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J Cancer Metastasis Treat 2019;5:5 I http://dx.doi.org/10.20517/2394-4722.2018.108 Page 15 of 27

and anogenital malignancies, mainly promoted by the high risk (HR) α-HPV16 E6E7 oncoproteins. Despite
scientific progresses and the development of vaccines, these tumors are still common and represent one of
the major causes of women’s death. Host’s cell replication fidelity depends by the DNA damage response
(DDR). Unlike from other DNA viruses, HR-HPVs encourage cells proliferation without inactivating
the DDR: the mechanisms at the basis haven’t been clarified yet. During HPV16 infection, E6 binds and
degrades p53 through the E6AP LXXLL domain. Similarly, E7 competes with E2F1-pRb interaction, thus
inactivating pRb, and promoting the linking the pRb-like proteins CBP/p300 and p107, that also harbor a
LXXLL sequence. Unfortunately, E6 E7 role in the DDR activation is not elucidated yet.

Experimental model: To gain new information, we reproduced an in vitro 3D HPV16-E6E7 infected
epithelium, already characterized for HPVs studies, and checked for cellular and viral markers, among
them HPV16E6E7 oncoproteins and the double strand breaks sensor 53BP1; we then made a Co-IF for E6
and E7 with 53BP1. Since E6 and E7 both interact with LXXLL containing proteins, we analyzed 53BP1
BRCT2 domain and we explored the binding hypothesis via the in situ PLA technique in 2D in CaSki and
E6E7HPV16 keratinocytes and in the 3D model.

Results: The in vitro infected epithelium resembles the in vivo tissue. E6E7HPV16, both in basal and
differentiated strata, induce a 53BP1 increase in nuclear foci. After highlighting E6 and E7 co-expression
with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the binding in all
the employed cellular models.

Conclusion: Our results add new information on HPV16 oncoproteins capability in overcoming cellular
defense strategies.

21. Iron metabolism regulates cancer related skeletal muscle wasting

Erica Mina, Elisabeth Wyart, Myriam Hsu, Paolo Ettore Porporato

Molecular Biotechnology Centre, Department of Molecular Biotechnology and health sciences, University of
Torino, Torino 10126, Italy.

Introduction: Cancer associated cachexia is a syndrome characterized by a significant weight loss, due
to metabolic changes affecting skeletal muscle and adipose tissue2018. Given the importance of iron in
controlling energy metabolism, we speculated that decreased iron availability occurring in cancer might
contribute to skeletal muscle atrophy.

Experimental model: In vitro experiments are performed with C2C12 myotubes, while in vivo experiments
are conducted using Colon-26 carcinoma bearing BALB-C mice.

Results: Interestingly, using Colon-26 carcinoma bearing mice, we found strong alterations in protein and
gene expression of iron homeostasis key players in skeletal muscle, notably a downregulation of transferrin
receptor 1 (TFR1, the main importer of iron) and an increase of the iron exporter Ferroportin. Coherently,
we observed a decreased iron content in both skeletal muscle and spleen while serum iron is increased,
suggesting a global mobilization of iron. To further confirm our hypothesis, we created iron-deprived
models in vitro using several iron chelators (Desferoxamine, BPS) or by TFR1 knockdown with siRNA and,
in line with our in vivo observations, iron depletion in C2C12 myotubes directly promoted atrophy. Finally,
we were able to prevent myotube atrophy by restoring iron transport.

Conclusion: Taken together, these results highlight a previously unknown role for altered iron homeostasis
in cancer-induced muscle wasting and provide a potential new therapeutic target.