, (4) GDC-0980 treatment (50nM) decreased pAKT-S473 and pP70S6K, and (5) GDC-0980 treatment time-dependently increased cleaved-caspase 3, 9, and cleaved-PARP in cells. Conclusion: Data indicate that GDC-0980 enhances the activity of ABT888, in the presence of carboplatin. We are currently pursuing the studies to delineate the mechanistic relationship between the anti-proliferative, pro-apoptotic, and anti-DNA-damage-repair effects of GDC-0980 in BRCA-competent TNBC cell lines in combination with ABT888 plus carboplatin, the results of which will be presented in the meeting. Citation Format: Nandini Dey, Jennifer Carlson, Yuliang Sun, Lori Friedman, Pradip De, Brian Leyland-Jones. Dual PI3K/mTOR inhibitor, GDC-0980 sensitizes BRCA-competent triple negative breast cancer cells to ABT888 in the presence of carboplatin by impairing DNA-damage repair: A proof of concept combination of PI3K/mTOR inhibitor with PARP inhibitor . [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 544. doi:10.1158/1538-7445.AM2013-544

Abstract P3-04-02: Absence of PTEN facilitates the anti-tumor efficacy of GDC-0980 in combination with ABT888 plus carboplatin in BRCA1-competent triple negative breast cancer

Introduction: PI3K pathway, in addition to its pro-proliferative and anti-apoptotic effects on tumor cells, is known to contribute to DNA-damage repair (DDR). We hypothesized that GDC-0980, a dual PI3K-mTOR inhibitor, will induce an efficient anti-tumor effect in BRCA-competent PTEN-null TNBC cells when combined with PARP inhibitor, ABT888 and carboplatin. We propose that in PTEN-null BRCA-competent TNBC model, the growth of TNBC tumor will be blocked due to the inhibition of (1) HR and NHEJ and (2) PI3K-mTOR pathway mediated survival signals following treatment with GDC-0980, when combined with PARP inhibitor (impaired DNA-SSB-repair) and carboplatin (increased DNA-DSB). Purpose: Here we tested the efficacy of a combination of GDC-0980 with ABT888 plus carboplatin in BRCA-competent PTEN-null model of TNBC. Methods: Athymic mice bearing PTEN-null TNBC xenograft tumors were treated with GDC-0980 alone or in combination with ABT888 and carboplatin. Results : Dual inhibition of PI3K and mTOR by GDC-0980 alone as well as in the presence of carboplatin plus ABT888 changed the state of the repair of DNA-damage in BRCA-competent PTEN null TNBC cells, which led to increased cellular apoptotic signals in addition to decreased survival/proliferative signals. GDC-0980 treatment led to DNA damage (increased pgH2AX), gain in PAR and a subsequent sensitization of BRCA-competent PTEN-null MDA-MB468 TNBC cells to ABT888 plus carboplatin with a time-dependent (1) decrease in proliferation signals (pAKT T308/S473, pP70S6K, pS6RP), PAR/PARP ratios, PAR/pgH2AX ratios, live/dead cell ratios, cell-cycle progression and clonogenic 3D growth and (2) increase in apoptosis markers (cleaved-caspase 3, 9, BIM, cleaved-PARP and annexinV positivity). These effects are more pronounced in MDA-MB468 than in RAS/RAF mutated MDA-MB231 cells. GDC-0980 alone and in combination with ABT888 plus carboplatin inhibited cell cycle progression, increased apoptosis, and decreased live/dead cell ratios in BRCA-competent PTEN null TNBC cells. GDC-0980 alone and in combination with ABT888 plus carboplatin attenuated anchorage -dependent and -independent clonogenic 3D growth comparatively more in BRCA-competent PTEN-null cells TNBC cells than MDA-MB231 cells. GDC-0980 in combination with ABT888 plus carboplatin blocked the growth of established PTEN-null TNBC tumors as compared to vehicle control(s) with a concomitant decrease in tumor Ki67 and CD31 IHC-stains. Conclusion : This is the first mechanism-based study to demonstrate that in BRCA-competent PTEN-null TNBC model, GDC-0980 enhanced antitumor activity of ABT888, in the presence of carboplatin by inhibiting DDR system in conjunction with the inhibition of PI3K-mTOR pathway-mediated proliferative, and anti-apoptotic signals. Considering (1) the importance of PARP as the target in TNBC, (2) the existence of a large percentage of BRCA-competent TN and/or basal type BC patients and (3) the high frequency of PTEN-null-ness in this subset of BC, this combination merits further investigation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-02.

Abstract P5-06-01: The PI3K inhibitor GDC-0941 combines with trastuzumab for superior anti-tumor efficacy in HER2+ breast cancer models

Background : PI3K-AKT-mTOR pathway signaling is important for the oncogenic function of HER2. Activating alterations of this pathway are frequently observed in HER2-enriched breast cancer and generally herald a poor response and resistance to trastuzumab (T). Purpose : Targeting the PI3K-AKT-mTOR pathway is an attractive strategy in HER2+ breast cancer that is refractory to trastuzumab. The hypothesis is that the suppression of this pathway by pan-PI3K inhibitor, GDC-0941 may lead to overcome trastuzumab-resistance. Experimental Design : The antiproliferative and HER2-mediated cellular signaling (pAKT, pP70S6K, pS6RP, p4EBP1 and p-ERK) effects of GDC-0941 alone and in combination with T were evaluated in HER2 amplified T-sensitive (BT474), T-resistant (BT474HR), and HER2 amplified/ PIK3CA mutated (HCC1954, UACC893) BT cell lines by MTT assay and Western blots. Clonogenic growth was tested by 3D ON-TOP assay and apoptosis markers were also tested. Athymic mice bearing BT474 and BT474HR xenograft tumors were treated with GDC-0941 and T (alone and in combination). Results : (1) GDC-0941 exhibited in vitro cell killing activity in MTT assay with IC509s ranging from 0.35 μM to 1 μm and potency was augmented by the addition of T, (2) inhibition of phosphorylation of AKT(S473, T308), P70S6K, S6RP, and 4EBP1(T37/46, T70) was observed following GDC-0941 treatment, and the combination of GDC-0941 and T more effectively blocked the PI3K-AKT-mTOR pathway, (3) GDC-0941 treatment increased apoptosis markers (CL-CASPASE3 and annexinV positivity), (4) GDC-0941 dose-dependently blocked 3D-ON-TOP clonogenic growth of HER2+ cells. This effect was potentiated in the presence of T and (5) in vivo, the combination of GDC-0941 and T significantly reduced established tumor growth in both sensitive (82%) and resistant (79%) models with concomitant decrease of different PD markers. Conclusions: Our data suggest that 1) therapeutic targeting of the PI3K-AKT-mTOR signaling should be effective in abrogating resistance to T therapy in HER2+ BT, and 2) targeting both the HER2 and the PI3K signaling pathways is an attractive strategy to enhance the clinical efficacy of T therapy, as well as to prevent or delay the development of resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-06-01.