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Thyroid hormone is a pleiotropic factor that controls many cellular processes in multiple cell types such as cancer stem cells (CSC). Thyroid hormone concentrations in the blood are stable, but the action of the deiodinases (D2–D3) provides cell-specific regulation of thyroid hormone activity. Deregulation of deiodinase function and thyroid hormone status has been implicated in tumorigenesis. Therefore, we investigated the role of thyroid hormone metabolism and signaling in colorectal CSCs (CR-CSC), where deiodinases control cell division and chemosensitivity. We found that increased intracellular thyroid hormone concentration through D3 depletion induced cell differentiation and sharply mitigated tumor formation. Upregulated BMP4 expression and concomitantly attenuated Wnt signaling accompanied these effects. Furthermore, we demonstrate that BMP4 is a direct thyroid hormone target and is involved in a positive autoregulatory feedback loop that modulates thyroid hormone signaling. Collectively, our findings highlight a cell-autonomous metabolic mechanism by which CR-CSCs exploit thyroid hormone signaling to facilitate their self-renewal potential and suggest that drug-induced cell differentiation may represent a promising therapy for preventing CSC expansion and tumor progression. Cancer Res; 76(5); 1237–44. ©2015 AACR.

The tumor microenvironment supplies proinflammatory cytokines favoring a permissive milieu for cancer cell growth and invasive behavior. Here we show how breast cancer progression is facilitated by IL4 secreted by adipose tissue and estrogen receptor–positive and triple-negative breast cancer cell types. Blocking autocrine and paracrine IL4 signaling with the IL4Rα antagonist IL4DM compromised breast cancer cell proliferation, invasion, and tumor growth by downregulating MAPK pathway activity. IL4DM reduced numbers of CD44+/CD24− cancer stem-like cells and elevated expression of the dual specificity phosphatase DUSP4 by inhibiting NF-κB. Enforced expression of DUSP4 drove conversion of metastatic cells to nonmetastatic cells. Mechanistically, RNAi-mediated attenuation of DUSP4 activated the ERK and p38 MAPK pathways, increased stem-like properties, and spawned metastatic capacity. Targeting IL4 signaling sensitized breast cancer cells to anticancer therapy and strengthened immune responses by enhancing the number of IFNγ-positive CTLs. Our results showed the role of IL4 in promoting breast cancer aggressiveness and how its targeting may improve the efficacy of current therapies. Cancer Res; 77(12); 3268–79. ©2017 AACR.

Colorectal cancer stem cells (CR-CSC) are responsible for the generation and maintenance of intestinal tumors and are highly resistant to conventional chemotherapeutic agents. Aurora-A, a serine-threonine kinase involved in mitosis regulation, plays multiple key functions in tumor initiation and progression. We found that Aurora-A is overexpressed in primary colorectal tumor cells, in the CR-CSC fraction, and in stem cell–derived differentiated cells, compared with normal colon tissue. Aurora-A expression was functionally linked to centrosome amplification in CR-CSC, as indicated by the decrease in cells with multiple centrosomes that followed Aurora-A silencing. Knockdown of Aurora-A resulted in growth inhibition of CR-CSC, alteration of cell cycle kinetics, and downregulation of the expression levels of antiapoptotic Bcl-2 family members, strongly sensitizing to chemotherapy-induced cell death. Moreover, Aurora-A silencing compromised the ability to form tumor xenografts in immunocompromised mice and reduced the migratory capacity of CR-CSC. Altogether, these results indicate that Aurora-A is essential for CR-CSC regeneration and resistance to cytotoxic stimuli and suggest that therapies directed against Aurora-A may effectively target the stem cell population in colorectal cancer. Cancer Res; 70(11); 4655–65. ©2010 AACR.

Colon tumors may be maintained by a rare fraction of cancer stem-like cells (CSC) that express the cell surface marker CD133. Self-renewing CSCs exhibit relatively greater resistance to clinical cytotoxic therapies and recent work suggests that this resistance may be mediated in part by an autocrine response to the immune cytokine interleukin 4 (IL-4). Blocking IL-4 signaling can sensitize CSCs to apoptotic stimuli and increase the in vivo efficacy of cytotoxic therapy. These findings suggest that inhibitors of IL-4 signaling may offer a new therapeutic tool in colon carcinoma. [Cancer Res 2008;68(11):4022–5]

Killing of tumor cells by cytotoxic therapies, such as chemotherapy or gamma-irradiation, is predominantly mediated by the activation of apoptotic pathways. Refractoriness to anticancer therapy is often due to a failure in the apoptotic pathway. The mechanisms that control the balance between survival and cell death in cancer cells are still largely unknown. Tumor cells have been shown to evade death signals through an increase in the expression of antiapoptotic molecules or loss of proapoptotic factors. We aimed to study the involvement of PED, a molecule with a broad antiapoptotic action, in human breast cancer cell resistance to chemotherapeutic drugs–induced cell death. We show that human breast cancer cells express high levels of PED and that AKT activity regulates PED protein levels. Interestingly, exogenous expression of a dominant-negative AKT cDNA or of PED antisense in human breast cancer cells induced a significant down-regulation of PED and sensitized cells to chemotherapy-induced cell death. Thus, AKT-dependent increase of PED expression levels represents a key molecular mechanism for chemoresistance in breast cancer.

Triple-negative breast cancer (TNBC) is characterized by an abundance of treatment-resistant cancer stem cells (CSC). The absence of a molecular target, coupled with its highly aggressiveness, leads to the lack of an effective therapy for TNBC. Norcantharidin (NCTD) is a synthetic demethylated small-molecule analog of the naturally occurring cantharidin isolated from blister beetles (Mylabris phalerata Pall). Unlike the conventional chemotherapeutics, NCTD toxicity is higher to cancer cells than normal ones, making this small molecule promising for cancer treatment. The aims of this work were: A) To study the effect of NCTD on 4T1 cell line proliferation in vitro. B) To analyze the effect of NCTD on 4T1 derived CSC on self-renewal and clonogenic capacity. C) To evaluate the effect of NCTD on 4T1 tumor growth in vivo. We employed the well-known 4T1 triple-negative breast cancer cell model, which presents a huge proportion of CSC. We observed that NCTD treatment during 96 h significantly reduced 4T1 cell proliferation in vitro. In addition, the IC50 value of NCTD was 27.35 ± 2.83 μM. Related to CSC, NCTD pre-treatment for 96 h impaired CSC self-renewal (Number of secondary mammospheres: Control: 276±39; NCTD: 163±18; p≤0.05) as well as the clonogenic capacity (Number of colonies: Control: 359±38; NCTD: 122±11; p≤0.05). By q-PCR, we observed that NCTD treatment for 48 h significantly induced an increase of Gli-1 and Smooth in CSC, keys member of Sonic Hedgehog pathway. Finally, we performed an in vivo assay, where 4T1 cells were orthotopically inoculated on mammary gland of BALB/c mice, and NCTD was i.p. inoculated twice a week (5mg/kg). We observed that NCTD treatment significantly reduced tumor growth in vivo. Our data suggest that NCTD treatment reduces tumor growth both in vitro and in vivo, possibly through the direct effect on CSC self-renewal and clonogenic capacity, by modulating Sonic Hedgehog pathway. Citation Format: Damian E. Berardi, Guido Cicuttin, Maria A. Taruselli, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffé, Alejandro J. Urtreger, Laura B. Todaro. Norcantharidin impairs tumor growth in vivo and inhibits stemness of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5111. doi:10.1158/1538-7445.AM2017-5111

Overexpression of MUC1 oncoprotein is frequently observed in cancer and contributes to confer resistance to genotoxic agents. Papillary, follicular, and anaplastic thyroid carcinomas are the three forms of thyroid epithelial cancer. Anaplastic tumors are less differentiated and extremely aggressive, characterized by a poor prognosis. Little is known about the role of MUC1 in thyroid cancer. We recently showed that autocrine production of interleukin (IL)-4 and IL-10 controls thyroid cancer cell survival, growth, and resistance to chemotherapy through activation of Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) and phosphatidylinositide 3′-OH kinase (PI3K)/Akt pathways. In the present study, we showed that MUC1 COOH-terminal subunit (MUC1-C) is overexpressed in all the histologic variants of thyroid cancer cells and localizes to mitochondria where it interferes with the release of mitochondrial proapoptotic proteins. Moreover, IL-4 and IL-10 promote the increase of MUC1-C expression levels in normal thyroid cells, whereas blockage of both cytokines or neutralization of JAK/STAT and PI3K/Akt pathways through the exogenous expression of SOCS-1 and AktK179M leads to a significant decrease of MUC1-C in primary thyroid cancer cells. Interestingly, down-regulation of MUC1 expression by direct targeting with RNA interference sensitizes anaplastic thyroid cancer cells to chemotherapy-induced apoptosis in vitro. Thus, MUC1 is a main component of the survival network acting in thyroid cancer and could be considered a key molecular target for sensitizing cancer cells to conventional or novel treatments. [Cancer Res 2007;67(11):5522–30]

Although CD95 and its ligand are expressed in thyroid cancer, the tumor cell mass does not seem to be affected by such expression. We have recently shown that thyroid carcinomas produce interleukin (IL)-4 and IL-10, which promote resistance to chemotherapy through the up-regulation of Bcl-xL. Here, we show that freshly purified thyroid cancer cells were completely refractory to CD95-induced apoptosis despite the consistent expression of Fas-associated death domain and caspase-8. The analysis of potential molecules able to prevent caspase-8 activation in thyroid cancer cells revealed a remarkable up-regulation of cellular FLIPL (cFLIPL) and PED/PEA-15, two antiapoptotic proteins whose exogenous expression in normal thyrocytes inhibited the death-inducing signaling complex of CD95. Additionally, small interfering RNA FLIP and PED antisense sensitized thyroid cancer cells to CD95-mediated apoptosis. Exposure of normal thyrocytes to IL-4 and IL-10 potently up-regulated cFLIP and PED/PEA-15, suggesting that these cytokines are responsible for thyroid cancer cell resistance to CD95 stimulation. Moreover, treatment with neutralizing antibodies against IL-4 and IL-10 or exogenous expression of suppressor of cytokine signaling-1 of thyroid cancer cells resulted in cFLIP and PED/PEA-15 down-regulation and CD95 sensitization. More importantly, prolonged IL-4 and IL-10 neutralization induced cancer cell growth inhibition and apoptosis, which were prevented by blocking antibodies against CD95 ligand. Altogether, autocrine production of IL-4 and IL-10 neutralizes CD95-generated signals and allows survival and growth of thyroid cancer cells. Thus, IL-4 and IL-10 may represent key targets for the treatment of thyroid cancer. (Cancer Res 2006; 66(3): 1491-9)

Introduction: Lung Adenocarcinoma (AC) is the most frequent lung cancer histological subtype and is a leading cause of cancer-related death worldwide. Hedgehog/Gli (Hh/Gli) signaling pathway regulates lung development and its aberrant activation contributes to tumor pathogenesis and play a role in cancer stem cells (CSC) control. We investigated oncogenic Hh/Gli signaling in AC-CSC. Methods: human AC-CSC were derived from primary tumors. For in vitro studies AC-CSC were maintained in serum-free medium supplemented with EGF/bFGF. For in vivo experiments, immunocompromised mice were injected with AC-CSC. Gli1 inhibitor GANT61 was used both in vitro and in vivo (IP 40 mg/kg twice/we) treatments. Results: Using mRNA datasets and tissue microarray of Non-small Cell Lung Cancer (NCSLC) samples we found that in AC Gli1 is expressed independently of its canonical activator Smoothened (Smo) expression level. Similarly, CSC were Gli1 positive regardless of Smo expression levels. Indeed, we found that SMO regulatory region is highly methylated in AC cells where Smo mRNA and protein are low. Looking for Smo-independent regulation of GLI activity we found that NRP2/Erk and IGF1R/Erk axes regulate Gli1 in AC-CSC. Suppression of Gli1 with GANT61 in CSC resulted in impairment of cell growth and stemness features. We performed in-vivo experiments: CSC-derived xenograft from GANT61 treated mice showed impaired tumor growth, reduced proliferation and increased apoptosis. We next highlighted a tumor-stroma crosstalk. A positive autocrine oncogenic feedback loop sustains Gli1 in AC-CSC since we found that the CSC expressed NRP2 and its ligands VEGF-A and VEGF-C. A paracrine positive loop also sustain Hh/Gli signaling in AC where Cancer-Associated Fibroblasts (CAFs) harbor Hh/Gli1 canonical pathway that regulates the expression of VEGF-A, VEGF-C and IGF2 ligands of NRP2 and IGF1R respectively. Conclusion: Our findings suggest a non-canonical activation of Hh/Gli pathway in AC. In this context, GLI1 transcription factor is effectors of NRP2/Erk and/or IGF1R/Erk signaling inputs. Our results have implications for the understanding of AC development and sustain the inclusion of target therapy acting downstream of Smo level in the design of AC treatment. Citation Format: Agnese Po, Marianna Silvano, Evelina Miele, Adriana Eramo, Matilde Todaro, Carlo Capalbo, Valentina Salvati, Giovanni Sette, Danilo Cucchi, Zein M. Besharat, Gianluca Canettieri, Lucia Di Marcotullio, Isabella Screpanti, Giorgio Stassi, Ruggero De Maria, Ann Zeuner, Enrico De Smaele, Elisabetta Ferretti. Non-canonical Hedgehog/Gli1 signaling drives lung adenocarcinoma stem cells survival and its targeting inhibits CSC-derived tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2484.

Background: Despite the advent of successful treatment of localized malignancies, metastatic cancer still lacks efficacious therapeutic approaches, including breast cancer. It is now well established that within malignant tumors Cancer Stem Cells (CSCs) constitute a unique cell subset that fuel and succeed at tumor growth and metastases formation. Tumor microenvironment sustains CSCs characteristics, making the molecular mechanisms driving tumor progression and recurrence more complex and difficult to elucidate. Furthermore, the interaction that occurs between CSCs and the nearby stroma has proven to enhance the aggressive behavior of several carcinomas through the secretion of microenvironmental cytokines. In this context, IL-4 has already been described to promote survival of cancer cells through the up-regulation of several anti-apoptotic factors. However, still little is known about its role in promoting breast cancer progression. Results: Here we show for the first time that autocrine and paracrine production of IL-4 regulates breast CSCs (BCSCs) features, including cell proliferation, motility and cytoskeletal organization via RAS/MAPK pathway. Interestingly, relief from IL-4 impaired BCSCs proliferation, colony forming efficiency and in vivo tumor formation, while it fostered the expression of the dual specificity phosphatase-4 (DUSP4) in triple-negative basal-like BCSCs, leading to the decrease of CD44+/CD24- population. DUSP4 is commonly deficient in the most aggressive breast cancers, such as the basal-like subtype. Likewise, we observed that the enforced expression of DUSP4 increases the CD24+ compartment in basal-like BCSCs, determining also a dramatic decrease of their proliferation, colony forming efficiency, invasiveness and metastases formation. Contrarily, in luminal-like BCSCs, DUSP4 suppression favors BCSCs cell traits, including their tumorigenic and metastagenic properties. Methods: Patient-derived BCSCs were obtained by digestion of breast cancer tissues and plated in serum-free media with bFGF and EGF. DUSP4 were inserted into the p-Lenti expression vector. Stable DUSP4 knockdown was produced by lentiviral transduction of the pGFP-C vector. IL-4 function was impaired by using a high affinity IL-4Rα antagonist. To assess tumorigenicity and metastases formation, BCSCs were suspended in matrigel and injected either orthotopically or intra caudal in NOD/SCID mice. Conclusions: These findings will shed light on the molecular basis of cancer progression and on the complex crosstalk occurring between tumor and its microenvironment. The identification of tumor-related molecular events, such as the IL-4 activated signaling, might be clinically exploited as therapeutic targets in the adjuvant setting and synergize the effect of conventional chemotherapy in patients affected by breast cancers with limited therapeutic options, such as triple-negative breast cancers. Citation Format: Alice Turdo, Miriam Gaggianesi, Tiziana Apuzzo, Antonina Benfante, Aurora Chinnici, Alessandro Giammona, Simone Di Franco, Giorgio Stassi, Matilde Todaro. Autocrine and paracrine IL-4 maintains breast cancer stem cells traits via RAS/MAPK/DUSP pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3311.

Lung cancer is the leading cause of cancer deaths worldwide and despite significant progress, current therapies are limited in efficacy. The CK2 Ser/Thr kinase has been historically linked with cancer. It is involved in cell proliferation, survival and apoptosis by modulating diverse signaling pathways, including Wnt and NF-κB among the most relevant. CIGB-300 is an antitumor peptide with a novel mechanism of action, capable of binding to CK2 substrates thus preventing the enzyme activity. Previously, we have determined that CIGB-300 induces apoptosis through caspase-3 activation in different lung cancer cell lines. Moreover, CIGB-300 strongly inhibited RelA/NF-κB (p65) nuclear translocation, even in the presence of a phorbol-ester activating stimulus. NF-κB activation is known to reduce chemotherapy efficiency in different malignancies, including lung cancer. Based on this evidence, we hypothesize that supplementing cisplatin with CIGB-300 would improve the treatment efficiency. Indeed, we observed by Western blot that nuclear p65 levels were highly increased after treating human NCI-H125 cells with cisplatin. Moreover, when cells were treated with cisplatin plus CIGB-300, NF-κB activation was completely abolished. Therefore, the CIGB-300 effect on NF-κB signaling pathway prevails over cisplatin. These promising results on NF-κB inhibition led us to evaluate the combined treatment in chemoresistant setting. For this purpose we developed a cisplatin resistant A549 lung cancer cell line (A549-Rcisp) by the chronic administration of cisplatin during six months. A549-Rcisp viability was 40% higher than parental cells, confirming the cisplatin-acquired resistance. Remarkably, cisplatin resistant cells showed a significant increase in CIGB-300 sensitivity as compared to the parental cell line (p Given that NF-κB dimer stability is regulated by the proteasome-selective proteolysis of its inhibitory proteins, we studied the effect of CIGB-300 on this process. Surprisingly, we observed a significant increase on protease activities associated with the proteasome after 30 minutes of CIGB-300 treatment. Thus, proteasome complex is a newly identified target of CIGB-300 that could be relevant for its mechanism of action and deserves further exploration in order to determine the association with the observed perturbation of different signaling pathways. Citation Format: Stéfano M. Cirigliano, María Inés Díaz Bessone, Carolina Flumian, Damián E. Berardi, Silvio Perea, Elisa Bal De Kier Joffé, Hernán Farina, Laura Todaro, Alejandro Urtreger. The synthetic peptide CIGB-300 inhibits nuclear factor κB (NF-κB) affecting the survival and chemoresistance of human lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1289.

Retinoids exert different effects on cell differentiation and malignant phenotype reversion through the modulation of Retinoid Acid Receptors (RAR). In this work we have analyzed the involvement of retinoid system induction on proliferation, self-renewal and differentiation of cancer stem cells (CSC) using the triple negative mammary cell line LM38-LP. In order to obtain a CSC enriched culture, cells where cultured under mammosphere conditions. We performed mammosphere cultures in order to enrich in CSC. The treatment with All-trans retinoid acid (ATRA1μM for 96 h) reduced CSC growth rate evaluated as mammosphere diameter (Control: 176±8 μm vs. ATRA: 129±10 μm; p≤0.05). Surprisingly, ATRA pre-treatment for 96 h increased CSC self-renewal evaluated as the number of secondary mammospheres (Control: 313±19; ATRA: 495±21; p≤0.05) and as the clonogenic capacity of LM38-LP CSC (Control: 6±0.5; ATRA: 11±1.2; p≤0.05). Concomitant with these biological effects, the same treatment showed, by RT-PCR, an increase in RARβ and RARγ levels in LM38-LP- CSC. Through the use of specific RAR antagonists, we found that RARγ inhibition impaired ATRA effects on self-renewal but RARβ inhibition increased the number of secondary mammospheres (Control: 382±27; ATRA: 735±11; ATRA/RARγ antagonist: 352±17; ATRA/RARβ Antagonist: 1260±177; p≤0.05). A similar result was observed when clonogenic capacity in RARγ blocked CSC was analyzed. Finally, we performed a matrigel 3D culture using cells from LM38-LP mammospheres. Under these conditions, colonies presented large irregular structures while ATRA treatment led to the formation of differentiated colonies with evidence of lumen. Our findings suggest that RARγ down modulation and RARβ induction could lead to CSC self-renewal inhibition and differentiation in triple negative mammary cancers. Citation Format: Damian E. Berardi, Carolina Flumian, Maria I. Diaz Bessone, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Involvement of Retinoic Acid Receptors RARβ and RARγ in growth, self-renewal and differentiation on mammary cancer stem cells. [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 2237. doi:10.1158/1538-7445.AM2015-2237

PKC is a serine-threonine kinase family that controls malignant transformation and metastatic dissemination. ATRA is the main active metabolite of vitamin A. Some evidences indicate that PKCδ may regulate the expression of some retinoid acid (RA) dependent genes, and others indicate that retinoids could alter PKCα intracellular localization; both processes would lead to cell differentiation. In this work we have developed human (MDA-MB 231) and murine (LM3) cell models overexpressing PKCα or PKCδ, in order to determine whether PKC expression alters the sensitivity to retinoids treatment (ATRA). The effect of ATRA was studied in vitro, analyzing biological responses related to tumor growth. In LM3 cells, only PKCα overexpression was able to reduce in vitro population doubling time (PDT) as compared to control (PDT: 14,8±2,3 h vs 21,2±3,1 h for LM3-PKCα y LM3-Vector respectively). Moreover, these cells also responded to retinoid treatment with a significant delay in cell proliferation (PDT: 24,3±4,2 h vs. 14,8±2,3 h for LM3-PKCα ATRA treated or not respectively. In MDA-MB 231 derived cell lines, PKC overexpression as well as ATRA treatment have no effect on proliferative potential. No differences were observed on migratory and invasive capabilities either. Interestingly, in LM3, PKCδ overexpression induced an important increase in proteolytic enzymes secretion, which correlates with its major invasiveness, but this increase had no impact on in vivo metastatic dissemination. Only PKCα overexpression increased this parameter (lung nodes, median (range): 55 (20-75) vs 0 (0-10) for LM3-PKCα y LM3-Vector respectively). Contrary to LM3-PKCδ in MDA-PKCδ cells we could detect a decrease on proteolytic enzymes production and invasive capability. Moreover, colonies growing in Matrigel as 3D cultures showed a small and branched structures. Finally we studied whether the overexpression of α and δ PKC isoforms is able to alter the activity of retinoic acid responsive elements (RARE) through a reporter gene assay. On LM3 model, the constitutive expression of PKCδ highly increased RARE dependent activity. Surprisingly, in MDA-MB231 derived sublines, we could detect a significant increase on RARE activity when cells were treated with ATRA, Altogether, these results suggest that PKCα overexpression confers a more aggressive phenotype in LM3 model, but also makes these cells sensitive to ATRA effects. We could hypothesize, that the absence of response to ATRA treatment displayed by MDA-MB 231 sublines can be explained by their lack of RARβ, which is implied in AP-1 transrepression in response to ATRA. Among others phenomena, AP-1 is involved in cell proliferation and proteolytic enzymes production. Regarding the differences of response to PKCδ overexpression of both models, it has been reported that this PKC isoform has a differential role, pro or anti tumorigenic, depending on cellular context. Citation Format: Maria I. Diaz Bessone, D. E. Berardi, S. M. Cirigliano, C. Flumian, E. D. Bal de Kier Joffe, L. B. Todaro, A. J. Urtreger. Differential response to retinoid treatment in mouse and human mammary tumor cell lines with alterations in protein kinase C (PKC) expression. [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 5203. doi:10.1158/1538-7445.AM2015-5203

Background: In the human mammary gland p63 isoforms control mammary epithelial cells fate and DNp63, which lacks the amino-terminal trans-activation domain, counterbalances the full-length isoform TAp63 to allow maturation of epithelia and the maintenance of the myoepithelial/basal cells features. Mammary gland tissue's adult stem cells retain self-renewal and multi-lineage differentiation ability and, by acquiring a malignant behavior, are in charge of tumor seeding. A correlation between Cancer Stem Cells (CSCs) in primary lesions and increased metastatic dissemination has been reported, however, still little is known about the unique profile of distant metastasis’ cell origin. Herein, we show for the first time that breast CSCs (BCSCs) expressing DNp63 are capable to generate metastasis in a preclinical model, while TAp63 can be solely tumorigenic and unable to serial xenografting. We anticipate novel farsighted implications for a prognostic role of p63 and for its targeting to prevent tumor growth and metastatic disease. Methods: BCSCs were freshly isolated from the histological uninvolved resection of breast cancer tissues. Obtained cells were plated in ultra-low flask in serum-free media in presence of bFGF and EGF. Cells were treated with 1mmol/l doxorubicin hydrochloride. Synthetic genes encoding DNp63 and TAp63 were inserted into the p-TWEEN EGFP lentiviral expression vector. Stable p63 knockdown was produced by lentiviral transduction of the pLentilox 3.7 vector carrying shp63 and scramble sequences. For the tumorigenic and metastagenic assay BCSCs (3×105) were suspended in matrigel and injected orthotopically or in the sub-renal capsule of NOD/SCID mice, respectively. Tumors were measured with a caliper each week and volume was calculated by the formula: π/6 x larger diameter x (smaller diameter)2. Metastasis formation was followed over time and visualized by in vivo imaging. Summary: Herein, we demonstrate that luminal and basal BCSCs differ in the content of p63 isoforms, DNp63expression was higher in the stem-like compartment of basal BCSCs, compared to TAp63 mainly expressed in bulk primary cells. DNp63 increased the G0-G1 phase in both luminal and basal BCSCs, enhanced doxorubicin resistance and induced a mesenchymal switch. Despite both TAp63 and DNp63 overexpressing BCSCs formed subcutaneous xenografts in NOD/SCID mice, DNp63 cells exclusively retain capabilities of serial xenografting. Thus, suggesting that exogenous expression of TAp63 could promote transition from stem cells to progenitor cells. In a metastatic preclinical model TAp63 overexpression hampered metastatic potential of BCSCs, while DNp63 overexpressing cells gained capabilities to colonize distant organs such as lung and kidney. Conclusions: These findings state DNp63 as a major regulator of stemness and invasiveness in malignant breast tissues and offer new insights on p63 role as a prognostic biomarker and therapeutic target. Citation Format: Alice Turdo, Simone Di Franco, Antonina Benfante, Maria Luisa Colorito, Marco Bonanno, Miriam Gaggianesi, Daniela Barcaroli, Francesco Dieli, Jan Paul Medema, Vincenzo De Laurenzi, Giorgio Stassi, Matilde Todaro. DNp63 governs metastatic outgrowth of breast cancer stem cells. [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 LB-143. doi:10.1158/1538-7445.AM2015-LB-143

Migration and adhesion are two critical processes highly related to metastasic dissemination and all trans-retinoic acid (ATRA) is known to diminished migration and metalloprotease (MMP) secretion in breast cancer cell lines. Our objective was to evaluate the effect of activating each retinoic acid receptor (RAR) isotype in adhesion, migration and MMPs secretion using two hormone-independent murine cell lines: LM38-LP y 4T1. First we determined that both lines express the different RAR isotypes with the exception of RARβ in 4T1 cell line. Cells were treated for 96h with AM580 (RARα agonist, 200nM), AC55649 (RARβ agonist, 2μM), BMS961 (RARγ agonist, 50nM) or vehicle (DMSO). The migratory potential was evaluated by a “wound healing” assay. LM38-LP cell line reduced its migratory capacity in response to AM580 and AC55649 (AM580: 108.0±24.3μm, AC55649: 114.1±12.8μm vs. vehicle: 186.9±31.0μm, p The same agonist treatments were used to obtain conditioned media in order to evaluate soluble MMPs activity by zymography. AM580, AC55649 and BMS961 pre-treatments decreased soluble MMP2 activity in LM38-LP cells (0.53±0.03au (arbitrary units), 0.47±0.01 au, and 0.75±0.04 au respectively, fold change vs. control). Conversely, AM580 pre-treatment increased MMP2 and MMP9 in 4T1 cells (1.57±0.10 au and 2.04±0.26 au respectively, fold change vs. control). Regarding adhesion assay, after the above mentioned treatments cells were detached, incubated 2h at 37°C in order to recover cell surface proteins and finally seeded. After 2h incubation, non-adherent cells were washed out with PBS and adherent cells were fixed, stained and quantified by densitometry. We observed that AM580 and AC55649 diminished LM38-LP adhesive capacity (0.51±0.02 au, 0.83±0.00 au respectively, fold change vs. control) while AC 55649 increased this parameter in 4T1 cells (1.28±0.06 au fold change vs. control). Finally we performed an experimental lung metastasis assay using LM38-LP cells. Cells where treated with the different agonist during 144h and then inoculated into the tail vein of syngeneic mice. Lungs were removed 21 days later and number of superficial lung metastasis was determined. AC55649 treatment induced an increase in the metastatic potential (Md [Rg]: 143, [86-314] vs. control Md [Rg]: [21.5, 2-73], p In conclusion, the activation of RARα and RARβ isotypes leads to opposite responses in different cell lines. We hypothesized that the differences in RARβ expression between this two cell lines should be responsible of this effect. Surprisingly, the activation of RARβ increased the metastatic potential of LM38-LP cells in spite of the negative regulation of parameters associated with the metastatic process. Citation Format: Carolina Flumian, Damián E. Berardi, Stefano M. Cirigliano, María I. Díaz Bessone, Elisa D. Bal de Kier Joffé, Alejandro J. Urtreger, Laura B. Todaro. Parameters associated with metastatic dissemination are differentially modulated by specific isotypes of the retinoic acid receptor. [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 5204. doi:10.1158/1538-7445.AM2015-5204

Breast cancer is still one of the most important public health problems in the entire world. Obesity and metabolic syndrome (MS) increases the incidence and aggressiveness of breast cancer. C-Terminal Binding Protein (CtBP1) is a transcriptional corepressor of tumor suppressor genes and is considered a molecular sensor of cell metabolic state due to is activated in high energy conditions (high NADH). In this work we studied the effects of the activation of CtBP1 pathway by metabolic syndrome on breast tumor development and progression. We generated a murine model of MS by chronic high fat diet (HFD) administration. By histological and whole mount methods, we found that breast tissue of animals receiving HFD presented higher levels of immature adipose tissue and an increased glandular area with more generation of lateral branches and terminal end buds of mammary ducts. Breast tissue of HFD animals also showed higher expression of the proliferation markers (cyclin D1) and epithelial markers (E-cadherin). Interestingly, HFD induced CtBP1 expression in the mammary ducts. Furthermore, the number and size of mamospheres generated with LM38-LP breast cancer cells were significantly increased when cells were incubated with serum from HFD fed mice compared to the serum of animals under control diet (CD). In addition, to investigate CtBP1 role in tumor progression we performed xenografts in nude mice fed with CD or HFD by subcutaneous injecton of breast tumor cells MDA MB 231 with depleted CtBP1 expression (shRNA CtBP1) or control cells (shRNA scramble). We found that CtBP1 depletion dramatically decreased tumor growth and KI67 expression relative to control tumors. Furthermore, xenografts developed in HFD fed mice were less differentiated compared to CD. Finally, CtBP1 diminished expression tumors showed lower mesenchymal markers expression, progenitor cells markers and markers involved in mammary development. Our studies demonstrated for the first time that gene transcription regulation by CtBP1 provides an important molecular link among MS, CtBP1 function and tumor growth. Hence, these results suggest an association to understand metabolism and breast cancer. Citation Format: Paola De Luca, Nicolás Dalton, Cristian Pablo Moiola, Carolina Flumian, Georgina Scalise, Juliana Porretti, Cintia Massillo, Edith Kordon, Laura Todaro, Elba Vazquez, Roberto Meiss, Adriana De Siervi. CtBP1 is the molecular link that associates breast cancer and metabolic syndrome. [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 1160. doi:10.1158/1538-7445.AM2015-1160

Stem-cell populations have been identified in a range of solid tumors and possess the capacity to self renew. It is thought that these cancer stem cells (CSCs) play an important role in cancer establishment, progression, and resistance to current treatments. CSCs have been identified in primary breast cancer tissues and cell lines derived from metastatic tumors. Traditional cancer therapies are effective at debulking some tumors but often fail to produce long-term clinical remissions, presumably due to their inability to eradicate the cancer stem cell population. Therefore, novel treatments aimed at targeting this population can potentially enhance treatment of both primary and metastatic tumors. Aminoflavone (AF; NSC 686288) is a novel anticancer agent. Previous work from our research group demonstrated that AF is a ligand of the aryl hydrocarbon receptor (AhR) and displays cytotoxicity in the MCF-7 breast cancer cell line. A prodrug formulation of AF (AFP464, NSC710464), recently entered phase II clinical trials. Moreover, we previously found that AF disrupts MCF-7 mammosphere formation via AhR signaling activation to inhibit alpha 6-integrin expression. To investigate the efficacy of AF in vivo, we used an estrogen dependent, Tamoxifen-sensitive spontaneous M05 model system involving 1-year-old virgin female BALB/c mice developed in our animal facility. The M05 tumor is a semi-differentiated adenocarcinoma that expresses estrogen and progesterone receptors. After AF treatment (12 mg/kg, i.p., QD × 5 days) tumor size and growth rate decreased substantially compared to control animals. Cytometric analyses of cells obtained from AF-treated and control animals revealed that Lin (-)/ CD29 (Hi)/CD24 (+) cancer stem cells were lower in animals treated with AF than in the vehicle control group. We also observed a diminished capacity for mammosphere formation in cells obtained from tumors treated with AF. These data suggest AF diminishes the quantity of breast cancer stem cells as well as their integrity and capacity to form mammospheres in vitro and in vivo. These findings shed additional insight into the molecular mechanism of anticancer action for this novel anticancer agent to strengthen the rationale for its inclusion in breast cancer treatment regimens. Citation Format: Mariana Callero, Damian Berardi, Laura Todaro, Marina Simian, Eileen J. Brantley, Ubaldo Soto, Andrea Loaiza-Perez. Aminoflavone exhibits in vivo efficacy on cancer stem cells in a spontaneous estrogen-dependent breast cancer murine model. [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 215. doi:10.1158/1538-7445.AM2014-215

Introduction. The activity of BET inhibitors in several models human hematologic disorders, including multiple myeloma (MM) has recently been demonstrated. Exposure of cancer cells to BET inhibitors results in a significant down-regulation of c-Myc expression, and suppression of transcriptional programs associated with proliferation and survival. OTX015 is a new selective orally bioavailable inhibitor of the BET family proteins in clinical development. We evaluated its activity in a panel of MM models with known upregulation of the c-MYC pathway. Materials and Methods. Human cell lines derived from MM (RPMI 8226, KMM1, JJN3, OPM2, KMS27, U266, KMS18) were treated with increasing doses of OTX015 (OncoEthix SA, Switzerland). Cell proliferation was evaluated by ATPlite and MTT assays over time, and cell cycle analysis by FACScan flow cytometer. RNA was extracted using TRIzol and reverse-transcribed with the Superscript First-Strand Synthesis System kit. RT-qPCR was performed using SYBR Green Master Mix on a Bio-RAD Real-Time PCR System. For Western blot analysis, lysates were fractionated on 8% polyacrilamide gels and membranes incubated with commercial antibodies. OPM2 cells were inoculated (1*10^6 cells both flanks) in NSG mice and 72h after treatments started (vehicle; OTX015 50 mg/kg bid, po, 5 days on-2 days off).Tumor growth was measured over time. Results. OTX105 treatment resulted in G1 cell cycle arrest in a dose-dependent manner in all MM cell lines. Increased percentages of G1 cells (∼20%) were found after 24h exposure, reaching a maximum after 72h. OTX015 treatment for 24h followed by drug washout induced an initial cell cycle arrest, which was then overcome at 72-96h. RPMI 8226 and KMS27 cell lines also displayed increased apoptosis after prolonged exposure (> 48h) at 500 nM of OTX015. In 7 out of 9 MM cell lines, OTX015 treatment resulted in rapid downregulation of c-MYC mRNA and protein, while two cell lines (U266 and KMM1) displayed dose and time dependent c-MYC upregulation. Equivalent activity was observed with OTX015 (250 nM) and the OTX015 analog and bona fide BET-inhibitor, JQ1. In OPM2-bearing tumor mice, OTX015 treatment showed impaired tumor growth respect to controls after 4 weeks of treatment. Conclusions. OTX015 is a novel potent BRD-inhibitor with cytostatic anti-proliferative or pro-apoptotic activity in MM demonstrated by in vitro and in vivo experiments. The different changes of c-MYC expression in MM cell lines after OTX015 exposure suggest that the biological effects could be due to multiple and/or alternative mechanisms, which are not strictly linked to down-regulation of this transcription factor. These findings support the ongoing clinical development of OTX015 in MM patients. Citation Format: Maria Todaro, Michela Boi, Valentina Vurchio, Elisabetta Ercole, Rodolfo Machiorlatti, Katia Messana, Indira Landra, Susanna Urigu, Sabrina Aliberti, Eugenia Riveiro, Francesco Bertoni, Giorgio Inghirami. OTX015, a novel BET inhibitor, is a promising anticancer agent for multiple myeloma. [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 5531. doi:10.1158/1538-7445.AM2014-5531

Background: Breast cancer is the leading worldwide cause of death among women due to the high metastatic spread of this disease. As by definition, Cancer Initiating Cells (CICs) are a fraction of primary tumor cells harboring tumorigenic potential and successful outgrowth at metastatic sites. Targeting molecular events affecting CICs peculiarities, as self-renewal and an innate chemoresistance, could improve the ineffectiveness of current therapies. Sam68, the major CD44 splicing regulator, is a multifunctional RNA-binding protein involved in multiple steps of RNA metabolism and its expression is aberrant in breast cancer. Herein, we highlight novel implications of Sam68 in the mammary tumor onset and progression. Material and methods: Tissue microarrays (TMA) were composed of n=155 histologically confirmed invasive breast carcinoma (T1-T2, N0, M0) and Sam68 expression was quantified as percent of immunoreactive tumor cells, a cut-off of 90% positive cells was chosen. Breast CICs were obtained from mechanically and enzimatically digestion of human breast tissues. Stable Sam68 knockdown was produced by lentiviral transduction and puromicin selection. Invasion assay was performed in 8µm pore size transwell coated with GF-depleted matrigel 1:3 in serum free medium. 3x104 cells were plated and 10% FBS medium was used as chemoattractant. Breast CICs (3 x 105) were suspended in 1:6 matrigel and orthotopically injected in NOD/SCID mice. Results: Univariate analysis of the TMA data showed that invasive breast carcinoma over-expressing Sam68 (≥ 90% positive cells) were associated with a significantly higher incidence of distant relapse (P = 0.011). Notably, in a cohort of screened breast cancer patients, breast CICs expressed higher Sam68 protein levels than tumor bulk, highlighting that Sam68 expression was likely restricted to cells with self-renewal activity. Stable knockdown of Sam68 in breast CICs significantly curtailed proliferation rate and was coupled with an increase in p27kip1 and reduction of Bcl-xL. Compared to control cells, downregulation of Sam68 was correlated with an attenuation of cell motility capability and Twist, Snail, CD44v6 and Met levels. Moreover, Sam68 modulated the expression of an oncogenic alternative splice variant of Met (Met-SM), probably through the ASF/SF2 splicing factor. In addition, depletion of Sam68 sensitized breast CICs to standard chemotherapy. Finally, Sam68 silencing ablated breast cancer xenograft formation in immunocompromised mice. Conclusions: Based on these results, we deem that Sam68 may promote pro-invasive signals by inducing and modulating the alternative splicing of oncogenic variants of several genes, such as CD44 and Met. Thus, further investigations on signaling pathways affecting CICs self-renewal and invasiveness could represent a crucial key to improve selective cancer treatment. Citation Format: Alice Turdo, Miriam Gaggianesi, Antonina Benfante, Mauro Piantelli, Matilde Todaro, Giorgio Stassi. Sam68 sustains self-renewal and invasiveness of breast cancer initiating cells. [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 3897. doi:10.1158/1538-7445.AM2014-3897

CK2 is a serine/threonine kinase involved in cell growth, survival and apoptosis. The CIGB-300 is a synthetic peptide capable of binding to CK2 substrates thus preventing the enzyme activity. Previously we have determined that CIGB-300 presented an inhibitory concentration 50 dose (IC50) of 119±2.4 µM in NCI-H125 human lung cancer cells. Throughout an Annexin V-FITC assay we could determine that CIGB-300 is a potent apoptosis inductor since the treatment with this drug for 60 minutes induced apoptosis at a level comparable with that observed with etoposide (10 µM). Concomitantly with cell death induction, we could observe caspase-3 activation and the decreased expression of c-myc and cyclin D1 and D2. In this work we analyzed whether CIGB-300 is able to alter the ability of cells to grow in 3D and to modulate signaling pathways involved in tumor progression. First, we developed stable spheroids of NCI-H125 cells, which were able to grow in culture for at least 14 days. After 5 days of treatment, CIGB-300 induced a significant growth inhibition (median volume: 4.07x107±0.70x107 µm3 in control spheroids vs 2.13x107±0.35x107 µm3 in spheroids treated with the IC50 dose p To analyze the effect of CIGB-300 on canonical Wnt signaling, this pathway was initially activated by incubating NCI-H125 cells with a conditioned media containing Wnt3a factor. This incubation led to a significant increase in the levels of cytoplasmic β-catenin. By Western blot we could observe that treatment with CIGB-300 blocked this increase, suggesting its role in the modulation of the Wnt pathway. In order to analyze the nuclear factor κB (NF-κB) dependent pathway, this transcription factor was activated by a phorbol ester treatment (PMA, 15 nM). Short treatment (15 min) with CIGB-300 induced an important reduction in NF-κB nuclear levels. However, this inhibition was reverted 24 h later as determined by Western blot and reporter gene expression assays. Our results indicate that the treatment with CIGB-300 induces a significant anti-proliferative response in a three-dimensional model, which resembles in vivo tumor growth conditions, also affecting key signaling pathways involved in tumor progression. Altogether, our data suggest that this peptide may become a new strategy for the treatment of lung cancer malignancies. Citation Format: Stéfano M. Cirigliano, María Inés Díaz Bessone, Carolina Flumian, Damián Emilio Berardi, Silvio E. Perea, Elisa Bal De Kier Joffé, Hernán Farina, Laura Todaro, Alejandro Urtreger. The inhibition of protein kinase CK2 activity by the CIGB-300 synthetic peptide impairs the three-dimensional cell growth, Wnt and nuclear factor қB signaling pathways in lung cancer cells. [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 3791. doi:10.1158/1538-7445.AM2014-3791

To study the interaction between PKCα and retinoic acid pathways on the biology of breast cancer, we employed a murine mammary triple negative cell model (LM38-LP, composed by luminal (LEP), myoepithelial (MEP) and cancer stem cells (CSC). We proposed to: A) Study the effect of ATRA (1μM) on the expression of PKCα and Retinoic Acid Receptors (RARs) in LEP, MEP and CSC (mammospheres); B) Evaluate the combined effect of ATRA and a PKCα pharmacological inhibitor (1 uM Go6976) on cell proliferation employing a method to determine the interaction type; C) Analyze the effect of ATRA/PKCα inhibitor on migration and MMP activity on LM38-LP;. D) Analyze the effect of ATRA/PKCα inhibitor on proliferation, self-renewal and differentiation of CSC; E) Study the expression profile of pluripotent genes in CSC and their modulation by treatments. By RT-PCR we found that ATRA (48 h) induced a decrease in PKCα in LEP, MEP and CSC. The same treatment increased RARβ2 and RARγ2 in CSC, and only RARβ2 in LEP. ATRA and PKCα inhibitor interaction results from the proliferation assay were analyzed by Chou-Talalay´s method. We found that the inhibitory effect exerted by ATRA/PKCα inhibitor was synergistic with CI=0.59 (Synergistic with CI ATRA/PKCα inhibitor treatment reduced LM38-LP migration more efficiently than each treatment alone, showing a synergic effect (% migration: Control: 80,1±6,9, ATRA: 55,4±5,6, PKCα inhibitor: 37,3±9,5, ATRA/PKCα inhibitor: 20,0±1,7; p≤0.05). Furthermore, MMP2 activity was strongly reduced by the combined treatment. Interestingly, we observed that only the combined treatment induced a decrease of RARγ2 expression in the highly migratory MEP cells. ATRA/PKCα inhibitor treatment synergized to reduce mammospheres growth (Diameter in µm at 96h: Control: 176±8, ATRA: 129±10; PKCα inhibitor: 130±11 ATRA/PKCα inhibitor: 103±5 p≤0.05). Pre-treatment with ATRA/PKCα inhibitor for 96h dramatically affected CSC self-renewal (Number of secondary mammospheres: Control: 313±19; ATRA/PKCα inhibitor: 69±21; p≤0.05). While in a 3D matrigel culture assay ATRA-pretreated CSC formed organized colonies with presence of lumen, the combined treatment led to the formation of small undifferentiated structures and evidence of cell death. By RT-PCR we determined that CSC expressed higher levels of pluripotent genes, such as Nanog, Sox2, Slug and Sox9, than the parental LM38-LP cell line. Besides, only the combined treatment for 96 h induced a decrease in the levels of Nanog and Slug in CSC. Our findings suggest that the pharmacological inhibition of PKCα and ATRA synergize to inhibit proliferation and migration possibly through the decrease of RARγ and the inhibition of MMP2 activity. Furthermore, the blockage of CSC expansion by the combined treatment, possibly occurred through the down regulation of Nanog and Slug. Citation Format: Damian E. Berardi, Maria I. Diaz Bessone, Carolina Flumian, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Pharmacological inhibition of protein kinase C alpha (PKCα) and all trans retinoic acid (ATRA) synergize to inhibit the proliferation, migration and cancer stem-like properties of a triple-negative mammary cancer model. [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 209. doi:10.1158/1538-7445.AM2014-209

Anaplastic large cell lymphoma (ALCL) is a subtype of peripheral T-cell lymphoma that includes two entities ALK+ and ALK- ALCL based on chromosomal translocations of the anaplastic lymphoma kinase (ALK). ALK- ALCL have very poor clinical outcome with conventional chemotherapy. Lack of representative cell lines and/or models has compromised the development of successful therapy. Toward this end, we report the generation and characterization of a group of novel ALCL tumor grafts. We implanted subcutaneously (s.c.) tumor tissue fragments, fresh or viable cryopreserved, from 11 cases of primary systemic ALCL (3 ALK+ and 8 ALK-) in six- to eight-week old severe immunocombined immunodeficiency NOD Cg-Prkdcscid/Il2rgtm1wjl/SzJ (NSG) mice. A single leukaemic ALCL sample was injected i.v. or s.c. Six cases led to successful lymphoma growth within 6-12 weeks as subcutaneous solid spheroid masses constituted by a uniform population of large pleomorphic cells CD30-positive. All tumor grafts were re-implanted in recipient animals, in same cases up to 21 consecutive implants with an analogous time growth rate. Two cases, in the same way of the original human neoplasia, were early associated with concomitant enlargement of multiple organs. All tumor graft lines eventually showed disseminate disease with involvement of distant lymph nodes and of spleen (with homig in peri-arteriolar spaces), liver (initially observed within the sinusoidal spaces) and kidney. The morphological and immunohistochemical (IHC) evaluation (expression of CD30, cytotoxic proteins and ALK) of the serially propagated ALCL tumor grafts demonstrated a high concordance between primary and derived animal tumors which showed highly stable profile along different passages from the early generation (T1) until the final ones (up to T16). The clonal relationship with primary and corresponding tumor grafts was confirmed by gene TCR rearrangement analyses and by ALK FISH studies. In addition we interrogated the model using gene expression profiling and single nucleotide polymorphism which established a close relationship among primary and derived tumors. Massive parallel sequencing on mRNA transcripts defines the presence of two novel chimeras involving NF1-MAZ and TRAF1-ALK1, which confirmes the evidence of ALK signaling and pinpoint a role of RAS in ALK- ALCL. The tumor grafts displayed, also, therapeutic responses with conventional therapy (CHOP) which reflected that seen in the donor patients: initial partial responses followed invariably by clinical relapses. To overcome this refractory we assayed the sensitivity of two tumorgraft lines, generated from ALK+ ALCL, to a new selective ALK inhibitor (CEP28122) that led a rapid and stable response. This study demonstrated the key role of these libraries of tumor grafts, especially in developing and testing new therapeutic regimens in refractory ALCL patients. Citation Format: Fabrizio Tabbo’, Antonella Barreca, Rodolfo Machiorlatti, Katia Messana, Indira Landra, Francesco Abate, Michela Boi, Maria Todaro, Cristina Abele, Domenico Novero, Alberto Zamo’, Marco Chilosi, Maurilio Ponzoni, Mangeng Cheng, Bruce Ruggeri, Pierpaolo Piccaluga, Stefano Pileri, Enrico Tiacci, Brunangelo Falini, Lenny Shultz, Roberto Piva, Enzo Medico, Francesco Bertoni, Raul Rabadan, Giorgio Inghirami. Humanized NOD/Scid/IL2g-/- tumor grafts recapitulate primary anaplastic large cell lymphoma. [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 3853. doi:10.1158/1538-7445.AM2013-3853

Retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through interactions with specific Protein Kinase C (PKC) isoforms. In this work we studied: A)- the modulation of PKCα and PKCδ expression when cells were treated with ATRA (1 μM), B)- The expression profile of Retinoic Acid Receptors (RAR) when cells are treated with ATRA and/or PKCα / PKC δ inhibitors (Gö6976 2.5 μM / Rottlerin 1 μM) and C)- the effect of combining ATRA and PKC inhibitors on in vitro cell growth, cell cycle distribution and senescence. We used as model the murine mammary tumor-derived cell line LM38-LP, composed by luminal (LEP) and myoepithelial (MEP) cells. By RT-PCR we could determine that 48 h ATRA treatment increased PKCδ only in the LM38-LP LEP population and decreased PKCα expression levels in both cellular components. Regarding RARs profile, the treatment with PKCα inhibitor Gö6976 reduced RARγ2 levels, while its combination with ATRA also increased RARβ2. Furthermore, 96 h treatment with ATRA, Gö6976, or Rottlerin or their combinations induced cell growth inhibition (cell number x105: control 22± 1.1; ATRA 11±0.1; Gö6976 3.6±0.6; Rottlerin 9±0.1; ATRA/Gö6976 2.3±0.03; ATRA/Rottlerin 6.7±1.3, p ≤0.05 vs control in all treatments). By flow cytometry we could determine that both ATRA and Rottlerin treatments induce LM38-LP cell cycle arrest in G1 phase, while Gö6976 increased the number of cells in the subG1 phase, correlating with increased apoptosis and confirmed by Annexin V-FITC staining. Employing β-galactosidase activity assay and bright field microscopy, we determined that 96 h ATRA treatment induced senescence mainly in the MEP component while Gö6976 does it in the LEP population. Interestingly, Rottlerin treated cells showed morphological alterations compatible with autophagy. We conclude that cell growth inhibition exerted by retinoids may be enhanced by the reduction in PKCα expression and / or activity. In addition the combination of ATRA plus Gö6976, induced differential responses on the different LM38-LP cell components, showing senescence in MEP cells, together with cell cycle arrest and apoptosis mainly in the LEP compartment. Consistently, all these biological effects correlated with an increase in RARβ2 (known to induce cell differentiation) and a decrease in RARγ2 (implicated in proliferation) mRNAs level. Citation Format: Damian E. Berardi, Maria I. Diaz Bessone, Carolina Flumian, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Proliferation, apoptosis and senescence differential modulation by combining the retinoid ATRA and Protein Kinase C pharmacological inhibitors, in a bicellular murine mammary tumor cell line. [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 555. doi:10.1158/1538-7445.AM2013-555

CK2 is a serine/threonine kinase involved in cell growth, survival and apoptosis. The CIGB-300 is a synthetic peptide capable of binding to CK2 substrates thus preventing the enzyme activity. In this work we have studied the effect of IGBC-300 on several signaling pathways involved in tumor progression, cell cycle, apoptosis and biological effects associated metastatic dissemination, using a model of human and murine lung cancer cell lines (H125 and 3LL respectively). CIGB-300 presented a lethal dose 50 (LD50) of 119±2.4 μM in H125 cells and 138.3±9.9 μM in 3LL cells. Throughout an Annexin V-FITC assay we could determine that CIGB-300 is a potent apoptosis inductor since the treatment with this drug induced apoptosis at a level comparable with that induced by 10 μM of etoposide. Besides, concomitantly with cell death induction, we could observe the activation of caspase-3 and decreased expression of c-myc and cyclin D1 and D2. The levels of nuclear and cytoplasmic components of the Wnt and NFB pathways were analyzed by Western blot after treatment with CIGB-300. In the Wnt pathway, a significant decrease in nuclear β-catenin levels could be detected. Related to NFB pathway, a decrease in the nuclear p65 levels was observed, without affecting cytoplasmic levels of the IBα inhibitor. Both phenomena could be associated with a reduction in cell survival. Related to metastatic dissemination, we studied in vitro the effect of CIGB-300 on the modulation of the migratory capacity using a wound healing assay. CIGB-300 induced a significant reduction in the migratory potential in a dose dependent way in both 3LL cells (18.86 ± 2.2% vs 32.99 ± 2.7% ¼ and ½ LD50 respectively p Our results indicate that the treatment with CIGB-300 induces apoptosis and negatively modulates several signaling pathways involved in malignant progression besides affecting cell motility. This drug may become in the future a new strategy for the treatment of lung cancer, based on the blockade of different signaling pathways in lung cells. Citation Format: Alejandro J. Urtreger, Stefano M. Cirigliano, Maria I. Díaz Bessone, Carolina Flumián, Damian E. Berardi, Elisa D. Bal de Kier Joffé, Laura B. Todaro, Hernan G. Farina. Effect of the CIGB-300 synthetic peptide in the modulation of signaling pathways involved with malignant progression of human and murine lung tumors. [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 5256. doi:10.1158/1538-7445.AM2013-5256

Some of the most promising signaling pathways for breast cancer treatment include the PKC family, involved in proliferation and apoptosis, and the retinoid system mainly involved in differentiation. In this work we have overexpressed α or δ PKC isoforms in two murine mammary models, a tumor-derived cell line (LM3) and a normal mammary gland-derived line (NMuMG), in order to analyze whether elevated expression of these kinases alter the sensitivity to retinoid treatment. The overexpression of α or δ PKC isozymes did not induce detectable morphological alterations either in LM3 or in NMuMG cell lines. In the transformed cell context, the overexpression of PKCα increased cell proliferation (population doubling time: 14.8±2.3 h vs 21.2±3.1 h in control cells p On the contrary, PKCδ overexpression did not affect either LM3 cells in vitro behavior or their response to ATRA. However, through a RARE-luciferase gene reporter assay, we could determine that the overexpression of PKCδ increased the activity of the retinoic acid receptors. In the normal cell context (NMuMG cells), the overexpression of both PKCs did not induce alterations in the proliferative potential (population doubling time: 14.8±0.8 h) or cell motility (wound coverage: 37.8±1.6%). Moreover, the subsequent treatment with ATRA neither affected the above mentioned parameters. Upon othotopic inoculation into syngeneic mice, LM3-PKCα cells formed tumors with higher growth rate and metastatic potential than LM3-vector ones. In vivo treatment with a subcutaneous ATRA pellet reduced both the local tumor growth and the number of spontaneous lung metastases (Md [Range]: 9 [0-27] vs 55 [20-75] for LM3-PKCα treated or not respectively p Our results show that in an already transformed context, PKCα overexpression induced a more aggressive phenotype but also conferred sensitivity to the retinoid treatment. On the other side high PKCδ levels increased the activity of retinoid receptors but did not modulate biological cell responses to this compound. In a normal cell context, both PKC isoforms as well as the retinoid treatment are innocuous thus ensuring the safeness use of retinoids in patients with aggressive breast tumors associated with high PKCα expression. Citation Format: María I. Díaz Bessone, Damian E. Berardi, Stefano M. Cirigliano, Carolina Flumian, Elisa D. Bal de Kier Joffe, Laura B. Todaro, Alejandro J. Urtreger. Differential response to retinoid treatment of normal and transformed murine mammary cells overexpressing protein kinase C (PKC) isoforms. [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 554. doi:10.1158/1538-7445.AM2013-554

Retinoids may exert some of their effects on cell differentiation and malignant phenotype reversion through interaction with different PKC isoforms. In this work we studied the crosstalk between retinoid acid system (RA) and PKC signaling pathway and its implication in malignant phenotype reversion using a murine mammary tumor cell line (LM3) and a human breast cancer-derived cell line (MDA-MB231). ATRA (all trans RA) treatment (1uM,72h) induced a significant growth inhibition in vitro in LM3 cell line. This result was confirmed in vivo when LM3 cells were injected orthotopically in female BALB/c mice while carrying subcutaneous slow release ATRA pellets (10 mg). This phenomenon was associated with the reduction of pErk1 levels (80±9%) and the increase of the cell cycle inhibitor p27 in the nuclear fraction without altering Cyclin D1 expression in LM3 cell line. None of these modulations were observed in ATRA unresponsive MDA-MB231 cell line. We determined by Western blot that 24h treatment with ATRA induced an increase of PKC∈ and PKC∈ levels, detecting alpha isoform only in the membrane fraction and delta isoform in the nuclear fraction. In contrast, in MDA-MB231 cells, PKC∈ and PKC∈ expression was reduced after ATRA exposure. Interestingly, pharmacological inhibition of PKC∈ and PKC∈ prevented retinoid receptors activation by ATRA in LM3 cells, as evidenced by a reporter gene assay (RARE-Luciferase). Western blot showed that pharmacological inhibition of PKC∈ (Rottlerin 1μM, 24h) impaired ATRA-induced RARα1 translocation to the nucleus while the pharmacological inhibition of PKC∈ (Gö6976 5μM, 24h) did not affect this translocation. Moreover, immunoprecipitation assays showed that only PKC∈ co-immunoprecipitated with RARα1 after ATRA treatment, suggesting a physical interaction between both molecules. Rottlerin-ATRA treatment (72h) reversed the effect on proliferation rate exerted by retinoid treatment. Gö6976-ATRA treatment (72h) significantly decreased cell duplication rate compared with ATRA or Gö6976 treatment alone, in an additive manner. Pre-treated LM3 cells with Gö6976 were intravenously injected in female BALB/c mice that carried subcutaneous slow release ATRA pellets in order to evaluate the importance of combination therapy in the last stages of metastatic spread. We noticed that this combined therapy produced a higher decrease in the number of lung metastases, suggesting an additive effect. Our results indicate that PKC/Retinoid Crosstalk is implicated in growth inhibition through the induction of RA transcription genes that lead to cell cycle arrest and differentiation. Finally we speculate that retinoid treatment combined with PKC∈ isoform modulators could be considered for treatment of PKC∈ positive breast cancer patients. 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 3015. doi:1538-7445.AM2012-3015

A personalized approach towards the therapy of colorectal carcinoma is critical for a successful outcome. In recent years, direct transfer xenograft models (or the “tumor graft”) have proven to be highly predictive and are being adapted in clinical practice. However, direct drug evaluation in tumor graft models for each individual patient is not feasible due to time and cost constraints. Predictive biomarker discovery is an extremely promising application of the tumor graft model, since the discovered biomarker signatures will direct therapy choice in a much broader patient population. In collaboration with the Catholic Health Initiatives Center for Translational Research we at Caliper Life Sciences have established a collection of tumor graft samples of primary human colorectal carcinomas. All samples were collected fresh from consenting patients following an IRB-approved protocol and implanted in vivo on the day of surgical tumor resection. Tumors were grafted either orthotopically or subcutaneously in female NIH-III mice. We present here the results of a comparative study of gene and protein expression profiles for the primary tumor (clinical samples) and the direct transfer xenografts. These data will be correlated with drug sensitivity profiles generated by using the same samples. The resulting data sets will be used to characterize predictive biomarker signatures that will be a valuable tool for selection of the most effective therapy tailored to the individual patient. 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 1319. doi:1538-7445.AM2012-1319

Some of the most promising signaling pathways for breast cancer treatment include the PKC family, involved in proliferation and apoptosis, and the retinoid system mainly involved in differentiation. In this work we have developed a murine model of mammary adenocarcinoma-derived cells (LM3) that overexpress α or ≤ PKC isoforms in order to analyze whether elevated levels of these kinases alter cell sensitivity to retinoid treatment (ATRA). LM3 cells are poorly responsive to ATRA. The overexpression of PKCα induced alterations in LM3 in vitro behavior that could be associated with tumor progression. In this sense, PKCα increased proliferation and motility (wound coverage: 69.3±8.3% vs 41.2±5.1% in control cells p 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 172. doi:1538-7445.AM2012-172